2008 Book IntegratedManagementOfDiseases

Integrated Management of Diseases Caused by Fungi, Phytoplasma
and Bacteria
Integrated Management of Plant Pests and Diseases
Published:
Volume 1
General Concepts in Integrated Pest and Disease Management
edited by A. Ciancio and K.G. Mukerji
ISBN 978-1-4020-6060-1
Volume 2
Integrated Management and Biocontrol of Vegetable and Grain
Crops Nematodes
ISBN 978-1-4020-6062-5
Forthcoming:
Volume 4
Integrated Management of Fruit Crops and Forest Nematodes
Volume 5
Integrated Management of Arthropod Pests
and Insect Borne Diseases
Integrated Management
of Diseases Caused by Fungi,
Phytoplasma and Bacteria
Edited by
A. Ciancio
C.N.R., Bari, Italy
and
K.G. Mukerji
University of Delhi, India
Editors
Aurelio Ciancio K.G. Mukerji
Consiglio Nazionale delle University of Delhi
Ricerche, Dipartimento Dept. Botany
Agroalimentare, New Delhi-110007
Istituto per la Protezione delle India
Piante
Via G. Amendola, 122/D
70126 Bari
Italy
a.ciancio@ba.ipp.cnr.it
Cover Illustration: Bacterial Spot Fruit lesions. (Courtesy Jeffrey B. Jones)
ISBN: 978-1-4020-8570-3 e-ISBN: 978-1-4020-8571-0
Library of Congress Control Number: 2008927634

c 2008 Springer Science+Business Media B.V.
No part of this work may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming, recording
or otherwise, without written permission from the Publisher, with the exception
of any material supplied specifically for the purpose of being entered
and executed on a computer system, for exclusive use by the purchaser of the work.
Printed on acid-free paper
9 8 7 6 5 4 3 2 1
springer.com
CONTENTS
Contributors ............................................................................................................ xv
Preface .................................................................................................................... xix
SECTION 1 - Diseases of Perennial Crops

1 Integrated Management of Stone Fruit Diseases ........................................... 3
A. Peter Sholberg and Frank Kappel
1. Introduction .................................................................................................... 3
2. Brown Rot ...................................................................................................... 5
2.1. Pathogen Identification and Disease Biology.......................................... 5
2.2. Integrated Management of Brown Rot .................................................... 8
3. Bacterial Canker........................................................................................... 12
3.1. Pathogen Identification and Disease Cycle ........................................... 12
3.2. Integrated Management of Bacterial Canker ......................................... 13
4. Leucostoma Canker...................................................................................... 15
4.2. Integrated Management of Leucostoma Canker .................................... 15
5. Powdery Mildew .......................................................................................... 16
5.2. Integrated Management of Powdery Mildew ......................................... 17
6. Postharvest Fruit Rots .................................................................................. 18
6.2. Integrated Control of Postharvest Fruit Rots ......................................... 19
References......................................................................................................... 21
2 Towards a Sustainable, Integrated Management of Apple Diseases .......... 27

Ralph L. Nicholson and Janna Beckerman
1. Introduction .................................................................................................. 27
2. The Spring Diseases ..................................................................................... 28
2.1. Apple Scab ............................................................................................ 28
2.1.2. Symptoms.................................................................................... 28
2.1.3. The Causal Pathogen ................................................................... 28
2.1.4. Disease Cycle .............................................................................. 28
2.1.5. Management ................................................................................ 29
2.2. Powdery Mildew .................................................................................. 33
2.2.1. Disease Cycle .............................................................................. 33
2.2.2. Management ................................................................................ 33
2.3. Fire Blight ............................................................................................. 34
2.3.1. Symptoms.................................................................................... 34
2.3.2. Management ................................................................................ 34
2.3.3. Root Stocks ................................................................................. 34
v
vi CONTENTS
2.3.4. Cultivars ...................................................................................... 35

2.3.5. Cultural Management .................................................................. 35
2.3.6. Chemical Management and Predictive Models ........................... 36
2.3.7. Removing Sources of Infection ................................................... 36
2.4. Rust Diseases......................................................................................... 37
2.4.1. Disease Cycle .............................................................................. 37
2.4.2. Management ................................................................................ 38
3. Summer Diseases ......................................................................................... 38
3.1. Bitter Rot ............................................................................................... 38
3.1.1. Symptoms.................................................................................... 38
3.1.2. Disease Cycle .............................................................................. 39
3.1.3. Management ................................................................................ 39
3.2. Flyspeck and Sooty Blotch .................................................................... 39
3.3. Disease Cycle ........................................................................................ 39
3.4. Management ......................................................................................... 40
4. Conclusions .................................................................................................. 40
References......................................................................................................... 41
3 Management and Ecology of Phytoplasma Diseases of Grapevine

and Fruit Crops............................................................................................... 43
Rita Musetti
1. Introduction .................................................................................................. 43
2. Phytoplasmas Diagnosis in Crops ................................................................ 46
3. Natural Transmission and Epidemiology ..................................................... 47
4. Plant Recovery ............................................................................................. 48
5. Phytoplasma Diseases and Management...................................................... 48
5.1. Grapevine Phytoplasmas ....................................................................... 49
5.1.1. Flavescence Dorée....................................................................... 50
5.1.2. Bois Noir ..................................................................................... 51
5.2. Fruit Trees Phytoplasmas ...................................................................... 52
5.2.1. Apple Proliferation .................................................................... 53
5.2.2. European Stone Fruit Yellows..................................................... 54
5.2.3. Pear Decline ................................................................................ 55
6. New Approaches and Perspectives............................................................... 56
References......................................................................................................... 56
4 Management of Citrus Diseases Caused by Phytophthora spp.................... 61

Santa Olga Cacciola and Gaetano Magnano Di San Lio
1. Introduction .................................................................................................. 61
2. Damages Caused by Phytophtora Root Rot ................................................. 62
2.1. Causal Agents........................................................................................ 64
2.2. Biology and Ecology ............................................................................. 65
2.2.1. Dissemination and Reproduction ............................................... 65
2.3. Epidemiology ........................................................................................ 65
CONTENTS vii
2.4. Symptomatic Diagnosis......................................................................... 67

2.4.1. Foot Rot or Gummosis ................................................................ 67
2.4.2. Fibrous Root Rot ........................................................................ 67
2.4.3. Brown Fruit Rot and Dieback of Twigs and Leaves ................... 68
2.5. Biological and Instrumental Diagnosis.................................................. 68
2.5.1. Baits............................................................................................. 68
2.5.2. Laboratory Analysis .................................................................... 68
2.5.3. Monitoring of Inoculum .............................................................. 69
2.5.3.1. Definition ....................................................................... 69
2.5.3.2. Critical Values of Inoculum Density.............................. 69
2.5.3.3. Sampling ....................................................................... 70
2.5.3.4. Population Dynamics ...................................................... 71
2.5.3.5. Molecular Methods ......................................................... 71
3. Disease Management.................................................................................... 71
3.1. Interventions on the Host-Plant ............................................................. 71
3.1.1. Rootstock..................................................................................... 71
3.1.2. Grafting ....................................................................................... 72
3.1.3. Sanitary Practices in Nurseries .................................................... 72
3.1.4. Pruning ........................................................................................ 74
3.1.5. Surgery ........................................................................................ 74
3.2. Cultural Practices................................................................................... 74
3.2.1. Soil Preparation ........................................................................... 74
3.2.2. Irrigation Management ................................................................ 74
3.2.3. Fertilising .................................................................................... 75
3.2.4. Soil Management and Weeds Control ........................................ 76
3.3. Chemical Control................................................................................... 77
3.3.1. Systemic Fungicides.................................................................... 77
3.3.1.1. Trunk Gummosis ........................................................... 78
3.3.1.2. Root Rot......................................................................... 79
3.3.1.3. Brown Rot of Fruit......................................................... 80
4. Conclusions .................................................................................................. 80
References......................................................................................................... 81
5 Biological Control and Management of Chestnut Diseases......................... 85

Tullio Turchetti and Giorgio Maresi
1. Introduction .................................................................................................. 85
2. Diseases Appearence, Epidemic and Actual Situation ................................ 87
2.1. Chestnut Blight...................................................................................... 87
2.2. Ink Disease ............................................................................................ 89
3. Symptomatology .......................................................................................... 90
3.1. Chestnut Blight and Types of Cankers .................................................. 90
3.2. Ink Disease ............................................................................................. 93
4. Evolution of the Diseases ............................................................................. 96
4.1. Spread and Effectiveness of Hypovirulence.......................................... 96
4.2. Morphology, Physiology and ds-RNA Presence and Transmission......... 99
viii CONTENTS
4.3. Mixed Inoculum .................................................................................. 101

4.4. Chestnut Resistance............................................................................. 102
4.5. Environmental Factors......................................................................... 103
4.6. Ecological Factors in Ink Disease........................................................ 104
4.7. Soil Microflora Action......................................................................... 106
6. Diseases Management ............................................................................... 107
6.1. Blight, Silviculture and Biological Control ......................................... 107
6.2. Ink Disease Control ............................................................................. 110
7. Perspectives and Conclusions .................................................................... 111
References....................................................................................................... 112
6 The Esca Disease Complex........................................................................... 119

Giuseppe Surico, Laura Mugnai and Guido Marchi
1. Introduction ................................................................................................ 119
2. The Pathogens of the Esca Disease Complex............................................. 120
2.1. Tracheomycotic Fungi......................................................................... 120
2.2. Basidiomycetes Causing White Rot .................................................... 122
3. Symptoms................................................................................................... 123
3.1. Brown Wood Streaking of Rooted Cuttings ........................................ 123
3.2. Petri Disease (or ‘Black Goo’) ............................................................ 123
3.3. Esca (Young Esca) .............................................................................. 124
3.4. White Rot ............................................................................................ 125
3.5. Esca Proper.......................................................................................... 126
3.6. Apoplexy ............................................................................................. 127
4. Source of Inoculum and Spread ................................................................. 127
4.1. Infection Routes and Disease Distribution in the Vineyard................. 128
5. Control ....................................................................................................... 130
5.1. Control in the Nursery ......................................................................... 130
5.2. Control in the Field.............................................................................. 131
6. Conclusions ................................................................................................ 133
References....................................................................................................... 133
7 Integrated Management of Rosellinia necatrix Root Rot on Fruit

Tree Crops .................................................................................................... 137
Leonardo Schena, Franco Nigro and Antonio Ippolito
1. Introduction ................................................................................................ 137
2. Taxonomy .................................................................................................. 138
3. Host Range and Geographic Distribution................................................... 138
4. Symptoms................................................................................................... 140
5. Disease Cycle and Epidemiology............................................................... 143
5.1. Survival ............................................................................................... 143
5.2. Dispersal.............................................................................................. 144
5.3. Infection Process ................................................................................. 146
CONTENTS ix
6. Control ....................................................................................................... 146

6.1. Healthy Propagative Materials ........................................................... 147
6.1.1. Current Legislation in Europe .................................................. 147
6.1.2. Diagnostic Tools ....................................................................... 148
6.2. Cultural Control Methods.................................................................... 149
6.3. Fumigation........................................................................................... 150
6.4. Chemical Control................................................................................. 151
6.5. Physical Control .................................................................................. 151
6.6. Biological Control ............................................................................... 153
References....................................................................................................... 154
SECTION 2 - Diseases of Annual Crops

8 Simulation Models for Potato Late Blight Management and Ecology ..... 161
G. A. Forbes, W. E. Fry, J. L. Andrade-Piedra and D. Shtienberg
1. Introduction ................................................................................................ 161
2. Plant Disease Simulation............................................................................ 162
2.1. Simulation vs. Forecasting .................................................................. 162
2.2. The Cornell Experience ...................................................................... 163
2.3. Globalizing LB1990 .......................................................................... 164
3. Other Simulation Models ........................................................................... 168
4. Innovation and Future Directions for Late Blight Simulation.................... 169
4.1. Comparative Epidemiology................................................................. 169
4.2. Biological Control ............................................................................... 172
4.3. Geographic Zonation and Impact Assessment..................................... 173
4.4. Plant Breeding - Predicting Resistance Performance .......................... 173
4.5. Training ............................................................................................... 174
References....................................................................................................... 174
9 An example of Integrated Forecasting System for Phytophthora

infestans on Potato ........................................................................................ 179
Jan Hadders
1. Introduction ................................................................................................ 179
2. Disease Forecasting Models & Principles .................................................. 181
2.1. Sub Model 1a - Unprotected Crop by Growth of New Leaves............ 181
2.2. Sub Model 1b - Unprotected Crop by Degradation
and Wear-Off of Chemicals................................................................. 182
2.3. Sub Model 1- Unprotected Part of the Crop ........................................ 182
2.4. Sub Model 2 - Infection Events of the Disease ................................... 182
2.5. Sub Model 2a - Formation of Spores on Each Infected Leaf .............. 183
2.6. Sub Model 2b - Ejection and Dispersal of Spores into the Air............ 183
2.7. Sub Model 2c - Spores Germination and Penetration into
Unprotected Leaves ............................................................................ 183
x CONTENTS
2.8. Sub Model 3: Combination of Unprotected Leaf Area

and Infection Events into Advice ........................................................ 185
3. Quality of Weather Forecasts ..................................................................... 186
4. Future Developments and Constraints........................................................ 187
References....................................................................................................... 188
10 Integrated Pest Management of Bacterial Fruit Blotch of Cucurbits ...... 191

Ron R. Walcott
1. Introduction ................................................................................................ 191
2. Background ................................................................................................ 192
2.1. Brief History........................................................................................ 192
2.2. BFB Etiology and Symptomatology.................................................... 193
2.3. Host Range and Geographic Distribution............................................ 195
2.4. Epidemiology ...................................................................................... 195
2.4.1. Seed Production......................................................................... 195
2.4.2. Transplant Production ............................................................. 196
2.4.3. Fruit Production Fields.............................................................. 197
2.5. Trends in Commercial Cucurbit Production........................................ 197
2.5.1. Hybrid Watermelon Cultivars .................................................. 197
2.5.2. Seedling Production .................................................................. 199
3. Integrated Management.............................................................................. 199
3.1. Avoidance............................................................................................ 199
3.2. Exclusion ............................................................................................. 200
3.2.1. Seed Health Testing................................................................... 200
3.2.2. Seed Treatments ........................................................................ 202
3.3. Protection............................................................................................. 204
3.3.1. Biocontrol Blossom Protection to Limit Seed Infection............ 204
3.4. Eradication........................................................................................... 204
3.5. Resistance............................................................................................ 205
4. Conclusions ................................................................................................ 206
References....................................................................................................... 206
11 Integrated Management of Tomato Bacterial Spot ................................... 211

A. Obradovic, J. B. Jones, B. Balogh and M. T. Momol
1. Introduction .............................................................................................. 211
2. Tomato Production .................................................................................... 212
2.1. Tomato Production Value.................................................................... 212
2.2. Cultivation Technologies..................................................................... 213
2.3. Tomato Bacterial Diseases .................................................................. 213
3. Bacterial Spot of Tomato ........................................................................... 214
3.1. Historical Perspective .......................................................................... 214
3.2. Host-Pathogen Interactions.................................................................. 215
3.2.1. Host Range ............................................................................... 215
3.2.2. Resistance and Avirulence Genes ............................................. 215
CONTENTS xi
3.3. Distribution of Pathogen Groups ........................................................ 216

3.4. Ecology and Epidemiology ................................................................ 216
4. Integrated Approach to Bacterial Spot Management.................................. 217
4.1. Bacterial Spot Control Practices and Recent Trials ............................ 217
4.2. Integrated Strategies ........................................................................... 219
References....................................................................................................... 221
12 Integrated Management of Verticillium Wilt of Tomato .......................... 225

Giovanni Bubici and Matteo Cirulli
1. Introduction ................................................................................................ 225
2. Integrated Control ...................................................................................... 228
2.1. Selecting Soil for Cultivation .............................................................. 228
2.2. Heat ..................................................................................................... 229
2.3. Solarization.......................................................................................... 229
2.4. Sanitation............................................................................................. 230
2.5. Tillage.................................................................................................. 230
2.6. Plant Residues ..................................................................................... 231
2.7. Weed Control....................................................................................... 231
2.8. Resistant Rootstocks............................................................................ 231
2.9. Cultivars .............................................................................................. 232
2.10. Fertilization ....................................................................................... 232
2.11. Irrigation............................................................................................ 233
2.12. Chemicals .......................................................................................... 234
2.13. Microbial Antagonists ....................................................................... 235
References....................................................................................................... 237
13 New Progress in the Integrated Management of Sclerotinia Rot

of Carrot ....................................................................................................... 243
Cezarina Kora, Mary Ruth McDonald and Greg J. Boland
1. Introduction .............................................................................................. 243
2. The Disease ................................................................................................ 244
2.1. Damage and Symptoms ....................................................................... 244
2.2. Causal Organism ................................................................................. 245
2.3. Etiology and Epidemiology ................................................................. 246
2.3.1. Preharvest Epidemics ................................................................ 247
2.3.2. Postharvest Epidemics............................................................... 249
3. Disease Management.................................................................................. 249
3.1. Field Practices ..................................................................................... 250
3.1.1. Cultural Control ........................................................................ 250
3.1.2. Host Resistance ........................................................................ 252
3.1.3. Biological Control ..................................................................... 253
3.1.4. Chemical Control ...................................................................... 255
3.1.5. Disease Forecasting................................................................... 255
xii CONTENTS
3.2. Storage Practices ................................................................................. 258

3.2.1. Cultural Control......................................................................... 258
3.2.2. Biological Control ..................................................................... 259
3.2.3. Alternative Methods ................................................................. 259
3.2.4. Chemical Control ...................................................................... 260
4. Recommendations on Integrated Management .......................................... 261
4.1. Reduction of Inoculum ...................................................................... 261
4.2. Reduction of Infection Rate ................................................................ 262
4.3. Reduction of Epidemic Duration ......................................................... 262
4.4. Proposed Integrated Disease Management Programs ......................... 262
5. Conclusions and Future Prospects.............................................................. 263
References....................................................................................................... 264
14 Integrated Management of Key Diseases of Cotton and Rice ................... 271

O. P. Sharma and O. M. Bambawale
1. Introduction ................................................................................................ 271
2. Identification of Diseases ........................................................................... 272
3. The Concept of Integrated Disease Management....................................... 273
4. Integrated Disease Management in Cotton................................................. 274
4.1. Seedling Diseases ................................................................................ 275
4.2. Bacterial Blight ................................................................................... 276
4.3. Alternaria Leaf Spot ............................................................................ 277
4.4. Grey Mildew........................................................................................ 277
4.5. Myrothecium Leaf Spot....................................................................... 278
4.6. Cercospora Leaf Spot ......................................................................... 279
4.7. Helminthosporium Leaf Spot ............................................................. 279
4.8. Macrophomina Leaf Spot and Stem Canker........................................ 280
4.9. Late Season Phoma Blight................................................................... 280
4.10. Rust ................................................................................................... 280
4.11. Leaf Crumple..................................................................................... 281
4.12. Cotton Leaf Curl Virus (CLCV)........................................................ 281
4.13. Tobacco Streak Virus ........................................................................ 281
4.14. Root Rot ........................................................................................... 282
4.15. Verticillium Wilt ............................................................................... 283
4.16. Fusarium Wilt ................................................................................... 283
4.17. New Wilt or Parawilt ........................................................................ 284
4.18. Boll Rots and Lint Diseases .............................................................. 285
5. Integrated Disease Management in Rice .................................................... 286
5.1. Blast..................................................................................................... 287
5.2. Brown Spot ......................................................................................... 289
5.3. Bacterial Leaf Blight .......................................................................... 290
5.4. Bacterial Leaf Streak .......................................................................... 291
5.5. Sheath Blight ...................................................................................... 292
5.6. Sheath Rot ......................................................................................... 294
5.7. Fusarium Wilt or “Bakanae” .............................................................. 294
CONTENTS xiii
5.8. Stem Rot ............................................................................................. 295

5.9. Tungro Virus ...................................................................................... 295
5.10. False Smut ........................................................................................ 296
5.11. Post-Harvest Diseases ....................................................................... 297
6. Conclusions ................................................................................................ 297
References....................................................................................................... 299
15 Biological and Integrated Means to Control Rust Diseases ...................... 303

Salvatore Moricca and Alessandro Ragazzi
1. Introduction ................................................................................................ 303
2. Biological Control ...................................................................................... 305
2.1. Suppression of Rust Agents................................................................. 306
2.1.1. Tuberculina spp......................................................................... 306
2.1.2. Verticillium spp. ........................................................................ 307
2.1.3. Cladosporium spp...................................................................... 308
2.1.4. Sphaerellopsis philum ............................................................... 310
2.1.5. Scytalidium uredinicola............................................................. 310
2.1.6. Aphanocladium album............................................................... 311
3. Diseases Suppression Mechanisms ............................................................ 311
3.1. Competition for Nutrients and Space ................................................. 311
3.2. Direct Parasitism ................................................................................. 312
3.3. Antibiosis............................................................................................. 312
3.4. Induction of Plant Resistance .............................................................. 312
3.5. Improvement of Host Fitness .............................................................. 312
4. Main Problems with Biological Control..................................................... 313
5. Eradication ................................................................................................. 317
6. Defining Hazard Areas............................................................................... 318
6.1. Quarantine ........................................................................................... 319
6.2. Cultural Practices................................................................................. 319
6.3. Chemical Control................................................................................. 320
6.4. Plant Breeding for Resistance.............................................................. 321
7. Conclusions ................................................................................................ 324
References....................................................................................................... 324
SECTION 3 - Advances in Management Tools

16 DNA Fingerprinting Methods for Microbial Pathogens:
Application to Diagnostics, Taxonomy and Plant Disease
Management.................................................................................................. 333
Keith R. Mitchelson and Salvatore Moricca
1. Introduction ................................................................................................ 333
2. Polymorphism Detection Methodologies ................................................... 335
2.1. Genetic Fingerprinting by Fragment Sizing ........................................ 337
xiv CONTENTS
2.1.1. Ribotyping................................................................................. 337

2.2. Ribosomal RNA Detection.................................................................. 338
2.3. Random Genetic Loci.......................................................................... 339
2.3.1. RAPD Fingerprinting ................................................................ 339
2.3.2. AFLPs........................................................................................ 340
2.4. STR Fragment Fingerprinting ............................................................. 341
2.4.1. DNA Shape Analyses ................................................................ 341
3. Combined Analyses.................................................................................... 342
3.1. Genetic Mapping ................................................................................ 343
3.2. PFGE Karyotyping of Fungi for Pathovar Identification .................... 344
4. Gene and Genomic Analysis ...................................................................... 345
4.1. Quantitative Real-Time PCR............................................................... 345
4.2. Microarrays for SNP Genotyping........................................................ 346
4.2.1. Microarray Chip-Based Automated Analysers.......................... 347
4.2.2. Microarray Analysis of Gene Expression.................................. 347
5. DNA Sequence Analysis ............................................................................ 349
5.1. Whole Genome Sequencing ................................................................ 349
5.2. Massively Parallel Sequence Analysis ................................................ 350
5.3. Metagenomic Sequencing.................................................................... 350
5.4. Analysis by Capillary Electrophoresis (CE)........................................ 352
5.4.1. CE Analysis by Size Separation ................................................ 352
5.4.2. CE Analysis by Fragment Shape ............................................... 353
5.4.3. Advanced Analytical Devices ................................................... 353
5.4.3.1. Miniaturized CE-Based Devices.................................. 353
5.4.3.2. Portable Microelectromechanical Systems (MEMS)
for On-Site Analysis ................................................... 354
6. Conclusions ................................................................................................ 355
References....................................................................................................... 355
17 Endophytic Fungi for Pest and Disease Management ............................... 365

Susheel Kumar, Nutan Kaushik, Ruangelie Edrada-Ebel, Rainer Ebel
and Peter Proksch
1. Introduction ................................................................................................ 365
2. Endophytic Fungi ....................................................................................... 366
3. Bioactivity of Endophytic Fungi ................................................................ 367
4. Endophytic Metabolites as Source of New Pesticides................................ 368
5. Conclusions ................................................................................................ 382
References....................................................................................................... 383
Index...................................................................................................................... 389
CONTRIBUTORS
Jorge L. Andrade-Piedra Santa Olga Cacciola

Papa Andina Initiative, Dipartimento di Chimica Biologica,
International Potato Center (CIP), Chimica Medica e Biologia
Apartado 17 21 1977, Molecolare,
Quito, Ecuador University of Catania,
Viale Andrea Doria 6,
95126 Catania, Italy
B. Balogh
Plant Pathology Department,
Matteo Cirulli
Institute of Food and Agricultural
Dipartimento di Biologia e Patologia
Sciences,
Vegetale, Università degli Studi di
University of Florida,
Bari, Via Amendola 165/A,
Gainesville FL 32611, USA
70126 Bari, Italy
O. M. Bambawale Ruangelie Edrada-Ebel,

National Centre for Integrated Pest Institut für Pharmazeutische Biologie
Management, IARI Campus, und Biotechnologie,
New Delhi-110 012, India Heinrich-Heine-Universität
Düsseldorf, Germany
Janna L. Beckerman
Department of Botany and Plant Rainer Ebel
Pathology, Purdue University Institut für Pharmazeutische Biologie
915 West State Street und Biotechnologie,
West Lafayette, IN Heinrich-Heine-Universität
47907-2054 USA Düsseldorf, Germany
Greg J. Boland G. A. Forbes

Department of Environmental International Potato Center,
Biology, University of Guelph, Apartado 1558,
Canada Lima 12, Peru
Giovanni Bubici W. E. Fry

Dipartimento di Biologia e Patologia College of Agriculture and Life
Vegetale, Università degli Studi di Sciences,
Bari, Via Amendola 165/A, Cornell University,
70126 Bari, Italy Ithaca NY, 14853 USA
xv
xvi CONTRIBUTORS
Jan Hadders Susheel Kumar

Dacom PLANT-Service BV, Plant Biotechnology,
P.O. Box 2243 Environmental and Industrial
7801 CE Emmen, The Netherlands Biotechnology Division,
The Energy and Resources Institute
(TERI), Darbari Seth Block, India
Antonio Ippolito Habitat Centre, Lodhi Road,
Dipartimento di Protezione delle New Delhi 110 003, India
Piante e Microbiologia Applicata
University of Bari,
Via Amendola 165/A Gaetano Magnano di San Lio
70126, Bari, Italy Dipartimento di Gestione dei Sistemi
Agrari e Forestali, Faculty of
Agriculture,
Jeffrey B. Jones Mediterranean University of Reggio
Plant Pathology Department, Calabria,
Institute of Food and Agricultural 89122 Reggio Calabria, Italy
Sciences,
University of Florida,
Guido Marchi
Dipartimento di Biotecnologie
Frank Kappel Agrarie, Sezione Patologia Vegetale,
Agriculture and Agri-Food Canada, Piazzale delle Cascine 28,
Pacific Agri-Food Research Centre, 50144 Firenze, Italy
Summerland, British Columbia,
Box 5000, 4200 Highway 97
Canada V0H 1Z0 Giorgio Maresi
IASMA Research Center,
Natural Resources Department,
Nutan Kaushik Via E. Mach 1,
Plant Biotechnology, 38010 San Michele all’Adige (TN),
Environmental and Industrial Italy
Biotechnology Division,
The Energy and Resources Institute
(TERI), Darbari Seth Block, India Mary Ruth McDonald
Habitat Centre, Lodhi Road, Department of Plant Agriculture
New Delhi 110 003, India University of Guelph, Canada
Cezarina Kora Keith R. Mitchelson

Pest Management Centre, The Medical Systems Biology
Agriculture and Agri-Food Canada, Research Center, Tsinghua
960 Carling Ave., Bldg 57 University School of Medicine,
Ottawa, ON, K1A 0C6, Canada Beijing 100084, China
CONTRIBUTORS xvii
M. T. Momol Aleksa Obradovic

Plant Pathology Department, Plant Pathology Department,
Institute of Food and Agricultural Faculty of Agriculture,
Sciences, University of Belgrade,
University of Florida, 11080 Belgrade - Zemun, Serbia
Peter Proksch
Salvatore Moricca Institut für Pharmazeutische Biologie
Dipartimento di Biotecnologie und Biotechnologie,
Agrarie, Sezione di Patologia Heinrich-Heine-Universität
Vegetale, Università di Firenze Düsseldorf, Germany
Piazzale delle Cascine, 28
50144 - Firenze
Alessandro Ragazzi
Dipartimento di Biotecnologie
Laura Mugnai Agrarie, Sezione di Patologia
Dipartimento di Biotecnologie Vegetale,
Agrarie, Sezione Patologia Vegetale, Università di Firenze
Piazzale delle Cascine 28, Piazzale delle Cascine, 28
50144 Firenze, Italy 50144 - Firenze
Rita Musetti Leonardo Schena

Dipartimento di Biologia e Dipartimento di Gestione dei Sistemi
Protezione delle Piante, Agrari e Forestali,
Università di Udine, Faculty of Agriculture,
Via delle Scienze, 208, Mediterranean University of Reggio
33100 Udine, Italy Calabria,
89122, Reggio Calabria, Italy
Ralph L. Nicholson†
Department of Botany and Plant
Pathology, Purdue University O. P. Sharma
915 West State Street National Centre for Integrated Pest
West Lafayette, IN Management, IARI Campus,
47907-2054 USA New Delhi-110 012, India
Franco Nigro Peter Sholberg

Dipartimento di Protezione delle Agriculture and Agri-Food Canada,
Piante e Microbiologia Applicata, Pacific Agri-Food Research Centre,
University of Bari, Summerland, British Columbia,
Via Amendola 165/A Box 5000, 4200 Highway 97
70126, Bari, Italy Canada V0H 1Z0
xviii CONTRIBUTORS
D. Shtienberg Tullio Turchetti

Department of Plant Pathology, Consiglio Nazionale delle Ricerche,
ARO, The Volcani Center, Istituto per la Protezione delle Piante,
Bet Dagan 50250, Israel Via Madonna del Piano,
50019 Sesto Fiorentino, (FI) Italy
Giuseppe Surico Ron R. Walcott

Dipartimento di Biotecnologie Department of Plant Pathology,
Agrarie, Sezione Patologia Vegetale, The University of Georgia,
Piazzale delle Cascine 28, Athens, GA 30607 USA
50144 Firenze, Italy
PREFACE
This volume focuses on integrated pest and disease management (IPM/IDM) and
biocontrol of some key diseases of perennial and annual crops. It continues a series
originated during a visit of prof. K. G. Mukerji to the CNR Plant Protection Institute
in Bari (Italy), in November 2005. Both editors aim at a series of five volumes
embracing, in a multi-disciplinary approach, advances and achievements in the
practice of crop protection, for a wide range of plant parasites and pathogens. Two
volumes of the series were already produced, dedicated to general concepts in IPM
and to management and biocontrol of nematodes of grain crops and vegetables.
This Volume deals, in particular, with diseases due to bacteria, phytoplasma and
fungi. Every day, in any agroecosystem, farmers face problems related to plant
diseases. Since the beginning of agriculture, indeed, and probably for a long time in
the future, farmers will continue to do so. Every year, plant diseases cause severe
losses in the global production of food and other agricultural commodities,
worldwide. Plant diseases are not limited to episodic events occurring in single farms
or crops, and should not be regarded as single independent cases, affecting only
farms on a local scale. The impact of plant disease epidemics on food shortage
ignited, in the last two centuries, deep cultural, social and demographic changes,
affecting million human beings, through i.e. migration, death and hunger. The effects
of severe epidemics, like those due to Phytophthora infestans, are well documented
in plant pathology and even in history treatises and literature, and their legacy is still
valid today. For this reason a disease causal agent should not only be regarded as a
noxious factor limiting crop production or lowering farmers’ incomes, but also as a
potential threat for the whole food production chain, worldwide. Global epidemics of
basic food crops are still a potential issue and a risk that should be considered when
planning the welfare of any community, at any scale.
This statement explains the attention devoted to plant diseases, and the efforts
deployed for their management and control. As for other disciplines concerning plant
protection, we reached today a mature stage in which the optimism initially
underlining the widespread use of chemicals and fumigants lent space to a more
pragmatic, comprehensive and integrated vision of control. There is, indeed, a
general concern about the negative consequences related to the widespread use of
chemicals, including not only environmental issues like pollution or contamination,
but also the insurgence of resistance in the target organism populations, as well as
the farmers’ health hazards represented by the use and manipulation of chemicals.
A wide literature already covers several aspects of chemical or biological
control, but there is a widespread interest for a more holistic vision of IPM. In this
series we tried to fill this gap, aiming at producing an informative coverage for a
wide range of cropping systems. Chapters are organized in a first Section dealing
with diseases of perennial crops, followed by a second one for annual crops, and a
third final Section dealing with advances in DNA application for management,
detection and diagnosis, and potentials of endophytes for disease control.
In the first chapter, disease management of stone fruit crops (apricot, cherry,
peach, nectarine and plum) is reviewed. These include important diseases like brown
xix
xx PREFACE
rot blossom blight and fruit rot. Research showed the importance of latent infections
in brown rot cycle, allowing options for a better disease management. Brown rot is
controlled by fungicides, but resistance to benzimidazoles is widespread and appears
to be developing further. Cultivars resistant to brown rot, although not yet
commercially available, could be helpful for selection of new resistant clones. Other
important stone fruit diseases like bacterial canker, Leucostoma canker, powdery
mildew and postharvest fruit rots are also reviewed. Both bacterial and Leucostoma
cankers cannot be controlled with chemicals, but they are managed using an
integrated approach relying on resistance, good horticultural practices and exclusion.
Resistance to fungicides in powdery mildew is developing, so the use of spray oils
with fungicides is examined. New fungicides are available for the postharvest
problems like fruit rots caused by Monilinia spp., Botrytis cinerea and Rhizopus spp.,
but they need careful management to avoid resistance. The development of new
molecular techniques for pathogens identification and their use in disease forecasting
and risk management is improving control of stone fruit diseases.
In the second chapter, the major diseases of apples, their management strategies
and the problems related to sustainable productions are discussed. Guidelines for
sustainable, integrated management of main apple diseases are reviewed, including
effective and sustainable tactics. Resistance plays a crucial role in the management
of apple diseases, and management problems include the development of fungicide
resistance as wel as breakdown of host resistance. Symptoms, causal pathogens,
disease cycles and management practices are reviewed for main spring diseases like
apple scab, powdery mildew, fire blight and rust diseases. Problems like fungicide
resistance and availability of plant resistance are discussed, together with
applications of cultural and chemical management with predictive models.
Symptoms, disease cycles and management issues are also reviewed for summer
diseases, like bitter rot, flyspeck and sooty blotch.
Third chapter follows dealing with the management and ecology of phytoplasma
diseases of grapevine and fruit crops. Management of phytoplasma-infected plants
focussed on controlling the insect vectors and on roguing infected crops and weeds.
Actual management concepts rely on environment compatible measures and on
cultural practices. The introduction of disease-resistance genes into cultivated crops
togheter with the use of resistance-inducing microorganisms represent potential tools
to control phytoplasma diseases.
The fourth chapter deals with citrus diseases caused by Phytophthora spp., with
reference to root rot, gummosis and brown rot of fruits. Some aspects of the biology
and ecology of P. citrophthora and P. nicotianae are revised, like dissemination,
reproduction and epidemiology. The symptomatic diagnosis of main diseases are
reviewed, including foot rot or gummosis, fibrous root rot, brown fruit rot and
dieback of twigs and leaves. Biological and instrumental diagnosis and laboratory
tests for monitoring, sampling and population dynamics studies are revised.
Management methods based on interventions on the host-plant, rootstock resistance,
grafting as well as sanitary practices in nurseries are shown, with pruning, surgery
and cultural practices, i.e. fertilization, irrigation, soil management and weeds
control. Chemical control methods are also reviewed, with reference to the use of
systemic fungicides for control of trunk gummosis, root rot and brown rot of fruits.
PREFACE xxi
In the following review of biological control and management of chestnut

diseases, the main strategies for efficient biological control and management of
chestnut blight and ink diseases caused by Cryphonectria parasitica, Phytophthora
cambivora and P. cinnamomi are discussed. The cankers of chestnut blight are
described, as well as the characters of the different infections caused by
C. parasitica. The diseases evolution, the spread and effectiveness of hypovirulence
are also revised, considering morphology, physiology, presence and transmission of
dsRNAs. Chestnut resistance, the role of environmental and other ecological factors
in ink disease, including soil microflora, are then discussed. The role of silviculture
and biological control strategies for blight and ink disease management are also
revised. Improvements in the management of chestnut disease need a better
understanding of the ecological dynamic of chestnut ecosystems. An holistic
approach including all the factors involved in chestnut trees ecology is proposed in
planning the management of such ecosystems, and in undertaking best conservation
and improvement measures.
Esca is a grapevine wood disease that seriously affects grapevine yield and
longevity, comprising a number of distinct diseases in which the main fungal agents
(primarily vascular pathogens) invade the vines, not only through field wounds but
also as a result of nursery practices. When vines become infected in the nursery, the
developing diseases may vary from Petri decline to full-blown esca, with or without
white decay. No chemical control is available and sanitary practices in the nursery
are suggested as the best way to eliminate or at least reduce pre-planting infections
by the tracheomycotic fungi. In absence of chemical prevention, preventive and
curative actions in the field can lower infections or hamper symptom appearance in
esca-infected vines.
In the following chapter, the integrated management of root rot caused by the
fungus Rosellinia necatrix on fruit tree crops is revised. This is a soil borne
pathogen causing a disease known as “white root rot”. The pathogen, widespread
in temperate and tropical climates, shows an increasing trend of attacks on several
host species. Economic losses are serious in the nurseries and on orchard trees, and
many field crops and weeds can also be severely damaged. The pathogen, mainly
disseminated by propagating material, can survive in soil for many years. Control
strategies, including cultural practices, soil disinfestations, chemical treatments,
soil solarization and biological control are expensive and not always resolutive.
White root rot control largely depends on pathogen exclusion through the use of
R. necatrix-free propagating material and planting in healthy soils. A fundamental
role is played by rules promoting trade of healthy propagating materials, and by
the availability of new molecular detection tools.
The second Section, on annual crops, begins with a review of simulation
models for potato late blight management. Potato late blight is widely studied and
particular attention was given to the mathematical description of its development.
Several simulation models are avilable and this chapter focuses primarily on
versions developed at Cornell University and other research centres. The most
recent version of the model was validated in the highland tropics and several other
countries and cropping systems. Late blight simulators, used to evaluate disease
management scenarios, were also used for other purposes, including sensitivity
xxii PREFACE
analysis of resistance components, comparative epidemiology, development of

forecasting models and education. The potential of disease simulation will continue
to improve, thanks to supporting technologies, both in computing power and weather
data acquisition.
A review about the potentials of the decision support systems approach then
follows, with an example of integrated forecasting system for management of
Phytophthora infestans on potato. The PLANT-Plus® system initially developed in
the Netherlands by Dacom, allowed management of P. infestans on-farm since 1994.
The system is based on the life cycle of P. infestans, combining infection events with
the unprotected part of the crop, and was extended with models for Alternaria solani
and several other fungi or insect pests, in different crops. Another modules include
the irrigation management system based on meteo data and soil moisture sensors,
and models for potato tuber infection and fertilizers management. The platform
enables data communication between farmer, consultants, processors and other users,
allowing the most appropriate interface to be chosen. Different output types include
SMS text messaging, fax and e-mail warnings. An integrated weather forecast
provides a predictive risk assessment for the coming days. The disease models
require the availability of automatic, on-farm weather data and were developed in
cooperation with experts from different areas and countries. The model will
recommend when to apply a new spray and what type of chemical to use, i.e. contact,
translaminar or systemic. The benefits were demonstrated in field trials and
evaluations all over the world, and provide safe spraying programmes with lowest
possible use of chemicals.
A subsequent chapter deals with the integrated management of bacterial fruit
blotch (BFB) of cucurbits, the most economically important bacterial disease of
cucurbits worldwide. The causal agent is Acidovorax avenae subsp. citrulli. This
chapter explores the current understanding of the biology and epidemiology of BFB
and the integrated management strategies currently available. BFB is a seed
transmitted disease, affecting all stages of cucurbit crops and causing destructive
fruit rots. Like many phytobacterial diseases, the chemical options for management
are limited and primarily include copper-based compounds. The unavailability of
resistant cucurbits cultivars makes management difficult. An integrated approach to
exclude primary inoculum through production of clean seeds is suggested, through
isolation of seed fields, inspection and certification, seed health testing, seedling
inspection and copper-based disease control. Despite the efforts to exclude the
pathogen from cucurbit production, BFB outbreaks occur sporadically, worldwide.
For a more effective integrated management, a better understanding of the disease
epidemiology and pathogenesis is needed in fruit and seed production. Additionally,
understanding the role of blossoms in seed infection revealed potential avenues for
integrated disease management.
In the following chapter, a review of the progress in the integrated management
of Sclerotinia rot of carrot is given. Bacterial diseases play an important role in the
world agriculture by reducing yields and marketability of crops or by limiting their
production in areas with environmental conditions conducive for disease
development. Plant pathogenic bacteria show several obstacles for efficient plant
protection practices. In spite of technological advances, there is no bactericide that
PREFACE xxiii
can be efficiently used to control plant bacterial diseases. Due to lack of chemicals,
plant pathologists search for alternatives i.e. the integration with preventive measures
to develop sustainable control strategies. Management of tomato bacterial spot
currently relies on use of pathogen-free seed and transplants, elimination of
volunteer tomato plants, resistant cultivars and application of a copper-based
bactericides. These practices are ineffective in hot and humid weathers that favor the
pathogen spread and the disease development. New technologies, i.e. systemic
acquired resistance inducers and biocontrol agents, integrated with conventional
practices, represent new options in plant protection and increased disease
management efficiency.
A review follows about the integrated management of Verticillium wilt of
tomato. The disease is caused by Verticillium dahliae and V. albo-atrum, and its
incidence and epidemiology are revised. Verticillium wilts are generally controlled
by a combination of measures aiming at reducing severity and delaying the disease
progress, including resistant cultivars or rootstocks, management of soil inoculum,
reduction of propagules spread and manipulation of epidemiological factors. Further
methods, including crop rotation, solarization, sanitation, tillage and weed control,
fertilization, irrigation, chemical treatments and use of microbial antagonists, are also
revised.
Sclerotinia rot, caused by the fungus Sclerotinia sclerotiorum, is an important
disease of carrot in the field and during storage. Chapter 13 describes control
methods, emphasizing emerging strategies supported by new information on its
etiology and epidemiology. Prospects and recommendations are given to integrate
current and emerging control methods for sustainable management. The primary
strategy to manage Sclerotinia rot is the integration of methods reducing within-field
sources of inoculum, suppressing the development of the fungus, and reducing the
infection rate in the field and/or storage. The integrated strategy recommended in this
review aims at achieving disease suppression through sanitation of soil and
equipment, monitoring the crop development and microclimate, modifying the
microclimate through canopy manipulation, predicting the disease and timing the
application of control practices, as required. Breeding carrot cultivars for an upright
and compact top growth may offer important contributions to the sustainable
management of Sclerotinia rot.
Chapter 14 describes the integrated management of key diseases of cotton and
rice. Issues related to disease identification and based on symptoms and presence of
pathogens are discussed, as they are very important for a successful management.
The main integrated management concepts are discussed, together with technologies
combining a variety of control measures, including the conservation of existing
natural enemies, crop rotation, intercropping and cultivation of pest-resistant
varieties. Cotton diseases considered include seedling diseases, bacterial blight,
Alternaria leaf spot, grey mildew and leaf spots caused by Myrothecium, Cercospora,
Helminthosporium, Macrophomina, stem canker, late season Phoma blight, rust
(Phakopsora gossypii), leaf crumple, Cotton Leaf Curl Virus, Tobacco Streak Virus,
root rot, Verticillium and Fusarium wilts, new wilt or parawilt, boll rots and lint
diseases. Rice diseases include rice blast, brown spot, bacterial leaf blight and leaf
xxiv PREFACE
streak, sheath blight, sheath rot, Fusarium wilt or “Bakanae”, stem rot, Tungro Virus,
false smut and post-harvest diseases.
A further review describes biological and integrated means to control rust
diseases. In Chapter 15 the strategies avilable in rust control, with a special emphasis
on biological control, are discussed in the light of evidence showing that disease
control is most effective when an integrated approach is followed. A survey of the
fungal antagonists (hyperparasites) most effective against rust pathogens is given.
Their mode of action is described, and the main problems concerning biological
control are discussed. The value and limitations of other control measures
(eradication, use of hazard areas, quarantine, cultural practices, chemical treatments,
and plant breeding for disease resistance) are also outlined. A consideration of all
control measures suggests that crop protection requires an holistic approach,
integrating a broad range of control techniques.
In the third and final section, two innovating research fields are revised: i) the
use of DNA fingerprinting methods for microbial pathogens diagnostics, with
potentials in taxonomy and plant disease and ii) the management of pests and
diseases through the exploitation of endophytic fungi and their metabolites.
Advanced DNA-based techniques improved the identification and charac-
terization of microbial pathogens, resulting in an accurate testing for pathogen identi-
fication, sub-species-level DNA fingerprinting, pathogen-load testing and disease
spread monitoring. These applications are instrumental to the study of plant disease
epidemiology, so that adequate control measures can be accordingly implemented. In
Chapter 16, a survey of the most popular DNA profiling techniques is given, together
with some new molecular methods. Combinations of different analytical techniques
are also proposed as a useful approach for low throughput bioassays. Advantages and
disadvantages of each single test are considered and key issues (i.e. sampling,
validation, large-scale testing) are discussed. An outline of emerging high-
throughput molecular technologies, improving diagnostic approaches and disease
management, is also provided.
In the final chapter a new field of investigation with exciting perspectives in
IPM/IDM is revised. Endophytes are non-pathogenic microorganisms inhabiting the
interior of healthy plants, with potentials for crop protection. Many cultivated and
wild type plants investigated showed presence of endophytic fungal metabolites
including guanidine and pyrrolizidine alkaloids, indole derivatives, sesquiterpenes or
isocoumarin derivatives. These metabolites show beneficial effects on crop plants
and many of them have pesticidal and antimicrobial activity against plant as well as
human pests and pathogens. Full potentials and efforts needed for their full
exploitation are discussed.
In conclusion, our attempts to provide new options in management solutions
available worldwide, in a broad range of agricultural systems, yielded a
comprehensive compilation. We acknowledge the Author’s contributions for their
outstanding work. Thanks to their experience, efforts and determination in seeking
and applying advanced solutions in their research and field work, we hope we were
PREFACE xxv
able to provide a further tool, useful in the comprehension and sustainable

management of plant pests and diseases. Our hope is that this volume, even if not
exaustive, will result helpful for any interested reader, inspiring and supporting the
research efforts today necessary in the field and laboratory work as well.
A. C.
K. G. M.
Section 1
Diseases of Perennial Crops

1
A. PETER SHOLBERG AND FRANK KAPPEL
INTEGRATED MANAGEMENT OF STONE FRUIT

DISEASES
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre,
Summerland, British Columbia, Canada V0H 1Z0
Abstract. Stone fruit crops (apricot, cherry, peach, nectarine and plum) are subject to many diseases
although only a few need yearly management. Brown rot blossom blight and fruit rot is one of these
important diseases and has been studied in detail. Recent research has elucidated the importance of latent
infection in the disease cycle of brown rot, allowing for better disease management. Brown rot is
effectively controlled by fungicides belonging to several chemical classes, but resistance to
benzimidazole fungicides is widespread and appears to be developing in demethylation inhibitor
fungicides. Cultivars resistant to brown rot have been identified although they are not used commercially,
but could be helpful in the selection of new resistant cultivars. Some other important stone fruit diseases
are bacterial canker, Leucostoma canker, powdery mildew and postharvest fruit rots. Both bacterial
canker and Leucostoma canker do not have adequate chemical controls but are managed using an
integrated management approach that depends on resistance, good horticultural practices and exclusion of
the pathogen from the orchard. Powdery mildew is controlled by fungicides but resistance is developing,
so a strategy integrating the use of spray oils along with fungicides from different classes is
recommended. Fruit rots caused by Monilinia spp., Botrytis cinerea, and Rhizopus spp. are always
important problems for storage and transit of stone fruit crops. Fortunately, new fungicides are available
for use during the postharvest phase that are very effective but need careful management to avoid
resistance. In conclusion, the development of new molecular techniques for identification of pathogens
and the use of them to aid in disease forecasting and risk management is leading to better management of
stone fruit diseases.
1. INTRODUCTION
Stone fruit crops consist of apricot, cherry (sour and sweet), peach, including
nectarine, and plum, including prune. The apricot, Prunus armeniaca L. has limited
production because it is subject to frost injury (Ogawa & Southwick, 1995). The
peach, Prunus persicae (L.) Batsch and nectarine, Prunus persicae (L.) Batsch var.
nucipersica are important to the agricultural economy in many countries with Italy
accounting for 19% and the United States accounting for 14% of the production
(Feliciano, 1995). Plums are classified into two groups, the European plum, Prunus
domestica L., and the Japanese plum, Prunus salicina Lindl. (Southwick & Ogawa,
1995). The leading producers of plums are the former Soviet Union, Romania,
China, the former Yugoslavia, and the United States. Prunes are dried plums and are
mainly produced from the French prune, Prunus domestica. The sweet cherry,
3
A. Ciancio & K. G. Mukerji (eds.), Integrated Management of Diseases Caused by Fungi,
Phytoplasma and Bacteria, 3–25.
© Springer Science+Business Media B.V. 2008
4 A. P. SHOLBERG AND F. KAPPEL
Prunus avium L. is widely planted in Europe, United States, and the former Soviet
Union (Rener & Southwick, 1995). Several of the newer cultivars have originated
from Canada and are planted around the world. Sour cherry, Prunus cerasus L. or
Table 1. Common diseases of stone fruit.
Disease Causal agent Comments
Armillaria root rot Armillaria spp. Occasional problem

Bacterial canker Pseudomonas syringae pv. Sporadic outbreaks that occur mostly
syringae; P. syringae pv. on weak trees
morsprunorum
Bacterial spot Xanthomonas campestris pv. Important in warm humid areas
pruni
Black knot of plum Apiosporina morbosa Important disease in Eastern North
America
Brown rot, American Monilinia fructicola Not found in Europe
Brown rot, European Monilinia laxa Found in most countries
Canker, Fusicoccum Phomopsis amygdali Minor disease of peach
Canker, Cytospora, Leucostoma cincta, L. Important disease of peaches in Eastern
Leucostoma, Peach persoonii N. America
Perennial
Cherry leaf spot Blumeriella jaapi Important disease of sour cherries
Crown gall Agrobacterium tumefaciens Important nursery disease
Fruit rot, Alternaria Alternaria spp. Important on sweet cherry
Fruit rot, Botrytis Botrytis cinerea Important on sweet cherry
Fruit rot, Rhizopus Rhizopus stolonifer Important postharvest disease
Gummosis Botryosphaera spp. Causes general tree decline in warm
areas
Little cherry disease Little cherry virus strains In Canada, Europe, Japan, and United
States
Peach leaf curl Taphrina deformans Common on peach and nectarine
Peach scab Cladosporium carpophilum Important in warm humid areas
Phytophthora root and Phytophthora spp. Causes severe losses at problem sites
crown rot
Plum pockets Taphrina pruni Not of economic importance
Plum pox Plum pox virus Causes serious losses in plum, peach,
nectarine, and apricot
Powdery mildew of Cherry Podosphaera clandestina Important disease of sweet cherry in
Pacific Northwest
Powdery mildew/ Rusty Sphaerotheca pannosa Occurs sporadically and of minor
Spot of Peach economic importance
Prunus necrotic ringspot Prunus necrotic ringspot virus Occasional high losses in sour cherry
strains
Prunus stem pitting Tomato ringspot virus Virus transmitted by nematodes
Silver leaf Chondrostereum purpureum Not of economic importance
Verticillium wilt Verticillium dahliae Occasional problem on cherry
X-disease Phytoplasma Serious disease of peach, nectarine, and
cherry
STONE FRUIT DISEASE MANAGEMENT 5
tart cherry, is produced around the world with Russia accounting for a third of the
world’s supply (Iezzoni, 1995).
This chapter will focus on economically important stone fruit diseases, especially
those that are important where stone fruit are grown under irrigation. Information on
diseases not found in this chapter but listed in Table 1 can be found in various
publications (Snowdon 1990; Ogawa & English, 1991; Ogawa et al., 1995a; Jones &
Sutton, 1996; Webster & Looney, 1996; Ram & Bhardwaj, 2004). The diseases
covered in depth with emphasis on integrated control are brown rot, bacterial canker,
Leucostoma canker, powdery mildew and fruit rots. It is our hope that this chapter
can be used as a general guide for the integrated control of these diseases and as a
source of information on selection of resistant cultivars.
2. BROWN ROT
2.1. Pathogen Identification and Disease Biology

Brown rot, a major disease of stone fruit throughout the world, is caused by three
closely related fungi: Monilinia fructicola (G. Wint.) Honey, M. laxa (Aderhold and
Ruhland) Honey and M. fructigena Honey in Whetzel (Ogawa, Zehr & Biggs,
1995b). M. fructigena is not found in North America, and M. fructicola has not been
detected in Europe.
Brown rot has two infection stages identified as blossom blight and fruit rot, with
an indeterminate stage known as latent infection. Blossom blight is associated with
prolonged wet weather during bloom. Although all flower parts, except the sepals,
are susceptible to infection by M. fructicola, only the infection of the stamens leads
to the development of peach blossom and twig blight (Gubler, Adaskaveg & Hasey,
2006a) (Fig. 1). Inoculation of blossoms showed no distinct differences in
pathogenicity between M. fructicola and M. laxa (Ogawa & English, 1960). Apricot
is most susceptible to blossom blight followed in order by prune, sweet cherry,
peach, sour cherry and plum. The disease, also known as European brown rot, is
caused by M. laxa and in North America is most important along the Pacific coast
(Ogawa & English, 1991). Monilinia laxa is identified by its growth habit on potato
dextrose agar (PDA) where it forms characteristic lobed margins and can be
distinguished from M. fructicola by the formation of distinct dark lines in the
medium (Hewitt & Leach, 1939; Sonoda, Ogawa & Manji, 1982).
In California, where apothecia are formed, blossom blight can also be caused by
ascospores. The apothecia are usually produced by M. fructicola and occur in
orchards at the time peach trees bloom (Ogawa & English, 1991). The optimal
temperature for discharge and germination of ascospores was 15 to 16°C, which is
similar to the optimum for development of apothecia (Hong, Michailides & Holtz,
1998). However, blossom infection due to release of ascospores is rare and in most
areas it is the result of conidia. The conidia are blown by wind and washed about by
rain (Corbin, Ogawa & Schultz, 1968). When the spores land on susceptible tissue
they germinate in 2 to 4 hrs if moisture is present and temperature is favourable.
Blossom blight will occur with as little as 3-5 hrs of wetting at 20°C (Weaver, 1950).
Figure 1. Brown rot blossom blight of apricot. Note the twig dieback.
Latent infections are important links between blossom blight and fruit rot.
Studies have shown that latent infections are not caused by conidia that have not
germinated but are the result of conidia that have germinated and stopped growing,
only to resume growth again when fruit began to ripen (Jenkins & Reinganum,
1965; Jenkins, 1968). A technique to identify the presence of latent infections in
stone fruit was developed by Northover and Cerkauskas (1994), that depended upon
dipping immature fruit in the herbicide paraquat. They found that plums at pit
hardening treated with paraquat developed brown rot in 80% of the plums. A
seasonal pattern of prune bloom and fruit susceptibility to latent infection was
determined (Luo, Morgan & Michailides, 2001). The highest levels of infection
were at pit hardening and the lowest level was at the early embryo growth stage.
Incidence of latent infection of immature peach fruit by M. fructicola was studied
in the state of Georgia in the United States (Emery, Michailides & Scherm, 2000).
Incidence was found to be low until 7-12 days before harvest, when it rose
dramatically. Due to the late development of these latent infections they would not
be useful for disease prediction. In sweet cherries, M. fructicola was isolated more
frequently than Botrytis cinerea from latent infections (Adaskaveg et al., 2000).
More infections were produced on cherry after 6-9, or 12 hrs wetness duration than
after 18 - 24 hrs, when active decay developed. Luo and Michailides (2001a; 2001b)
conducted a risk analysis for latent infection of prune and provided quantitative
relationships between latent infection and wetness duration, in California. The risk
of infection was higher in March and April than May and lowest in June (Luo,
Morgan & Michailides, 2001). Bloom risk was highest at the popcorn and full
bloom stages, rather than at later bloom stages. Optimal temperature for blossom
infection was 22 and 26°C while blossom blight did not occur below 10 or above
30°C and with less than 4 hrs wetness duration. In summary, conditions that lead
latent infection to fruit rot are: 1) latent infection level; 2) fruit developmental stage;
3) inoculum concentration; 4) hours of relative humidity greater than 90% and 5)
total hours of dew period from mid-July to mid-August. A preliminary decision
support model to guide fungicide application was then developed, based on this
information (Luo & Michailides, 2003).
The fruit rot stage of brown rot is of most concern to growers because visibly
infected fruit cannot be sold. Information that relates more to postharvest decay is
discussed in the following postharvest fruit rot section. Under optimum conditions,
decay of ripe peaches infected with M. fructicola may be visible within 48 hrs of
infection (Ogawa et al., 1995b).
Usually, the fruit rot phase of brown rot is caused by M. fructicola in North
America although M. laxa is also relatively common in parts of North America and
is the chief pathogen in other countries especially in Europe. Techniques to identify
species of Monilinia have been important, due to quarantine restrictions on M.
fructicola in Europe and M. fructigena in North America. Primers to detect DNA of
both M. laxa and M. fructicola were first used in a rapid test to detect early and late
latent infections in sweet cherry (Förster & Adaskaveg, 2000). Species-specific
primers were designed for each M. fructicola, M. laxa, and M. fructigena and
successfully tested on a collection of these fungi in which they were used to amplify
a 356 base pair fragment from each of the three species (Ioos & Frey, 2000). This
simple and rapid method is particularly useful to detect M. fructicola, which is a
quarantine fungus in all European countries.
In the United States, inoculum sources for peach have been identified and studied
in South Carolina (Landgraf & Zehr, 1982). Nonabscised, aborted fruits, infected
thinned fruits on the ground and plum infections appear to be more important
sources of inoculum that affect ripening peach fruits, than blighted blossoms. In
Ontario, Canada, the most important source of inoculum was from thinned fruits on
the ground and nonabscised aborted fruits in the tree (Biggs & Northover, 1985).
In a 2 year study fruits were susceptible for 2 to 3 weeks in June, while they
became resistant at pit hardening, and then susceptible 2 weeks before full ripening
(Biggs & Northover, 1988a). Infection in both cherry and peaches increased with
wetness duration with greater than 80% infection after 15 h at 20-22°C (Biggs &
Northover, 1988b).
Insects can be important vectors of M. fructicola during fruit ripening, carrying
conidia to injuries produced by moths, beetles, bees, or ants (Ogawa et al., 1995b).
Wounds become resistant to fungal infection in 6 hrs because nutrients are absorbed
by underlying cells (Wade & Cruickshank, 1992). Structural barriers based on
suberin are formed later in the wound. The conidial concentration is important in
appearance of lesions on sweet cherry, reducing time of appearance from 5 to 2 days
(Northover & Biggs, 1995). The response time of ripe sour cherries was very similar
with initial lesion appearance advanced from 4 to 2 days. Polynomial models were
used to describe the responses produced in detached cherries that were based on the
inoculum concentrations, wetting durations, and incubation times encountered in
real cherry orchards (Northover & Biggs, 1995).
2.2. Integrated Management of Brown Rot

Cultural practices, such as removal of mummies and pruning infected twigs, reduce
inoculum level but do not eliminate the disease. Insect control is essential for
effective brown rot management, along with protective fungicide treatments (Ogawa
et al., 1995b). Orchard sanitation in which thinned fruits are removed from the
orchard floor will reduce brown rot in most cases. For example, removal of thinned
nectarines resulted in less brown rot in six California orchards (Hong et al., 1997).
Furthermore, proper thinning can reduce disease in prune plums because fruit-to-
fruit contact predisposes prunes to infection (Michailides & Morgan, 1997).
It might be possible to reduce fruit losses from brown rot in prune orchards by
thinning fruit, to reduce fruit clustering. Harvesting fruit at correct maturity and
avoiding wounding or bruising during harvest will result in less decay. Hong and
Michailides (1998) showed that measures to avoid injuries during harvest reduced
brown rot on plum at any spore load, but only when less than 50 spores per
millimetre were found on peach. Nectarine was even more susceptible when injured
requiring only 5 spores for decay to occur. Excessive fertilization can increase
brown rot in stone fruit orchards. Fertililization with 360 kg/ha ammonium nitrate in
‘Flavortop’ nectarines increased disease to 12.5%, compared to 4.2% when fertilized
with 280 kg/ha (Michailides, Johnson & Morgan, 1992).
Fungicides available for control of brown rot belong to at least 12 different
chemical classes, and provide fair to excellent control (Table 2). Timing of fungicide
application is critical for blossom blight control, because susceptible flower parts
must be protected before occurrence of prolonged wetness and mild temperatures
that allow infection. Timing for application to ripening fruit does not need to be as
precise. In cherries, host resistance increases with pit hardening and decreases
around 3 weeks before harvest (Northover & Biggs, 1990). Fungicide application is
recommended at shuck fall and before harvest for both sweet and sour cherry, with
an additional mid-season spray for sweet cherries.
Control of fruit brown rot with less traditional materials has focused on products
such as calcium, vapour of acetic acid or thymol, or use of antagonistic
microorganisms. Calcium propionate and calcium silicate were the best material for
the control of brown rot on detached peaches (Biggs et al., 1997). The incidence of
brown rot on detached peaches was reduced to 3 and 32% when ‘Manch’ apricots
were fumigated with thymol or acetic acid, respectively (Liu, Chu & Zhou, 2002).
Epiphytic fungi reduced brown rot of sweet cherry in three separate years in a
study conducted in Oregon from 1990 to 1993 (Wittig, Johnson & Pscheidt, 1997).
The potential for biocontrol of M. laxa with Penicillium frequentans has been shown
in the laboratory, in experimental peach plots and in commercial orchards (De Cal &
Melgarejo, 1994). Two isolates of Trichoderma atroviride, one isolate of T. viride,
and one of Rhodotorula sp. were effective in controlling brown rot on harvested
stone fruit (Hong, Michailides & Holtz, 1998). New control options that substitute
pesticides considered risky for organic or biorational materials for control of brown
rot remain a high priority. As stated by Sutton (1996) concerns over pesticide use
and risk will generate opportunities for new environmentally safe fungicides with
novel modes of action.
Fungicide resistance is an important concern when new fungicides are introduced
for disease control (Table 2). Benomyl was first used for the control of brown rot in
the United States in 1972 (Ogawa et al., 1988). Benomyl resistance was documented
in a California cling peach orchard where mixed populations of benomyl-resistant
and sensitive to M. fructicola were located (Sonoda et al., 1983). Use of benomyl in
this orchard increased the proportion of benomyl-resistant isolates on blighted
blossoms, but not on unsprayed border trees.
Populations of M. laxa resistant to benomyl were not detected before 1980 in
surveys conducted in apricot orchards sprayed with benomyl (Ogawa et al., 1984).
Experience with benomyl resistance by Monilinia spp. has led to management
strategies for stone fruit crops (Ogawa et al., 1988). It is suggested that a resistance
monitoring system be used to detect benomyl resistance, as well as only using the
minimum number of applications of the at-risk fungicide.
Dicarboximide resistance was first found in New Zealand in 1985, where
resistant isolates were as much as 300 times less sensitive to iprodione than sensitive
isolates (Elmer & Gaunt, 1994). The resistant strains were aggregated in orchard
blocks supporting the belief that they had not acquired the necessary characteristics
to remain in or dominate the field population when not selected by iprodione
application (Elmer et al., 1998).
Iprodione is considered a medium to high risk for fungicide resistance.
Dicarboximide resistance does not appear to be increasing because, in general,
resistant isolates are not as fit as sensitive isolates and disappear from the population
when this class of fungicide is not used. Resistance management strategies that
stress the use of fungicides with a different mode of action have been successful in
prolonging the use of these fungicides.
Demethylation (sterol) inhibiting (DMI) fungicides were introduced after both
benomyl and dicarboximide-containing fungicides. Reduced sensitivity by M.
fructicola to propiconazole, a DMI fungicide, was reported in the United States in
South Carolina peach orchards, beginning in 1995 (Zehr et al., 1999). However,
failure to control brown rot had not occurred as of 1998. It appears that the
resistance is developing slowly and will be influenced by resistance management
strategies that have been put in place, such as the use of fungicides having other
modes of activity during the susceptible bloom and ripening periods.
Results from Georgia, United States, suggest that isolates with reduced
sensitivity to propiconazole have also developed there (Schnabel et al., 2004). It
appears that these isolates are more difficult to control in the field, as well as having
reduced sensitivity. New products containing fungicides from the strobilurin (QoI)
class may be viable disease control alternatives or rotation partners. Recent results
from California show that none of the M. fructicola isolates tested was resistant
to either iprodione or tebuconazole, although resistance to the benzimidazole,
Table 2. Efficacy and resistance risk of fungicides used on stone fruit crops
Resistance
Fungicide Common name Class Estimated disease control 1
risk
Brown rot Mildew Gray mold
Abound azoxystrobin Strobilurin Good Good Not used High
Benlate benomyl Benzimidazole Good Good Good Very high
Botran dichloran Aromatic Fair Not used Fair High
hydrocarbon
Bravo chlorothalonil Aromatic nitrile Fair Not used Fair Low
Cabrio pyraclostrobin Strobilurin Fair Fair Not used High
Captan captan Phthalamide Fair Not used Good Low
Elevate fenhexamid Hydroxyanilide Good Not used Excellent High
Elite tebuconazole DMI-Triazole2 Excellent Good Good High
Flint tryfloxystrobin Strobilurin Good Good Not used High
Funginex triforine DMI-Piperazine Good Good Not used High
Indar fenbuconazole DMI-Triazole Excellent Not used Not used High
Maneb maneb Carbamate Fair Not used Fair Low
Orbit propiconazole DMI-Triazole Good Good Not used High
Penbotec3 pyrimethanil Anilino- Good Not used Excellent High
pyrimidine
Pristine boscalid + Strobilurin + Excellent Excellent Good Medium
pyraclostrobin Carboxyanilide
Procure triflumazole DMI-Imidazole Good Good Not used High
Quintec quinoxyfen Quinoline Not used Excellent Not used Medium
Rally/Nova myclobutanil DMI-Triazole Fair Excellent Not used High
Rovral iprodione Dicarboximide Good Not used Good Medium
Rubigan fenarimol DMI-Pyrimidine Good Excellent Not used High
3
Scholar fludioxonil Phenylpyrrole Excellent Not used Excellent Low
Thiram thiram Carbamate Minimal Not used Minimal Low
Topsin- thiophanate- Benzimidazole Good Good Fair Very high
M/Senator methyl
Vangard cyprodinil Anilino- Excellent Not used Excellent High
pyrimidine
Ziram ziram Carbamate Minimal Not used Minimal Low
1
Estimated rating for disease control when the fungicide was applied at the correct rate and timing
according to label directions.
2
DMI = demethylation (sterol) inhibitor
3
Used only for postharvest treatment of stone fruit crops.
thiophanate-methyl was found, characterized as both high and low level resistance
(Yoshimura et al., 2004).
Fungicide mixtures, although useful for delaying resistance, will not be used in
practice for this purpose unless they are synergistic and reduce the concentration of
product needed to control disease (Emery, Scherm & Savelle, 2002). The absence of
synergism between most common fungicides when mixed indicated that a rotating
schedule for control and resistance management is most likely to be used by the
grower. This scenario might change when pre-packaged mixes are more commonly
made available. For example, Pristine®, a mixture of boscalid and pyraclostrobin,
promotes resistance management because it contains two fungicides with different
modes of action and is considered synergistic for control of at least three stone fruit
diseases (Table 2).
Fungicide applications for control of most stone fruit diseases could be reduced
by incorporating a forecasting method that considers such parameters as wetness
duration, temperature, inoculum level, and fruit maturity (Ogawa et al., 1995a).
Latent infections can be monitored on developing fruit and could act as predictors of
disease. This possibility was tested by Emery, Michailides and Scherm (2000) in
Georgia. They found that incidence of latent infections may be useful for providing
an estimate of fruit storability but was not conducive to forecasting disease in the
field. This may not be true for other crops and in different geographical areas.
Studies in California have shown that in prune, chemical control of blossom blight is
needed only in orchards that historically show a high inoculum potential under
favourable weather conditions during bloom (Luo, Morgan & Michailides, 2001).
Epidemiological studies with accurate information on inoculum concentration, now
possible using molecular techniques, should make it possible to reduce the use of
fungicides when low levels of inoculum are present. For example, Sholberg,
O’Gorman and Bedford (2005) showed that apple diseases could be identified and
monitored using a DNA macroarray for use in disease prediction. Furthermore, use
of molecular techniques to detect fungicide resistant isolates is a possibility that
could lead to a better understanding of fungicide resistance at the population level
(Ma & Michailides, 2005).
The use of resistant cultivars for management of stone fruit brown rot has been
an important goal of many research programs. Studies at our laboratory showed that
‘Staccato’, ‘Stardust’, and ‘Sweetheart’ cherry cultivars were the least susceptible
out of 16 that were tested on cherry fruit (Table 3). Research has shown that the
‘Bolinha’ cultivar from Brazil is more resistant than ‘Conserva 144’ as shown by
reduced rate of lesion development and sporulation of peach fruit (Feliciano,
Feliciano & Ogawa, 1987). Field trials also showed that this cultivar is less
susceptible than other commercial cultivars. Cultivars from almond have been the
most promising for brown rot resistance in peach (Gradziel, Bostock & Adaskaveg,
2003). Resistant progeny had thicker cuticles, and more waxes, pectin, phenolics
and chlorophyll. Selection for resistance using epidermis-based resistance, combined
with high flesh colour, was successful in breeding resistant genotypes. In sweet
cherry, thickness of the epidermal cell wall of the fruit also correlated with increased
resistance to brown rot (Biggs & Northover, 1989).
Table 3. Susceptibility of cherry cultivars to brown rot in controlled laboratory tests
Cherry, sweet Susceptibility to Monilinia fructicola

in cherry fruit
Bing Medium to High

Cristalina High
Lambert High
Lapins Medium to High
Samba High
Sandra Rose High
Santina Medium
Skeena Low to Medium
Sonata Medium to High
Staccato Low
Stardust Low
Stella High
Sweetheart Low
Symphony Medium
Van High
3. BACTERIAL CANKER
3.1. Pathogen Identification and Disease Cycle

Bacterial canker caused by Pseudomonas syringae pv. syringae van Hall may cause
cankers on any fruit crop, whereas P. syringae pv. morsprunorum (Wormald) Young
et al. infects only stone fruit (Ogawa & English, 1991). A third pathovar, P. syringae
pv. persicae causes disease symptoms on peach in France and bacterial decline of
nectarine, peach and Japanese plum in New Zealand (Young, 1987; 1988). Strains of
P. syringae pv. syringae isolated from stone fruit formed a cluster distinct from most
of strains isolated from other hosts, when characterized by using enterobacterial
repetitive intergenic consensus (ERIC) primers (Little, Bostock & Kirkpatrick,
1998).
Bacterial canker is also known as gummosis, because it causes gumming in
infected trees (Fig. 2) and also as blossom blast, because it causes blackened wilted
blossoms in the spring. Disease outbreaks are sporadic and more frequent on sweet
cherry than on sour cherry (Jones & Sutton, 1996). The higher populations of the
pathogen occur in early summer, and lowest populations in midsummer and during
the coldest weeks of winter (Cameron, 1970). Stress factors that predispose trees to
bacterial canker are freeze damage, wounds, nematode damage and dual infections
of Pseudomonas spp. and plant pathogenic fungi such as Cytospora and Nectria
(Hatting et al., 1989). Weaver (1978) found that the sour-sap odor developed only
on twigs that were frozen at -10°C after inoculation with P. syringae pv. syringae
and incubated at 15°C.
Pathogenic Pseudomonas spp. have been isolated from many apparently healthy
buds of stone fruit trees, with a higher number of active expanding buds than
dormant buds containing the pathogen (Roos & Hattingh, 1986b). Weeds may also
serve as sources of inoculum for bacterial canker of stone fruit (Roos & Hattingh,
1986a). The fall population is considered the most important in terms of disease,
because the onset of dormancy wounds will take a long time to heal and bacteria
will be able to establish an infection (Jesperson & Bedford, 2001). Trees are
particularly susceptible in sandy as well as in waterlogged soils, and during
prolonged periods of drought. Young peach trees on sandy soils that were irrigated
in the fall developed more severe bacterial canker than control trees (Ogawa &
English, 1991). The severity of bacterial canker is also markedly increased by the
ring nematode, Mesocriconema xenoplax but not by other common nematodes.
Attempts to reduce the severity of bacterial canker by raising the soil pH with lime
or other alkaline materials have met with variable results (Ogawa & English, 1991).
The type of rootstock and time of pruning have an effect on the severity of bacterial
canker and are discussed below.
3.2. Integrated Management of Bacterial Canker

Major outbreaks of bacterial canker in young orchards are often attributed to poor
management practices. No single management practice will indeed adequately
control bacterial canker. Several factors must be taken into consideration: orchard
sites should not have acidic or sandy soils; trees should be purchased from nurseries
known to be free of bacterial canker; rootstock and cultivar selections should be
appropriate for the geographical area; tree vigour should be maintained by using
proper fertilization and irrigation practices; and trees should be pruned to limit
chance of spread by pruning in early summer rather than autumn and winter
(Hattingh & Roos, 1995).
Lovell peach rootstocks are more tolerant to bacterial canker than most other
peach rootstocks. Plums on Lovell and French prune on both Lovell and Nemaguard
rootstocks were damaged less than those on Myrobalan or Marianna rootstocks.
‘Schmidt’, ‘Windsor’, and ‘Hardy Giant’ sweet cherry cultivars are susceptible and
should be avoided in disease prone areas (Jones & Sutton, 1996). Cherry rootstocks
considered resistant to bacterial canker are F12-1 and Mazzard. In California,
‘Mahaleb’ is the most tolerant, ‘Colt’ is moderately susceptible, and ‘Mazzard’ is
susceptible.
Chemical control of bacterial canker is based on sprays with fixed copper or
Bordeaux mixture in autumn and in spring before blossoming. Copper sprays protect
initial infection but cannot prevent the canker phase, once infection has occurred
(Hattingh & Roos, 1995).
Figure 2. Bacterial canker of peach with gumming from cankers.
Olson and Jones (1983) reduced P. syringae pv. morsprunorum populations to

a low level on ‘Montmorency’ sour cherry with tribasic copper sulphate, although
several applications were needed. Pseudomonas syringae pv. syringae resistant to
copper has been isolated from orchards in Michigan, California, and Oklahoma
(Hattingh & Roos, 1995). The wide distribution of copper-resistant P. syringae pv.
syringae on cherry in Michigan show that copper bactericides are no longer effective
in this area (Sundin, Jones & Fulbright, 1989). In light sandy soils, preplant
fumigation for nematodes has been initially successful, but only lasts a few years
(Adaskaveg & Gubler, 2006). Following planting, the use of fenamiphos in orchards
on a yearly basis until trees are 8 years old has been effective. Pruning in early
summer, rather than autumn and winter, lessens the chance that trees will become
infected (Hattingh & Roos, 1995).
Bacterial canker can be disseminated in a multitude of ways such as in dormant
buds, on weeds, in cankers, from blighted blossoms, by insects, from seed,
systemically in twigs, and by pruning shears (Hattingh, Roos & Mansvelt, 1989).
Emphasis for the control of bacterial canker needs to be on selection and breeding
for disease resistance. Techniques have been devised to screen for cherry resistance.
For example, cherry genotypes were screened by an in vitro leaf bioassay which
showed that ‘Corum’, ‘Royal Ann’ and ‘Rainier’ were very susceptible (Roche &
Azarenko, 2001). A detached leaf bioassay was developed that showed ‘Sweetheart’
was more susceptible than ‘Merchant’ and ‘Merpet’ to bacterial canker, as
previously found. It thus could be a useful technique for screening new cultivars for
bacterial canker resistance (Bedford, Sholberg & Kappel, 2003).
4. LEUCOSTOMA CANKER

Leucostoma canker, also known as perennial canker of peach, Cytospora canker, or
Valsa canker is caused by two related fungi, Leucostoma cincta (= L. cinctum) (Fr.
Ex.:Fr.) Höhn and L. personii Höhn. These pathogens can only invade wounded or
dead tissue and over winter as pycnidia in diseased tissue under bark (Biggs, 1995).
Conidia are resistant to desiccation when contained in the pycnidium, but die within
6 hrs once they are released by water and subsequently dried. Most infections take
place in late fall or early winter and in late winter or early spring (Ram & Bhardwaj,
2004). The most common infection sites are pruning cuts, insect injuries, twigs
weakened by shading in tree centres, winter-killed buds, and bark killed or injured
by low winter temperatures (Rosenberger, 1982). Most conidia are spread by
splashing rain although boring insects, birds, and pruning tools are important
alternate routes (Biggs, 1989a). In California, the disease is caused by L. persoonii
and causes serious damage on young trees as a secondary invader of cankers caused
by P. syringae. In Washington, the disease is caused by L. cinctum which is reported
to cause approximately 5% in losses to the cherry and peach crops of that state
(Grove & Biggs, 2006).
4.2. Integrated Management of Leucostoma Canker

Control of Leucostoma canker requires integrated practices for all aspects of orchard
management, from planning new plantings to care of bearing orchards. Control is
based on preventative measures with the object of decreasing winter injury, insect
damage, and other wounds that act as infection sites (Rosenberger, 1982). For best
management of Leucostoma canker the following practices should be observed: 1)
control insects and diseases; 2) protect the bases of trees; 3) train trees according to
recognized horticultural practices (Westwood, 1993); 4) avoid rodent injury; 5)
prevent cold injury; 6) prune correctly and at the proper time (Westwood, 1993;
Flore, Kesner & Webster, 1996); 7) remove cankers from trunks and scaffold limbs;
8) cover pruning cuts with an effective paint; use chemicals to cover leaf scar
wounds and 9) apply nitrogen fertilizer in late winter or early spring (Biggs, 1989a).
The type of irrigation used in the orchard can predispose stone fruit trees to
disease. Grove and Biggs (2006) showed that over-the-canopy and under-tree
sprinkler irrigation can promote sporulation and disperse conidia of L. cinctum
during the growing season. Because the irrigation practice can be important in
disease spread, it may be beneficial to convert to a system that is less likely to wet
canker surfaces such as underground drip or microsprinkler irrigation. Common

used sealants retard the wound response whereas fungal cell wall extracts and
cellobiose enhance the wound response (Biggs & Peterson, 1990). Biggs and Miles
(1988) demonstrated a correlation between suberin accumulation in peach and
disease resistance for several cultivars. Further research confirmed these results for
suberin accumulation, inhibition of canker length, days for resistance to develop,
and field performance in the most resistant to the least resistant cultivars as follows:
V68101 > Redhaven > Vanity > Candor > Madison and Earlired (Biggs, 1989b).
Studies on leaf abscission and harvest date for various peach cultivars was not
associated with susceptibility to Leucostoma canker (Biggs, 1991). Chemical control
of the disease by fungicides is problematic and no fungicides have been registered
for its control. Biggs (1989a) recommended controlling leaf scar infections with fall
or spring sprays for peach leaf curl likely, because sporulation is highest during
spring and summer. This involves the use of copper sprays (Bordeaux mix) in the
fall at leaf fall, and again before the buds swell in the spring.
5. POWDERY MILDEW

Powdery mildew on stone fruit is caused by at least three different fungi.
Cleistothecia of Podosphaera clandestina (Wallr:Fr.) Lév. and P. tridactyla (Wallr.)
de Bary are relatively common; those of Sphaerotheca pannosa (Wallr.:Fr.) Lév. on
stone fruit are rare (Ogawa & English, 1991). Thus S. pannosa over winters
primarily in host buds whereas the other two pathogens over winter in leaves.
Apricot and plum are infected by P. tridactyla or S. pannosa originating from
infected roses or peaches, near apricot and plum plots. Powdery mildew of peach is
also known as rusty spot of peach. Peach buds serve as over wintering structures for
S. pannosa, with leaves becoming infected as they emerge from buds.
Peach fruit are susceptible from the early stages of growth to about the beginning
of pit hardening (Fig. 3). Sphaerotheca pannosa can spread from rose to peach, as is
the case for apricot. However, mildew from rose causes only fruit blemishes (Kable,
Fried & MacKenzie, 1980). Manji (1972) reported that Podosphaera leucotricha,
the causal agent of apple powdery mildew, could cause rusty spot of peaches.
Cherries are primarily infected by P. clandestina, whose growth is favoured by
dry summer with intermittent periods of high humidity and moisture. Cleistothecia
develop in leaves in midsummer and the fungus over winters in fallen leaves. Grove
and Boal (1991a) showed that P. clandestina survived winter as cleistothecia on
senescent cherry leaves on the orchard floor or trapped in tree crotches.
Ascospores lose viability over the spring, going from 55-90% in February to 5-
33% in mid-May. In controlled environment studies ascospore release occurred in
water at 5 to 30°C, and was optimal after 8 hrs wetness duration at 15-20°C (Grove,
1991). Ascospore germination was observed after 8 hrs at 25°C or 16 hrs at 15 and
20°C. Germination on immature (green) fruit increased with incubation time, but
was lower on ripening fruit and was suppressed by increasing soluble solid
concentration (Grove & Boal, 1991b). Fruit infections appear if rain occurs near
harvest, but it is unclear whether the moisture promotes fruit infection or profuse
sporulation of pre-existing colonies (Grove, 1995).
Figure 3. Powdery mildew of nectarine, showing white mildew growth on fruits.
5.2. Integrated Management of Powdery Mildew

Control of mildew on stone fruit depends on the use of fungicides, removal of
alternate hosts, and use of resistant cultivars. Properly timed fungicide sprays are
effective for control of powdery mildew on stone fruit (Table 2). The use of DMI
fungicides has improved powdery disease control, compared to most other fungicide
classes especially sulphur (Grove, Boal & Bennett, 2000) (Table 2). Sulphur
compounds have a short residual activity, are effective over a limited temperature
range, pose significant phytotoxicity risks and may interfere with beneficial insects.
Timing applications of fungicides for control of powdery mildew is very
important even though most DMI fungicides are considered systemic (Adaskaveg
et al., 2006). Applications should commence at shuck fall (detachment of flower
parts) or early popcorn (petals showing) through to pit hardening. The early
applications are the most important. Up to three applications may be necessary on
peach in seasons when there is cool weather, with occasional rain (Gubler,
Adaskaveg & Hasey, 2006b).
Management of powdery mildew on cherry requires the use of several strategies.
Powdery mildew inoculum is reduced by removal of infected water sprouts between
bud burst and leaf fall. Cultural practices that promote low humidity in the orchard
will reduce infection. Chemical control depends on using materials with three
different modes of action as represented by oils, DMI and strobilurin fungicides to
provide for resistance management, as well as effective disease control (Grove, Boal
& Bennett, 2000). Development of resistance to DMI fungicides by P. clandestina
has occurred in the state of Washington, where as many as eight applications of
DMI fungicides are made each growing season. Oil products such as JMS Stylet Oil
are used no later than the pit hardening stage, to prevent tissue damage. A second
approach is to use a temperature based disease forecasting system, that utilizes oils
for control and requires fewer fungicide applications.
Apricot cultivars with resistance to powdery mildew are available, although most
commercial cultivars are susceptible. In apricot ‘Blenheim’, ‘Rival’, and ‘Tilton’ are
susceptible as well as ‘Kelsey’, ‘Graviola’, and ‘Wickson’ plum (Grove, 1995).
In peach, susceptibility to powdery mildew varies considerably and is highest in
the nonglandular pubescent cultivars. ‘Flame Crest’, ‘Flavour Crest’, ‘Red Lady’,
‘Elegant Lady’, ‘O’Henry’, ‘Davidson’, ‘Yakima Hale’, ‘Peak’, and ‘Palor’ are
susceptible whereas ‘Angelis’, ‘Walton’, ‘Johnson’, ‘Halford’, and ‘Stuart’ are more
resistant. Most commonly grown nectarine cultivars are susceptible and is
particularly severe on ‘Red Supreme’ and ‘Laurie Red’.
The use of cherry cultivars with resistance to powdery mildew has been studied
in Washington. Olmstead, Lang and Grove (2001) developed a technique for rating
susceptibility in sweet cherry using digital image analysis and compared it to visual
assessment. These authors found that the standard visual assessment is an accurate
method for estimating disease severity. Using image analysis cherries were ranked
from least susceptible to most susceptible as follows: 1 (Chelan, Lambert, Moreau,
and Venus), 5 (Black Tartarian, PMR-1, and Van), 8 (Tieton), 9 (Lapins, Stella), 11
(Ranier), 12 (Sam), 13 (Black Republican) and 14 (Bing).
6. POSTHARVEST FRUIT ROTS

Stone fruits are susceptible to decay by several different pathogens immediately
after harvest (Fig. 4). The most common pathogens causing decay are: Monilinia
fructicola (Wint.) Honey, Rhizopus stolonifer (Ehr.:Fr) Vuill., Botrytis cinerea Pers:
Fr., Penicillium expansum Lk., Alternaria alternata (Fr.) Keissl., and Mucor
piriformis Fischer. Brown rot is the most common disease of harvested stone fruit,
although in some years gray mold and Rhizopus rot are just as important. Conditions
that lead up to postharvest storage problems often originate in the orchard. Injury to
fruit during harvest can result in increased postharvest losses. Cherry fruit that have
been bruised are much more susceptible to increased decay by B. cinerea and R.
stolonifer (Ogawa et al., 1972).
Rhizopus spp. are soilborne and require a wound for infection, but once growing
on containers soiled with fruit juice they can spread to healthy fruit (Adaskaveg,
1999). Initial stages of fungal decay can be mistaken for bruised fruit. Fungal
infections in sweet cherry fruit can also be facilitated by cuticular fractures (Børve,
Sekse & Stensvand, 2000). This may be important when cherry fruit are immersed
in water on the packing line and come in contact with pathogenic fungal spores in
the water.
Drying of prune plums immediately after harvest was found to be important in
the prevention of a postharvest slip-skin maceration disorder (Sholberg & Ogawa,
1983). Fruit held in bins for 24 hrs or more after harvest, developed the disorder
after drying in proportion to the occurrence of prior Rhizopus spp. infection.
Mucor rot caused significant losses of stone fruit in California in 1977 when an
unusual amount of decay developed during cold-temperature transit at 5°C
(Michailides & Spotts, 1990). Infested soil and debris are the major sources of
inoculum for Mucor piriformis Fischer.
Figure 4. Soft rot of peaches caused by Monilinia and Rhizopus spp. Note Rhizopus spp. in
the dark areas and Monilinia spp. in the light areas on infected fruit.
6.2. Integrated Control of Postharvest Fruit Rots

Sweet cherries are used in this section as an example for postharvest decay control
in stone fruit. Losses from decay during transit and storage has been a limiting factor
in the shipment of sweet cherries to both local and distant markets (Ogawa &
English, 1991). Control of decay in sweet cherry begins in the orchard. Fungicides
effective for control of brown rot, are applied as discussed in the previous brown rot
section. If fruit are cracked due to rain, effective control is not possible. Cherries are
picked into padded buckets to prevent bruising, dumped into bins, and trucked to the
packing shed. The use of a reflective tarp once the bins are filled could help keep the
fruit cool, improve fruit quality, and reduce decay.
Improved quality of blueberries was attributed to the lower fruit pulp

temperatures and higher humidity in the airspace surrounding tarp-covered fruit
(Toivonen et al., 2004). The fruit is dumped into chlorinated hydro-cooled water
(50-100 mg ⋅ l -1) at the packing shed. Chlorination of the hydro-cooled water kills
pathogens that may be in the water, preventing them from infecting fruit that may
have been wounded during harvest. Protectant fungicides can be applied as a spray
as the cherries go over the packing line.
New postharvest fungicides (Table 2) have been developed for use on stone fruit,
because the older materials such as benomyl and iprodione are no longer available
for postharvest use. The reduced-risk fungicides, fludioxonil, fenhexamid and
pyrimethanil belong to three different chemical classes and should be used in
alternation to prevent fungicide resistance (Adaskaveg, Kanetis & Förester, 2005).
Fungicide resistance can be avoided by management strategies in the packinghouse
that include monitoring the air, equipment, and recycled water and treating solution
systems for pathogen spore load and sensitivity, as well as cull and fruit residue
analysis (Goodwine 2005). The use of the correct fungicide for the pathogen causing
the postharvest problem at recommended rates and the alternation of different
chemical classes, should always be practiced in the packinghouse.
Several alternative treatments not based on commercial fungicides have been
proposed for the control of stone fruit decay. Hot water treatments for control of
fungal decay in fresh produce looks promising (Fallik, 2006). Of particular interest
to stone fruits was the control of brown rot in peaches and nectarines, by dipping
them in hot water at 46 or 50°C for 2.5 min (Morgosan et al., 1997). Sholberg and
Gaunce (1996) evaluated acetic acid vapour on apricot, cherry, peach and nectarine
to control fruit rot caused by M. fructicola, R. stolonifer and A. alternata. The results
showed that the treatment was effective, but burned and blackened the fruit in some
cases.
Chu et al., (1999) showed that thymol and acetic acid vapour were effective
treatments for gray mold decay caused by B. cinerea on modified atmosphere
packaged sweet cherries. In this study thymol was more effective than acetic acid
but imparted a medicinal odour on the cherries. Further studies on thymol and
acetic acid vapour showed that they were also effective treatments for brown rot of
apricots and plums (Liu, Chu & Zhou, 2002). Peracetic acid solution used to treat
stone fruit (sweet cherry, apricot, peach and nectarine) reduced the incidence of
brown rot caused by M. laxa and soft rot caused by R. stolonifer (Mari, Gregori &
Donati, 2004). Peracetic acid efficacy on pre-existing infections could be very useful
for the control of stone fruit diseases that spread during transit.
Biological control has been used for postharvest diseases of cherry. Utkehede
and Sholberg (1986) used Bacillus subtilis and Enterobacter aerogenes as
antagonists to control postharvest brown rot and Alternaria rot on detached cherry
fruit. Zhou, Northover & Schneider (1999) used Pseudomonas syringae as a
bacterial antagonist to control brown rot and soft rot caused by R. stolonifer on
peaches. The commercial product, Bio-Save (Jet Harvest Solutions, Longwood, FL),
has a strain of P. syringae (ESC-10) as its active ingredient and is registered in the
United States for the control of postharvest blue mold (Penicillium expansum) and
gray mold on cherries. In general, these biological treatments are not being widely
used by the industry, probably because they are relatively expensive, produce
variable results, and must be carefully used so that the correct number of viable cells
are present to control pathogen levels found at harvest. However, there remains a
need for this type of product and research should continue to find a robust biological
control effective for postharvest diseases of stone fruit.
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2
RALPH L. NICHOLSON1 AND JANNA BECKERMAN
TOWARDS A SUSTAINABLE, INTEGRATED

MANAGEMENT OF APPLE DISEASES
Department of Botany and Plant Pathology,
Purdue University, IN, USA
Abstract. Current understandings and guidelines for sustainable, integrated management of apple diseases
are reviewed, and currently effective and sustainable tactics are discussed. Disease management in apples
faces several critical problems not seen in other agronomic systems. As long-lived, clonal crops, fungicides
and disease resistance play crucial roles in the management of apple diseases. Unfortunately, the pressure
placed on these two strategies results in the development of fungicide resistance, and breakdown of host
resistance. This review discusses the major diseases of apples, their management strategies, and the problems
that have developed to impact sustainable apple production. Symptoms, causal pathogens, disease cycles and
management practices are reviewed for primary diseases affecting apples in spring like apple scab, powdery
mildew, fire blight and rust diseases. Problems due to fungicide resistance and availability of root stocks and
cultivars for exploitation of plant resistance are discussed. Applications of cultural and chemical management
with predictive models are also shown. Symptoms, disease cycles and management are also reviewed for
summer diseases of apple, like bitter rot, flyspeck and sooty blotch.
1. INTRODUCTION
Plant disease management, and apple management in particular, has reached a
critical juncture: control is no longer an option, and the recognition that management
may not even be attainable to the desired degree is changing our approach to plant
health problems. In fact, our repeated attempts at controlling diseases through the
use of pesticides, and the temporary elimination of disease problems has left our
management options limited.
Fungicides, once commonly used for disease control, are no longer effective, and
cannot be incorporated into a successful management strategy, even though
management is a more realistic approach of containment rather than the past policy
of zero tolerance. Today, perhaps more than ever before, is an understanding that
cultural management is the foundation of good disease management, and that
chemical management provides added benefits in quality and production, but cannot
be relied upon for control of plant health problems.
This paper focuses on our current understanding of sustainable, integrated, plant
disease management, and seeks to discuss currently effective and sustainable tactics
1
Born Aug. 25, 1942, died Oct. 10, 2007.
27
28 R. L. NICHOLSON AND J. BECKERMAN
in the management of major diseases of apple, including scab, fire blight, and
emerging problems of fly speck/sooty blotch, collar rot, and bitter rot.
2. THE SPRING DISEASES

2.1. Apple Scab
Apple scab, caused by the fungus Venturia inaequalis, is one of the most
economically devastating diseases of apple. The scab pathogen can infect both
leaves and fruit, and can cause severe defoliation of apple trees if poorly managed.
The disease negatively affects fruit size and quality, due to blemishes and poor
ripening. Overtime, repeated defoliation caused by the disease reduces tree vigor,
growth and yield.
2.1.2. Symptoms
Apple scab is most commonly observed on leaves, but fruit, blossoms, sepals,
petioles and pedicels, can also become infected. On leaves, lesions first appear on
the undersides, as they emerge and are exposed to infection in the spring. These
young lesions are often diffuse, and can be mistaken for sooty mold, or even leaf
“fuzz”. As leaves mature, lesions become more distinct, turn brown to olive green
and have feathery margins. Leaf yellowing commonly precedes leaf drop, and both
are common symptoms of scab, regardless of the pattern of foliar symptom
development.
Fruit lesions appear similar to those on leaves, but as the infected fruit matures,
lesions become brown and corky. These lesions are often smaller, have distinct
borders, and enlarge more slowly than foliar lesions. Early season infection can
cause uneven, cracked, or deformed fruit. Late summer fruit infections may not be
visible until the fruit are in storage. Although unusual, fruit may drop if an infected
pedicel becomes girdled.
2.1.3. The Causal Pathogen

Recent research suggests that V. inaequalis exists as a complex species, capable of
infecting apples (Malus spp.), cultivated flowering crabapples, wild apples (M.
iowensis and M. coronaria), mountain-ash (Sorbus), Cotoneaster, hawthorn
(Craetagus spp.), and Pyracantha. Scab isolates that infect pears and flowering
pears (Pyrus spp.) were found to be genetically distinct (LeCam, Parisi & Arene,
2002). The fungus reproduces sexually every spring, releasing ascospores from
perithecia (a flask shaped structure embedded in decaying leaf litter). Upon
successful infection, the fungus reproduces asexually on the host plant surface.
2.1.4. Disease Cycle

The pathogen generally over winters in leaves and fruit on the orchard floor.
Ascospores are the major source of primary inoculum. They are produced within
SUSTAINABLE MANAGEMENT OF APPLE DISEASES 29
pseudothecia that develop in fallen leaves during the winter months. In the spring,
ascospores are shot from the perithecia to infect developing leaves. Successful
infections result in the production of copious amounts of spores, called conidia.
Conidia are disseminated to developing leaves and fruit by splashing rain and wind,
and secondary cycles of conidial infection can occur during the growing season,
depending upon the susceptibility of host tissue and conducive environmental
conditions, generally in about 9-17 days (Table 1).
2.1.5. Management
Chemical management of apple scab began with the use of the Bordeaux mixture in
1887. However, its efficacy was not truly demonstrated until 1890, during an
unusually wet year. Even with the benefit of scab control was the recognition that
Bordeaux mixture caused fruit russeting, reducing appearance and perceived
consumer quality.
Early extension publications recognized cultivar sensitivity to the Bordeaux
mixture, and adjusted the rates accordingly, going so far as to recommend lime-
sulfur for particularly sensitive varieties (McCue, 1912). These same publications
also recognized the role of weather in scab epidemics, the difficulty of timing
delayed-dormant sprays, and the need for reliable weather forecasts.
Beginning with the development of inorganic (protectant) fungicide schedules in
the late 1880’s, management changed with the incorporation of epidemiological
data, in particular the Mill’s Table in 1944 (Table 1), which provided growers with
one of the first models to predict when to apply sulfur fungicides (Mill, 1944). In
that same year, the introduction of ferbam changed management yet again, and
ushered in the beginning of the synthetic fungicides. Within the decade, improved
sprayer technology followed, along with new classes of fungicides that prevented
tissue invasion, after infection had occurred.
The introduction of the systemic fungicides benomyl and dodine dramatically
changed apple scab management — at least until the first reports of resistance in the
late 1960’s and early 1970’s (Jones, 1981).
Resistance to fungicides develops due to selective pressure (in this case, the
fungicide) that results in a genetic mutation. Resistance is a phenotype that may
result from single or multiple gene mutations. Single-gene mutations, that confer
resistance to site-specific fungicides (like the benzimidazole fungicide, benomyl),
are more likely to develop than the simultaneous multiple gene mutations that are
needed to confer resistance to multi-site inhibiting fungicides. The mechanisms of
resistance usually depend on reduced fungicide uptake or detoxification of the
pesticide.
Fortuitously, the development of dodine and benomyl resistance coincided with a
new class of fungicides: the sterol-inhibiting (SI) fungicides (with active ingredients
like propiconazole, triademefon or myclobutanil). These fungicides still provided
the desired “kick-back” that was lacking in the protectant class of fungicides, (e.g.,
copper products, captan and mancozeb), and still allowed growers the luxury of “not
quite perfectly timed” delayed-dormant sprays. Unfortunately, the phenomenon of
fungicide resistance repeated itself in the 1990’s (Koller & Wilcox, 1999), when
developing resistance to SI fungicides emerged, but coincided with the introduction

of strobilurins (Koller et al., 2005).
Table 1. The Mill’s table providing the approximate wetting period necessary for primary
apple scab infection at different air temperatures, and time required for the subsequent
development of conidia a.
Average temperature Wetting Incubation

period (hr) b period (days) c
Farenheit Celsius Light Moderate Heavy
infection infection infection
78 25.6 13.0 17 26 -
77 25.0 11.0 14 21 -
76 24.4 9.5 12 19 -
63 - 75 17.2 - 23.9 9.0 12 18 9
62 16.7 9.0 12 19 10
61 16.1 9.0 13 20 10
60 15.6 9.5 13 20 10
59 15.0 10.0 13 21 12
58 14.4 10.0 14 21 12
57 13.9 10.0 14 22 13
56 13.3 11.0 15 22 13
55 12.8 11.0 16 24 14
54 12.2 11.5 16 24 14
53 11.7 12.0 17 25 15
52 11.1 12.0 18 26 15
51 10.6 13.0 18 27 16
50 10.0 14.0 19 29 16
49 9.4 14.5 20 30 17
48 8.9 15.0 20 30 17
46 8.3 15.0 23 35 -
45 7.8 16.0 24 37 -
44 7.2 17.0 26 40 -
43 6.7 19.0 28 43 -
42 6.1 21.0 30 47 -
41 5.6 23.0 33 50 -
40 5.0 26.0 37 53 -
39 4.4 29.0 41 56 -
38 3.9 33.0 45 60 -
37 3.3 37.0 50 64 -
36 2.8 41.0 55 68 -
33 - 36 0.6 - 2.2 48.0 72 96 -
a
Adapted from Mills, 1944, as modified by A. L. Jones.
b
The infection period starting at the beginning of rain.
c
Approximate number of days needed for development of conidia after the infection period begins.
It was almost 50 years ago that the protectant fungicides, e.g., captan, ferbam
and mancozeb, were relegated to a secondary status, with most growers opting for
the systemic properties of dodine, benomyl, or the sterol-inhibitors. In areas where
fungicide resistant populations are established, apply protectant fungicides such as

copper, captan, and mancozeb, starting at green tip, through seven day intervals to
effectively manage scab, and other foliar fungal pathogens. Today, a new reliance on
protectant fungicides is developing, due to the fact that scab has never developed
resistance to these products. These protectant class fungicides remain effective when
applied as a prophylactic treatment, and arrest developing infections if applied in
conjunction with a Mill’s, or modified Mill’s Table (Table 1). However, coinciding
with the new dependence on the protectant classes is the loss of these products due
to human and environmental health issues, with Ferbam losing its label on apples in
the United States in 2005. Captan, a protectant fungicide used for over sixty years, is
currently classified by the EPA as a “probable human carcinogen” using their 1986
guidelines for cancer risk assessment. In 2001, the Captan Task Force (CTF)
requested that EPA re-evaluate this fungicide under its current cancer risk
assessment guidelines.
In 2005, under the Guidelines for Carcinogen Risk Assessment (EPA, 2005),
captan would no longer be considered a B2 carcinogen, although it is likely to be
carcinogenic over long-term, high level exposure (Wilkinson, Arce & Gordon,
2004). In areas where resistance has not yet developed, most fungicide programs
today recommend incorporation of the protectant fungicides in rotation or tank-mix
with SI-fungicides, strobilurins, and the newer anilopyrimidine class (AP) of
fungicides. Starting at green tip, through seven-day intervals (or prior to predicted
rain), protectant fungicides such as copper, captan, and mancozeb, can be used to
effectively manage scab, and other foliar fungal pathogens.
As the outlook for chemical management appears bleak, the continued breeding,
and genetic engineering of scab-resistant apples holds the tremendous promise that
fungicide applications would not be needed for scab, although it must be stressed
that fungicides will still be necessary to control other fungal diseases, such as
powdery mildew, rust, flyspeck, sooty blotch, and bitter rot, to name but a few!
Some resistances are known and incorporated (Table 2). It is important to note that
no apple cultivar is resistant to every disease problem.
In 1943, Hough identified the segregation of scab resistance in Malus floribunda
‘821’ by crosses done in 1926 by C. S. Crandall. To date, numerous breeding
programs continue a modified backcross program to introgress genes for apple scab-
resistance from M. floribunda ‘821,’ while retaining commercially-accepted traits
like flavor, color, quality, and yield. This cluster of resistance genes, termed Vf, is
used in approximately 90 percent of the scab-resistant apple cultivars available, and
include varieties like ‘Enterprise’, ‘Freedom’, ‘Gold Rush’, ‘Jonafree’, ‘Liberty’,
‘Pristine’, and ‘Redfree.’ (Crosby et al., 1992).
Despite over 75 years of breeding for resistance, and the production of dozens of
scab-resistant cultivars, no variety to date has met with much commercial success.
When compared with such commercially popular varieties such as Ida Red, Red
Delicious, and Jonathan, the fruit-quality traits of color, size, and flavor of these
scab-resistant cultivars are less accepted by the general public. For this reason, the
direct transfer of the Vf gene to apple varieties currently popular may provide
consumers with the product they desire, with a reduced use of fungicides.
Table 2. Resistance ratings for some common commercial apple cultivars a.
Fire Cedar-Apple Powdery

Cultivar Apple Scab
Blight Rust Mildew
Red Delicious S R VR MR
Fuji S VS VR—VS R
Gala R VS R—S R
Golden
Delicious S S S S
Honey Crisp MR R VS S
McIntosh VS S VR MR
Mutsu
(Crispin) VS VS S R
a
S= susceptible; VS= very susceptible; R= resistant; VR= very resistant; MR= medium resistance.
In 2004, Belfanti et al. successfully transformed the susceptible cultivar ‘Gala’

with the HcrVf2 gene cluster that possesses an extracellular leucine-rich repeat
domain and a putative transmembrane domain, homologous to previously identified
resistance genes, including the tomato Cf gene allowing resistance to the tomato leaf
mold caused by Cladosporium fulvum. Transformation of the susceptible apple
cultivar Gala resulted in four independent transformed lines resistant to apple scab,
demonstrating that HcrVf2 cluster is sufficient to confer scab resistance to a
susceptible cultivar (Belfanti et al., 2004).
Despite the excitement of both the breeding and biotechnology programs, scab
was observed on resistant cultivars possessing the Vf cluster in 1984, in Germany,
and later, in other parts of Europe and New Zealand (Parisis et al., 1993). What this
means for the long-term use and durability of the Vf cluster in apple breeding and
biotechnology programs remains to be seen. Numerous other resistance genes have
been identified and mapped: the Vm gene from M. atrosanguinea 804, the Vr gene
from a Russian apple seedling from the Caucasas Mountains, Vm from M.
micromalus; and several candidates with polygenic resistance. With the public
sentiment strongly against genetically modified organisms (GMOs), however, the
future of transgenic apples remains uncertain, at best.
On the cultural side of apple scab management, the goal of sanitation is to
eliminate, or at least minimize, the development of primary inoculum, thereby
preventing the primary inoculation event from occurring. If ascospores cannot
successfully infect early in the season, the secondary cycle of conidia cannot become
established. Conversely, ascospore-incited infections that occur early in the season
(before tight cluster) can cause significant losses, due to the subsequent production
of conidia when leaves and fruit reach maximum susceptibility.
Sanitation is best directed at the production of pseudothecia in the leaf litter.
Examples of this include mulching or flail mowing leaf litter, applications of 5%
urea to trees just prior leaf drop, or applied to fallen leaves in autumn (Sutton,
MacHardy & Lord, 2000). These practices facilitate leaf decomposition, thereby
preventing the development of pseudothecia in the following season. In addition to

sanitation, pruning trees to enhance air movement and increase sunlight, reduces
foliar wetness on leaves and fruit.
2.2. Powdery Mildew

Powdery mildew has the unique distinction of being the only fungal disease of apple
capable of infecting in the absence of free moisture. Caused by the fungus
Podosphaera leucotricha, symptoms on fruit include russeting, whereas leaf
symptoms include chlorosis, necrosis, and the presence of a characteristic white
powder covering the leaf surface (Jones & Aldwinkle, 1990).

The fungus overwinters in terminal buds of apples, which became infected during
the previous growing season. In the spring, under drier conditions, conidia develop
and are released from leaves as they emerge from infected buds at about tight cluster
stage. They germinate in the absence of free water, but high relative humidity and
cool temperatures (10-25 °C). Early-season mildew development is affected more by
temperature than by relative humidity, with sporulation resulting in secondary
disease cycles that continue until susceptible, new growth is no longer available
(upon cessation of shoot growth). At this phase, the overwintering buds on infected
plants are also infected after bud initiation, setting the stage for next years infection
cycle. The window for fruit infection occurs between pink to bloom, and results in
russetting (Jones & Aldwinkle, 1990).
2.2.2. Management
Trees planted in sunny locations with good airflow reduce the humidity around
branches, thereby lowering the risk of disease. Protecting plants from powdery
mildew is important, as heavily infected shoots and buds possess reduced vigor,
resulting in winter damage and dieback. Ironically, this same phenomenon of
dieback eradicates much of the primary inoculum when winter temperatures drop
below –24 °C. In areas with warmer winter temperatures, infected branches should
be pruned, if feasible.
Numerous varieties are resistant to powdery mildew. These include Braeburn,
Delicious, Enterprise, Fuji, Gala, Grimes, Golden, Jonafree, Pricilla, Sir Prize and
Winesap. Pl2, a major resistance gene to apple powdery mildew, introgressed from
Malus zumi, is the primary resistance source used in apple-breeding programs. In an
experimental orchard in France, an increase of susceptibility to powdery mildew was
observed on apple genotypes carrying Pl2 (Caffier & Laurens, 2005). This increase
of susceptibility could not be explained by an effect due to i.e. the age of the trees,
or by an effect related to the amount of inoculum on Pl2 resistance expression. It
was demonstrated, by tests of pathogenicity in controlled conditions, that isolates of
P. leucotricha sampled in this orchard were virulent to Pl2.
When mildew-susceptible varieties are grown, the use of fungicides is required

to prevent russeting. Fortunately, most, but not all, fungicides effective against scab
are effective against powdery mildew. Successful management of powdery mildew
requires early season sprays from tight cluster to petal. However, the frequent
application of SI fungicides has resulted in a loss of efficacy against powdery
mildew. Nova®, which is increasingly ineffective against scab due to the widespread
nature of SI resistance, still provides excellent control against powdery mildew, as
do the strobilurins.
2.3. Fire Blight

Fire blight, caused by the bacterium Erwinia amylovora, attacks over seventy
members of the Rosaceae family, and is a devastating disease of apples and pears.
2.3.1. Symptoms
Twigs, branches, and leaders on infected trees wilt, forming a characteristic
“shepherd’s crook,” as the infected area discolors from tan to black. The name “fire
blight” was based upon the blackened leaves that are characteristic of infections on
pears. Infected apple branches turn reddish-brown to brown. In both hosts,
discolored leaves and flowers remain attached to the infected portion of the tree.
There are several distinct stages of fire blight, including blossom blight, shoot
blight, and rootstock blight. In the shoot blight phase, cankers develop rapidly,
resulting in scattered dead branches throughout the canopy. Tree death can result if
infections spread into the main stem or the rootstock. The younger the tree, the more
likely it will die, following infection (Jones & Aldwinkle, 1990).
2.3.2. Management
Effective management of fire blight is difficult because management options are so
limited. More so than any other disease of apple, fire blight requires an integrated
approach that combines the following tactics:
1. incorporation of resistant root stocks (and varieties, in the case of organic
growers);
2. cultural management of trees growth, through the use of judicious pruning
or plant growth regulators;
3. timely application of copper and streptomycin (if allowed), and
4. sanitation to reduce inoculum, when successful infection has occurred.
2.3.3. Root Stocks

Up until about 50 years ago, scion wood of desired cultivars were propagated on
seedlings. These grafts produced large trees that came late into bearing, often 10
years after planting. Today, root stocks can be chosen to produce a desired size,
from EMLA 27 (M27) that produces a tree about 1/5 the size of a standard apple tree
(approximately 8-10 feet) to P-18, or Antonovka 313, which is similar in size to a
seedling graft, but provides better fire blight and collar rot resistance. However,
most commercial orchards primarily use dwarfing rootstocks from the Malling
station (M): M7, M.9 and M.26. These dwarfing rootstocks produce trees that are
30-55% of the height of a standard tree, come into bearing early, and the small size
readily lending itself to high-density orchards. Unfortunately, the use of these
rootstocks in high-density orchards, coupled with their susceptibility to fire blight,
and the use of scions of susceptible varieties like Gala, often results in rootstock
infections, and catastrophic losses when fire blight occurred. Growers with a history
of fire blight should consider the use of rootstocks M.111, M.7, B.9, Geneva (G.) 30,
Alnarp 2, all of which provide better resistance to fire blight than M.9 and M.26
(Norelli, Jones & Aldwinkle, 2003).
2.3.4. Cultivars
In addition to highly susceptible rootstocks, newer varieties of apples, like ‘Pink
Lady,’ ‘Gala,’ ‘Braeburn,’ and ‘Fuji,’ are even more susceptible to fire blight than
many of the previously available commercial varieties, with infection of the
scionwood portion of these cultivars rapidly spreading to the rootgraft union,
resulting in complete loss of the infected tree (Longstroth, 2000). Scionwood
resistant varieties include ‘Red Delicious’ or ‘Empire’, that are commercially grown,
and varieties like ‘Liberty,’ ‘Pricilla’ or ‘Gold Rush,’ bred for resistance, that are
more a fixture in smaller orchards, organic orchards, and backyard growers. Organic
growers are strongly encouraged to plant these resistant varieties, grafted on
resistant rootstocks.
2.3.5. Cultural Management

Even resistant varieties can succumb to fire blight under high disease pressure, or
when plants are poorly managed. Managing tree vigor, particularly reducing
excessive vigor, and promoting early cessation of tree growth, are important factors
in minimizing the risk of fire blight. Excessive fertilization, particularly nitrogen,
encourages succulent growth while suppressing defense response, and can
predispose even resistant plants to fire blight.
Recently, the growth regulator prohexadione calcium (Apogee®) has been
effectively used to manage fire blight (Yoder et al., 1999). Apogee works by
inhibiting gibberellin biosynthesis, resulting in early cessation of the terminal.
Therefore, it has no effect on the blossom blight stage of the pathogen, but does
effectively reduce the incidence of shoot blight by inhibiting shoot growth. As there
is less susceptible tissue that can serve as an infection court, there is a corresponding
reduction in infection. It is important to stress that Apogee decreases host
susceptibility, and does not directly impact the pathogen.
2.3.6. Chemical Management and Predictive Models

Of all the prophylactic treatments available, the judicious application of copper
applied prior to the 1/4-inch green tip stage has been shown to play a crucial role at
reducing the amount of inoculum on the outer surfaces of infected trees. Essentially,
this application acts as a disinfectant, chemically “sterilizing” the surface of the tree,
and reducing the population of epiphytic bacteria that can colonize future wounds.
As for integrated management, this application of copper can double as the first
protectant scab spray of the season, and can be tank mixed with dormant oil for mite
and scale control, as long as the copper in question is not copper sulfate (e.g., Tri-
Basic Copper Sulfate, C-O-C-S). The adhesive properties of the Bordeaux mixture
can provide weathering ability not found to the same degree in the fixed coppers (C-
O-C-S, copper oxychloride + copper sulfate) or Kocide (copper hydroxide). All
application of copper should cease by ½ inch green to minimize the possibility of
phytotoxicity and russetting. Other chemicals, such as those that contain
phosphorous acid as the active ingredient, or agents of biological control, have
provided inconsistent levels of efficacy. The prophylactic use of copper, and the
reduction in inoculum is critical, as weather conditions and susceptible host plants
conducive to disease development can quickly result in epidemics, due to the
ubiquitous nature of the pathogen.
The destructive potential of fire blight necessitated the use of predictive models.
Certain weather events, and the timing of these events, were known as early as the
1950’s to exacerbate fire blight epidemics. Bloom is one of the most susceptible
phases in the fire blight disease cycle, and the use of copper-based sprays,
streptomycin sulfate, or oxytetracycline is critical at this time. Effective blossom
blight management prevents disease establishment, thereby reducing both the
incidence and severity of fire blight (Van der Zwet, Zoller & Thomson, 1988).
Trauma blight, when heavy rains, warm weather, and hail occur during the fire
blight infection period, is another key event in fire blight, and warrants the use of
antibiotics the day before, until 24 hrs after the trauma event occurred. When the
pathogen is already established, as in the case of canker and shoot blights, antibiotic
use (if legally allowed) is not recommended, to delay the additional development of
streptomycin-resistant strains of the bacterium (Steiner, 1990).
2.3.7. Removing Sources of Infection

Perhaps the most critical step in fire blight management is dormant pruning:
eradication of over wintering infections reduces inoculum levels next season,
thereby reducing infection rates. Pruning cuts should be made four to six inches
below any canker, or obviously dead bark, and all pruned material should be
removed from the orchard or burned.
Unlike dormant pruning, for which a positive consensus exists, the pruning of
fire blight strikes during the growing season is controversial. For growing season
pruning of fire blight to be effective, prompt pruning must coincide with early
symptom development, and the pruned material must be removed from the orchard.
At this time, fire blight strikes should be removed by making cuts 12 to 15 inches
below visible symptoms. Although theoretically sound, pruning during the growing
season can exacerbate the incidence and severity of fire blight by creating additional
wounds that serve as infection courts, by potential spreading the pathogen through
contaminated tools, and by excessive pruning that encourages the growth of
vegetative tissue, providing new infection courts for the pathogen. For this reason,
growers are encouraged to remove fire blight strikes if the infections sites are few. If
the damage is extensive, the grower should refrain from pruning until the dormant
season, where there is little risk of actually facilitating the spread of the pathogen
(Van der Zwet & Beer, 1995).
2.4. Rust Diseases

The cedar rusts standout due to the conspicuous nature of the disease, and the fact the
fungus completes its life cycle on two plant hosts — one in the cypress or juniper
(Cupressaceae) family and one in the rose family — with both commonly planted in
the urban landscape. There are three common cedar rust diseases in the Midwest:
- Cedar-apple rust, caused by the fungus Gymnosporangium juniperi-virginianae,

which requires two hosts to complete its life cycle: the fungus must infect apple
or crabapple in the spring, and an alternate host, Eastern red cedar (Juniperus
virginiana) or Rocky Mountain juniper (J. scopulorum) in the late summer.
Symptoms and signs of the infection can be found on leaves and fruit.
- Cedar-hawthorn rust, caused by Gymnosporangium globosum, which alternates
between junipers and hawthorn, crabapple, and apple in addition to several other
rosaceous hosts. Hawthorn rust causes leaf lesions but rarely infects apple fruit.
- Cedar-quince rust, caused by Gymnosporangium clavipes, that infects junipers
and a wide range of rosaceous hosts, namely hawthorn, but also serviceberry,
quince and pear. In apple, quince rust causes fruit lesions, but almost never
infects the leaves of apple.

The disease cycles of the three rust fungi are similar and surprisingly complex. In
the spring, orange gelatinous horns develop from gray to brown colored fungal galls
on the branches of infected junipers. These telial horns produce wind blown
basidiospores that infect apple trees. Symptoms on apple initially appear on the
upper leaf surface as small yellow spots that later enlarge and turn orange. Unlike
apple scab, cedar-apple rust lesions on apple leaves will not produce spores that
reinfect apple leaves and fruit. As the fungus continues to grows, fungal mating
occurs in rust lesions which result in the formation of aecia, yellow-brown lesions
develop on the underside of the leaf and form small, whisker-like structures
containing rust-colored aeciospores which are carried by wind to cedar trees where
they infect and complete the disease cycle. Galls start to develop on cedar shortly
after infection but do not exude telial horns until the second spring after infection.
The following spring, these galls produce orange gelatinous horns that release spores
and continue the infection cycle. Dead galls on cedar and juniper may remain
attached for a year or more.
2.4.2. Management
Some apple cultivars are resistant to cedar-apple rust (Table 1). All three rust fungi
require infection of eastern red cedar, or related species of Juniperus, to complete
their life cycles. Therefore, removing junipers within a 2-mile radius of an orchard
will disrupt the disease cycle, and fungicides may not be needed.
The rust diseases are usually kept in check by fungicides aimed at scab, although
captan, dodine, and benomyl do not control rust diseases. Where fungicide use is
minimal (e.g., on scab-resistant cultivars or in organic orchards), rust diseases,
especially cedar-apple rust, can severely spot leaves and damage fruit.
Quince rust causes fruit lesions but rarely affects leaves of apple. Hawthorn rust
causes leaf lesions but rarely affects apple fruit. To reduce the severity of rust,
growers should avoid planting susceptible juniper varieties near apple trees. If
juniper galls have already formed, the galls may be pruned from infected trees to
help reduce the number of spores available for infection in the following spring
(Jones & Aldwinkle, 1990).
3. SUMMER DISEASES
3.1. Bitter Rot
Bitter rot, caused by the fungi, Glomerella cingulata and Colletotrichum acutatum,
is a frequently occurring disease of apples wherever they are commercially grown,
and is particularly severe in the southeastern United States (Biggs & Miller, 2001).
Although historically considered a disease of warmer climate orchards, this disease
is developing into an emerging problem in more northern regions of the United
States. Of the three major fruit rot diseases on apple (bitter rot, black rot and white
rot), bitter rot regularly causes the most damage, and will be the only one discussed
here. One reason for the increasing incidence of this disease is due to the 77-day pre-
harvest interval (PHI) restrictions surrounding the use of EBDC fungicides.
3.1.1. Symptoms
Initial symptoms produced by perithecial or conidial strains are similar. Lesions
begin as small, slightly sunken areas, which are light brown to dark brown. Lesions
caused by conidia are often sunken and light brown, with concentric rings of
radiating pink-colored spore masses (acervuli) under humid conditions. Ascospore-
incited lesions are darker than those caused by conidia, and rarely sunken, with
radiating rings of brown to black acervuli.
Bitter rot decay extends in a cone shape toward the core, which helps distinguish
bitter rot from other fruit rots. Perithecia are found in dark brown to black clumps
scattered on the surface. A key diagnostic feature of bitter rot lesions (regardless of
which spore type or species causes infection) is the cone-shaped lesion that extends
to the core of infected apples. Infected fruit will eventually mummify, and some
may remain attached to the tree through the winter.

The fungus overwinters in mummified fruit infected during the previous season, and
in bark cracks and cankers of infected trees. Conidia serve as the primary source of
inoculum in over wintering sites, although ascospores are infective as well.
Interestingly, symptoms differ depending upon the inoculum type that is responsible
of infections. Dispersal of conidia occurs by splashing, wind-blown rain, insects
and birds. Warmer temperatures between 26.6 or 32.2 °C favor the disease
development, with fruit susceptible to infection soon after petal fall until harvest.
Historically, entire crops were lost to bitter rot during warm, wet summers. It is
unknown if fungicide resistance and pesticide restrictions may play a role in the
resurgence of this disease problem, in northern orchards.
3.1.3. Management
Mummies that remain in the trees from the previous season serve as a source of
inoculum, and should be removed. Trees should be monitored from mid-season
through harvest. Fungicide applications to control scab, powdery mildew, flyspeck
and sooty blotch should be effective against bitter rot. Fungicide application from
petal fall through harvest on a 10- to 14-day schedule should provide effective
disease control.
3.2. Flyspeck and Sooty Blotch

Sooty blotch and flyspeck are the terms used to describe “two” of the most common
late summer diseases of apples, symptoms of which appear as a “sooty blotch” or
small specks of black. Current research has identified more than 20 genera of fungi
that cause ‘flyspeck’ and ‘sooty blotch’ (Batzer et al., 2005). Although caused by
more than 20 different organisms, the appearance of this disease complex coincides
with both occurring on the fruit surface, and both being favored by similar
environmental conditions and cultural practices. It is important to note that this
problem results in economic losses due to the diminished appearance, and that the
disease complex has no effect on flavor or quality. Infection may shorten storage life
due to a loss of moisture in storage, but does not contribute directly to decay.
Flyspeck and sooty blotch symptoms are more pronounced on light colored fruit,
like Golden Delicious or Granny Smith, but no differences in resistances or
susceptibility have been identified.
3.3. Disease Cycle

The pathogens that cause these two diseases both overwinter through the production
of sexual or asexual sporocarps, depending upon genus. Spores of these pathogens
are released during the petal fall stage on apples, when mainly infect non-hosts like
nearby wild brambles. Orchard establishment of inoculum is often prevented due to
the application of scab fungicides at this time. These primary infections on cane fruit
and non-apple hosts later produce conidia during summer that cause most of the
apple fruit infections. Upon infection, the incubation period prior to the development
of signs can require several weeks or even months. The fungus develops a discrete
network of fine hyphal strands that eventually develops numerous pycnidia or
pseudothecia, that continuously produce spores to sustain the infection process.
3.4. Management
As previously stated, no known resistance exists to these pathogens. Therefore
cultural practices consist of proper pruning to open up the tree canopy to allow for
both drying, and access of fungicides needed to manage this problem. Clustered fruit
and overly vigorous trees with dense canopies prevent good fungicide applications
(for any disease problem). Proper pruning and hand thinning are critical to flyspeck
and sooty blotch control during unusually wet years.
For years, sooty blotch and flyspeck have been adequately controlled by
mancozeb. Restrictions on the use of benzamidazole fungicides like mancozeb,
captan, and maneb have resulted in an increasing incidence of flyspeck and sooty
blotch. However, trials have demonstrated that Topsin-M, Flint and Sovran are all
very effective for controlling flyspeck, and all three of these fungicides provide
some post-infection activity (Rosenberger, Meyer & Ahlers, 2000; Rosenberger
et al., 2001)
4. CONCLUSIONS
The greatest challenge facing agriculture today isn’t creating a set of technologies to
manage pest and disease problems, but educating consumers about food production
practices while at the same time educating growers to incorporate and use the
technologies that encourage a “long view” of stewardship, and natural resource
management. Growers readily adopt new apple varieties (contrary to the opinions
held only 25 years ago, that the public would not adopt apple varieties without name
recognition) and in the last two decades the variety of apples being sold has
increased to fulfill customers’ demand for novel, and sweeter apples. The
incorporation of these apples or “new technology”, was driven by customer demand.
Customer demand has also driven a wider adoption of “organic” practices. For new
policy adoption, a program of scientific education as to what is required to produce
“blemish-free” apples, must be developed and disseminated to the general public if
acceptance of future disease management practices is going to happen.
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and flyspeck complex on apples based on analysis of ribosomal DNA gene sequences and
morphology. Mycologia, 97, 1268-1286.
Belfanti, E., Silfverberg-Dilworth, E., Tartarini, S., Patocchi, A., Barbieri, M., Zhu, J. et al. (2004). The
HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proceedings
of the National Academy of Sciences of the USA, 101, 886-890.
Biggs, A. R., & Miller, S. S. (2001). Relative susceptibility of selected apple cultivars to Colletotrichum
acutatum. Plant Disease, 85, 657-660.
Brown E. M., & Sutton, T. B. (1986). Control of sooty blotch and flyspeck of apple with captan,
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Caffier, V., & Laurens, F. (2005). Breakdown of Pl2, a major gene of resistance to apple powdery
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Crosby, J. A., Janick, J., Pecknold, P. C., Korban, S. S., O’Connor, P. A., Ries, S. M., et al. (1992).
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Jones, A. L., & Aldwinckle, H. S. (1990). Compendium of Apple and Pear Disease. APS Press, 110 pp.
Jones, A. L., & Walker, R. J. (1976). Tolerance of Venturia inaequalis to dodine and benzimidazole
fungicides in Michigan. Plant Disease Reporter, 60, 40-44.
Jones, A. L., Lillevik, S. L, Fisher, P. D, & Stebbins, T. C. (1980). A microcomputer-based instrument to
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virulent to apples with resistance due to the Vf gene. Phytopathology, 83, 533-537.
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3
RITA MUSETTI
MANAGEMENT AND ECOLOGY OF PHYTOPLASMA

DISEASES OF GRAPEVINE AND FRUIT CROPS
Dipartimento di Biologia e Protezione delle Piante,
Università di Udine, via delle Scienze, 208, 33100 Udine, Italy
Abstract. Some aspects of the biology and management of phytoplasma diseases of grapevine and other
fruit crops are revised. Management of phytoplasma-infected plants has mainly focussed on controlling
the insect vectors and on roguing infected crops and weeds. The actual concept of “management” implies
the application of measures compatible with the environment, and of cultural practices essential for the
crops and economic thresholds. The production of genetically engineered plants by introducing disease-
resistance genes into cultivated crops togheter with the use of resistance inducer microorganisms to
reduce the disease symptoms, represent potential tools to control phytoplasma diseases.
1. INTRODUCTION
Phytoplasmas are an important group of plant pathogens representing a distinct
monophyletic clade within the class Mollicutes (ord. Acholeplasmatales, fam.
Acholeplasmataceae) (IRPCM, Phytoplasma working team 2004). The association
of these pathogens with plants exhibiting “yellows” symptoms was demonstrated for
the first time by Doi et al. (1967) using Transmission Electron Microscopy (TEM).
Until then, yellows diseases were though to be caused by viruses, since
phytoplasmas show certain characteristics in common with them. For example, they
could not be grown on culture media, are obligate parasites, cannot survive away
from a host, and grow and reproduce in the phloem of the host plants or within
insect vectors.
Phytoplasmas are wall-less prokaryotes, similar to bacteria but without a rigid
cell wall. They are pleomorphic in shape, looking like sacks or blobs, ranging from
70 to 1000 nm in diameter, or roughly the size of a plant cell’s chloroplast.
Phytoplasmas are bounded by a trilaminated unit membrane, containing ribosome
and fibrils of DNA (Musetti & Favali, 2004). Their shape may be helical,
filamentous, beaded or simply spheroid (Fig. 1). They are localized exclusively in
the sieve tubes of the host plants, where they are capable of active multiplication
(Favali & Lombardo, 1970). They are transmitted by phloem-sap-feeding insects
such as leafhoppers and psyllids (Kummert & Rufflart, 1997), or by vegetative
propagation, such as grafting. Phytoplasmas are responsible worldwide of hundreds
43
44 R. MUSETTI
of diseases (McCoy et al., 1989; Lee, Gundersen-Rindal & Bertaccini, 1998; Lee
et al., 2000), affecting different plants belonging to 98 families including many
economically important crops.
These plant-pathogens contain a minimal genome and lack genes coding for
ATP synthases and sugar uptake and use, making them completely dependent on
their host. As with other Mollicutes, phytoplasmas lack several genes that bacteria,
such as Escherichia coli, need for metabolism: for example, Mollicutes do not
possess genes for the synthesis of amino acids, fatty acids or nucleotides
(Christensen et al., 2005). Phytoplasmas stand out in lacking many genes considered
essential for cell metabolism, therefore they must rely on the uptake of nutrients by
membrane-transport processes. In fact, many important transporters are retained
compared with those present in other Gram-positive bacteria. On the other hand, the
sequenced phytoplasmas revealed a repeat-rich genome, indicating that these
prokaryotes have compensated for their constraints with horizontal gene transfer,
rearrangement of DNA and recombination between the chromosome and the
plasmids (Lee, Zhao & Bottner, 2005).
Figure 1. Transmission electron microscopy image showing pleomorphism of

phytoplasma cells in host plant sieve tubes.
In the host plants, these prokaryotes induce low growth rate, stunting, yellowing
or reddening of the leaves, reduced leaf size, shortening of internodes and loss of
apical dominance. These effects lead to stunting of the plant, proliferation of shoots
or roots, witches’ brooming, reduced yields, general decline and, sometimes, death
of the plant. Several symptoms affect flowers, including virescence, phyllody and
sterility. In phytoplasma-infected plants, symptom appearance is preceded by
cellular modifications, visible only by TEM, such as: callose deposition near sieve
plates and plasmodesmata; starch accumulation in the chloroplasts and their
disorganization; phloem necrosis (Musetti, 2006).
Symptoms of phytoplasma diseases are due to interactions with the hormonal
balance of the host (Pertot et al., 1998), but physiological relationships between
PHYTOPLASMA DISEASE MANAGEMENT 45
phytoplasma and host plant have remained largely unknown. Recent developments
have improved our knowledge on the effect of phytoplasma infection on host
secondary metabolites, mainly in herbaceous host-plants (Musetti et al., 1999;
Musetti, Favali & Pressacco, 2000; Tan & Whitlow, 2001; Choi et al., 2004), but the
literature available is still scarce regarding the physiology of phytoplasma infections
in fruit crops (Musetti et al., 2004) and grapevine (Bertamini et al., 2002; Musetti
et al., 2006). Phytoplasma infection can lead to production of defence proteins,
increase of phenolic compounds, and involvement of important signal molecules
such as Ca2+ and H2O2 (Musetti & Favali, 2003; Musetti et al., 2004). Moreover,
using different display of mRNA, genes involved in photosynthesis and amino acid
transport were found to be downregulated, while other genes, particularly those
involved in stress response, were upregulated (Jagoueix-Eveillard et al., 2001).
The progress of knowledge in phytoplasma research has mainly depended on
tools aiming at their characterisation, since they are not culturable in vitro. This fact,
together with the great difficulty in defining different phenotypic characters, has
caused many problems with the classification of these microorganisms. Molecular
characterisation of conserved genes codifying for the 16S rRNA, has presented a
partial solution to the problem, thus allowing the first classification scheme based on
10 major groups and 15 subgroups (Gundersen et al., 1994). This classification was
later expanded to 14 groups and 38 subgroups (Lee et al., 1994). Subsequently, the
use of restriction fragment length polymorphism (RFLP) analysis permitted the
identifications of unknown phytoplasmas, allowing the classification of 57
phytoplasma strains in 20 distinct groups (Seemüller et al., 1998).
Since, according to conventional Latin binomial nomenclature, the description
of organisms cultured in vitro is required for naming of species (and phytoplasmas
cannot be isolate in artificial media) a classification comprising “Candidatus (Ca.)
Phytoplasma species” has been recently proposed (IRPCM, Phytoplasma working
team 2004).
The provisional “Ca. Phytoplasma” species were then defined according to the
available 16S rRNA gene sequences. More than 200 different length sequences of
16S rRNA genes have been obtained and compared, permitting a delineation
between different strain clusters. A strain can be described as a novel “Ca.
Phytoplasma” species if its 16S rRNA gene sequence has less than 97.5% similarity
to that of any previously described “Ca. Phytoplasma” species. Phytoplasmas
presenting 16S rRNA gene sequences that share more than 97% similarity must be
considered as ecologically separated populations. Moreover, since some
phytoplasmas, sharing high similarity, show different biological, phytopathological
and molecular properties, the description of two different species is recommended
only when the following three conditions are verified: 1) the two phytoplasmas are
transmitted by different vectors, 2) they have different natural host plants or their
behaviour is different in the same host plant, 3) serological tests or polymerase chain
reaction (PCR)-based assays show significant molecular diversity. Following these
rules, around 20 “Candidatae species” have been formally described to date
(IRPCM, Phytoplasma working team 2004; Firrao, Gibb & Streten, 2005).
46 R. MUSETTI
2. PHYTOPLASMAS DIAGNOSIS IN CROPS

An early and accurate diagnosis of plant diseases is a crucial component of any crop
management system. Plant diseases, in particular phytoplasmoses, can be managed
most effectively if control measures are applied at an early stage of disease
development. Reliance on symptoms is often not adequate, since their expression
can be highly variable. In the past, the detection and identification of phytoplasmas
was quite imprecise, due to the impossibility of their isolation from the host. The
observation of phytoplasma cells in sieve elements of affected trees with TEM was
the main criterion used to ascertain the possible phytoplasmal origin of a disease.
Moreover, this method was laborious and time-consuming, and not adequate to
distinguish differences between phytoplasmas or the relative phytoplasma strains
(Musetti & Favali, 2004). The most recent advances in molecular biology and
biotechnology have been applied to the development of rapid, specific and sensitive
tools for the detection of phytoplasmas in the infected plants.
Serological tests such as enzyme-linked immunosorbent assay (ELISA) and
immunofluorescence, performed by the use of specific antibodies, provided sensitive
methods for the detection and identification of the different phytoplasma strains
(Chen, Lei & Lin, 1992; Lee et al., 2000). Obtaining monoclonal antibodies against
phytoplasmas, associated with important diseases, such as grapevine Flavescence
dorée (FD) and Apple proliferation, improved the use of serological methods for the
detection of these pathogens to the extent that several commercial kits are now
available (Boudon-Padieu, Larrue & Caudwell, 1989; Loi et al., 2002).
Dot and Southern hybridisation, using cloned phytoplasma DNA probes,
permitted not only the precise detection of various phytoplasmas associated with
plants and insects, but also enabled studies about their genetic relationships. Several
different groups and subgroups have been thus recognised (Lee et al., 1992).
Restriction fragment length polymorphism (RFLP) analyses of the phytoplasma
genomic DNA permitted further differentiations of groups. The first genotype-based
classification of phytoplasma was thus established (Lee et al., 1992).
New molecular tools, based on PCR assays, further advanced diagnostics for
phytoplasma diseases, providing a much more sensitive means than serological tests
or DNA hybridisation assays. In fact, PCR methods also permitted the detection of
low titre of phytoplasmas, that were not easily revealed by the techniques described
above. Several universal or group specific primers were designed, both based on
highly conserved 16S rDNA gene sequences and 16S-23S intergenic spacer region
sequences, or on conserved rp gene and elongation factor EF-Tu (tuf) gene
sequences (Lee et al., 2000).
Recently, real time PCR (RT-PCR) technologies opened new opportunities for
the detection and the studies of phytoplasmas: in fact they combine the sensitivity of
conventional PCR with the generation of a specific fluorescent signals providing
real time analysis of the reaction kinetics, also allowing quantification of specific
DNA targets. Moreover, the absence of post PCR manipulations prevents carryover
contamination. The application of this method to phytoplasma determination
increased in recent years (Bianco, Casati & Marziliano, 2004; Torres et al., 2005;
Marzachì & Bosco, 2005), showing that it is also suitable for monitoring the
progress of the infection in the hosts. Finally, DNA microarray systems could
represent a versatile new technology to detect different plant pathogens, including
phytoplasmas, and allowing a simultaneous screening that should distinguish a
broad range of microorganisms (Hadidi, Czosnek & Barba, 2004).
3. NATURAL TRANSMISSION AND EPIDEMIOLOGY

Phytoplasmas reside in the phloem tissues of the infected plants, invading primary
sieve tubes elements. They can be transmitted to the host plants by three
mechanisms: i) vegetative propagation (grafting included), ii) vascular connections
between infected and non-infected host plants provided by parasitic plants such as
dodder (Cuscuta spp.), and iii) phloem-feeding insect vectors. In nature, the third
mechanism is the most common.
The single most successful order of phytoplasma vectors is the Hemiptera, most
commonly leafhoppers, although also planthoppers and psyllids can be active
vectors of phytoplasmas. The transmission of phytoplasmas is persistent, circulative
and propagative. There are specific interactions between phytoplasmas and their
insect vectors (Weintraub & Beanland, 2006), including the possibility that some
phytoplasmas are transmitted by several species of leafhoppers (peach-X disease
phytoplasma), while others (elm yellows phytoplasma) are transmitted by one or
only a few species. However, in general, phytoplasmas transmission tends to be
specific.
The molecular mechanisms that regulate this strict pathogen-insect specificity
are poorly understood. Recently, the involvement of a phytoplasma membrane
protein (named antigenic membrane protein, Amp) that is able to recognize and bind
insect vector proteins (particularly actin, actin myosin heavy chain, actin myosin
light chain) has been hypothesised (Suzuki et al., 2006).
The efficiency of phytoplasma transmission by insect vectors is due to several
biological characteristics. Vectors may be hemimetabolous, with nymphs and adults
feeding similarly and in the same physical location. They may feed selectively on
certain plant tissues, their feeding being not destructive and promoting pathogen
inoculation in the plant vascular system without damage. In alternative the vectors
may host obligate symbiotic prokaryotes that are passed to the offsprings by
transovarial transmission, also enabling the transovarial transmission of
phytoplasmas.
The presence of other prokaryote microorganisms inside the phytoplasma-
vector tissues could indeed reduce the vector competence, resulting in pathogenicity
to the insect or interfering with the presence of the phytoplasma (Marzorati et al.,
2006). The effects of phytoplasmas on their insect vectors, however, are not clear.
Bressan et al. (2005) reported about negative effects on the insect vector
Scaphoideus titanus when infected by FD phytoplasma.
The complex interactions between insects and phytoplasmas also play a very
important role in determining the spread of phytoplasma diseases. Polyphagous
vectors could have the capacity to inoculate a wider range of plants even if it was
demonstrated that some of them cannot acquire, in equal manner, phytoplasmas
48 R. MUSETTI
from different infected plants (Bosco et al., 1997). On the other hand, several studies
reported that insects that do not feed on certain plant species in nature are able to
acquire and transmit phytoplasmas under experimental conditions (Weintraub &
Beanland, 2006).
4. PLANT RECOVERY
Recovery in diseased plants is a spontaneous remission of symptoms that has been
reported in grapevine, apple and apricot plants affected by phytoplasmas (Osler
et al., 2000; Carraro et al., 2004). The physiological basis for this phenomenon is
not yet completely known. On the basis of phytoplasma-closely-related-pathogens
(Gram+ bacteria), we can correlate recovery to various biological events. These
include the presence and dominance of hypovirulent strains, the presence of
antagonists or phytoplasma parasitoids (Marzorati et al., 2006), the activity of
particular substances or plant secondary metabolites, or the induction of systemic
acquired resistance (SAR). Recently, the involvement of H2O2 and some ROS-
related metabolites and enzymes in the recovery phenomenon has been hypothesised
(Musetti et al., 2004; 2005b). These observations, together with the fact that
recovered plants can be re-infected in nature to a lesser extent than non-infected
plants (Osler et al., 2000), indicate that a type of SAR could be involved in the
induction of recovery.
In grapevines, the phenomenon appears to depend on different factors including
the type of pathogen (phytoplasma), host plant variety (e.g. cv Prosecco allows
recovery whereas cv Perera does not), type of rootstocks, environmental conditions,
and agronomic practices (eg. pruning, transplanting). Recovery can be complete or
partial, temporary or permanent, common or rare and, consequently, it can be
significant or not in an infected crop. A very convincing case is that of the grapevine
cv. Prosecco where more than two million grapevines completely recovered from
FD between 1995 and 1998 in North-East Italy (Province of Treviso), and normal
production has been re-established (Osler et al., 2003). However, recovery was not
observed on phytoplasma-infected Perera grapevines (Pavan et al., 1997).
5. PHYTOPLASMA DISEASES AND MANAGEMENT

Phytoplasmas have been associated with diseases in several hundred plant species
including vegetable and fruit crops, ornamental and forest trees, and many other
emerging diseases are being identified. Phytoplasma infections are the most
important limiting factor for production of important crops all over the world. For
example, “grapevine yellows” contributes to the loss of grapevine production in
Europe and Australia, as well as “apple proliferation” and other fruit phytoplasma
diseases cause the loss of fruit production in Europe and North America. Because of
these and other important diseases, the movement of affected plant species is
restricted by quarantine rules.
The relatively low titres, associated with irregular distribution of phytoplasmas
inside host tissues, and the inability of their cultivation in vitro has previously made
difficult the development of accurate diagnostic procedures for phytoplasmas.

Today, the application of rapid and sensitive diagnostic tools for the precise
detection and identification of these prokaryotes in plant material allowed a very
important contribution towards control strategies. In fact, control measures can be
effective if applied at an early stage of disease development. Unfortunately, no
effective means of curing phytoplasma diseases are available at present.
Phytoplasma diseases are complex and their progress is also highly variable.
Their development depends on many factors including: 1) the state of the host
plants; 2) the pathogen and its different biotypes; 3) the tendency for mutation; 4)
the presence and dynamics of the vectors; 5) the concentration on the host-plants of
both phytoplasma and vectors; 6) the environmental conditions as well as 7) the
agronomical practices being used. As a consequence, no single fixed control strategy
can be adopted. In practice, management and control are based on the elimination of
sources of phytoplasmas and on the control of insect vectors.
However, before intervening, some important observations must be made,
including: 1) disease severity; 2) whether or not to rogue infected trees; 3) roguing
strategies; 4) possibility and relevance of recovery phenomena; 5) advisability of
replacing young plants after roguing; 6) strategies to control the insect vectors (Osler
& Carraro 2004).
In the following paragraphs, some aspects of the phytoplasmas induced diseases
are reviewed, focusing on grapevines and fruit trees, which are the most severely
affected crops.
5.1. Grapevine Phytoplasmas

Grapevine yellows (GY) diseases are a group of disorders of Vitis vinifera
associated to phytoplasmas belonging to different groups (Prince et al., 1993; Conti
2001). In fact, GY include: Flavescence dorée (16SrV, Elm Yellows) reported in
France, Italy, Spain, Portugal and Serbia; Palatinate grapevine yellows (16SrV, Elm
Yellows) in Germany; Bois noir (16SrXII-A, Stolbur) in Europe, Israel, Lebanon;
Australian grapevine yellows (16SrXII-B, Stolbur), Tomato big bud (16SrII,
Stolbur) and Buckland valley grapevine yellows (16SrI, Aster Yellows) in Australia;
Grapevine Yellows (16SrI-A, Aster Yellows) in Italy, Slovenija and Croatia;
Grapevine Yellows (16 SrIII-B, X-disease) in USA, Northen Italy, Israel.
In spite of the diversity of the pathogens, the symptoms caused by different GY
are not distinguishable. They are characterised by growth reduction, leaf
discoloration (such as yellowing or reddenish and downward curling of leaves on
stunted shoots), reduced fruit quality and yield. Symptoms are not uniform, and may
appear on some, or even all, shoots of infected vines. A few rootstock varieties are
tolerant to GY and can be symptomless carriers of the disease. Leaf symptoms in
white grapevine cultivars appear as small, yellow spots along the main veins. These
spots enlarge to form yellow bands along the veins, which gradually extend over
large parts of the leaf. Red cultivars develop a similar pattern on the leaves, but the
discolorations are reddish. Infected shoots often fail to lignify, and appear thin and
rubbery. They later become brittle, sometimes with bud necrosis. Affected branches
50 R. MUSETTI
blacken and die during the winter. Numerous small black pustules form along the
diseased branches of susceptible cultivars. Fruit set is reduced on grapevines
infected early in the season, as the inflorescences dry out and fall off. In later
infections, bunches may become brown. Premature berry drop occurs in some
cultivars (Fig. 2).
FD, reported for the first time in France (Caudwell, 1957) and Bois noir (BN),
also described in France (Caudwell 1961), are the most important GY diseases in
Europe.
Figure 2. Symptoms of phytoplasma disease on grapevine leaves and shoots.
5.1.1. Flavescence Dorée

Among phytoplasmas affecting grapevines, FD is considered one of the most severe
threats for viticulture where its presence, or that of its insect vector, Scaphoideus
titanus Ball, has been reported. It is known to occur in several European countries,
from Portugal to Serbia (Carraro & Ermacora 2004), destroying large viticultural
areas in Europe (it has been defined “catastrophic” in France), and it is still
spreading despite mandatory control programs. Impacts include reduced vitality of
grapevines, yield reductions, and reduced wine quality, due to high acid and low
sugar contents of fruit harvested from infected plants. Without control measures, the
disease spreads rapidly, affecting up to 80-100% of grapevines within a few years.
The phytoplasma associated to this disease, for which the novel designation
“Candidatus Phytoplasma vitis” (“Ca. P. vitis”) has been suggested (IRPCM,
Phytoplasma working team 2004), belongs to the elm yellows group (EY, 16S r V
group) (Daire et al., 1993, 1997; Seddas et al., 1996). Different Ca. P. vitis strains
have been characterised, showing different geographic distributions. For example,
strain FD70 is present in France, FD92 in France and Spain (Daire et al., 1997),
whereas FD-C and FD-D have been reported in Italy (Martini et al., 2002).
Cultivars such as “Chardonnay”, “Cabernet Sauvignon”, “Pinot noir”,
“Riesling”, “Prosecco”, “Merlot”, “Barbera” are highly susceptible to FD, while
others, such as “Garganega”, “Perera”, “Sangiovese” are extremely susceptible and
are quickly killed (Borgo 1996).
FD is spread by the leafhopper S. titanus, which is native to eastern North
America, and spends its whole life cycle on grapevines. It is a highly mobile and
efficient vector that is largely responsible for the epidemic spread of FD. Both
nymphs and adults are able to acquire the phytoplasma while feeding on infected
grapevines. After a latent period they are able to transmit the disease until they die.
This leafhopper overwinters as eggs that are inserted (laid) into the bark of
grapevines.
FD is a quarantine disease and its significance is emphasised by regulations
such as the EPPO certification scheme for grapevine propagation materials or the
CEE plant health directive (2000/29/CE) that requires phytoplasma-free grapevine
material and mandatary control of the vector.
In fact, the disease can be controlled by applying insecticides against the vectors
with one treatment per year, thus reducing the population level of leafhoppers by
almost 96%, (Pavan et al., 2004). The effectiveness of this treatment against S.
titanus can be explained by a number of reasons. The insect is monofagous, feeding
exclusively on grapevines and cultivated grapevines are their only hosts. Moreover,
the vector produces only a generation per year. Insecticide treatments against S.
titanus represent a method to prevent disease spread in non infected areas. In areas
where the disease is present and spreading, elimination of infected plants must be
performed, but if there is a real possibility of recovery, roguing is not advisable. For
example, grapevine cv. Prosecco is known to recover frequently (Osler et al., 2003).
Furthermore, the phytoplasma itself disappears from the crown of the recovered
grapevines, so they are no longer a dangerous source of inoculum for successive
transmission (Musetti et al., 2006).
5.1.2. Bois Noir

BN is a grapevine disease characterised by symptoms that are nearly identical to
those induced by FD. In contrast to FD, BN is endemic to Europe where it is
widespread in almost all vine-growing regions (Maixner, Langer & Gerhard, 2006).
The BN associated phytoplasma belongs to the 16SrXII-A group (stolbur), for
which the novel designation “Candidatus Phytoplasma solani” (“Ca. P. solani”) has
been suggested (IRPCM, Phytoplasma working team 2004). Three different isolates
of the phytoplasma can be distinguished by molecular traits and by the different
52 R. MUSETTI
association to different natural host plants (Langer & Maixner, 2004). The most
susceptible cultivar is “Chardonnay”, but also “Pinot”, “Merlot”, “Cabernet” and
“Barbera” are highly susceptible to the disease.
In addition to grapevines, this phytoplasma occurs in many herbaceous and
woody plants in several European countries (Seemüller et al., 1998).
“Ca. P. solani” is transmitted to grapevine by Hyalesthes obsoletus Sign.
(Cixiidae) (Maixner, 1994; Sforza et al., 1998). This insect is ubiquitous, being able
to feed and complete its cycle on several spontaneous erbaceous plants, such as
Urtica dioica, Convolvolus arvensis, Setaria viridis, Potentilla reptans, Cirsium
arvense, Solanum nigrum and Plantago lanceolata (Credi et al., 2002). These
weeds, when growing in the proximity of vineyards, play an important role in the
epidemiology and spread of the disease (Alma & Conti 2002), representing a
dangerous source of inoculum. Populations of H. obsoletus show on these plants
differences regarding feeding preferences and the time required to complete the life
cycle. This is of particular interest since, according to the different insect
behaviours, it can be hypothesised that different epidemiological cycles of the
phytoplasma occur in Europe (Maixner, Langer & Gerhard, 2006). The disease can
be also transmitted to different herbaceous hosts by Pentastiridius beierii Wagner
(Gatineau et al., 2001), although the spread of the disease in areas where these
vectors do not occur suggests the existence of other potential insect vectors
(Maixner, Langer & Gerhard, 2006).
The insecticide treatment to control BN influences neither the population
density of the vector H. obsoletus nor disease incidence. The poor effectiveness of
chemical sprays can be explained by the vector behaviour. In fact, the natural
transmission of BN to grapevines involves several weeds (i.e. U. dioica, C.
arvensis), that represent dangerous source of inoculum. Thus, the selective
elimination of weeds hosting the phytoplasma appears to be an important procedure
to control the vector, and disease, spreading. In infected vineyards elimination of
symptomatic grapevines is advisable, but an evaluation of recovery could avoid
drastic measures, thus limiting the eradication only to weaker plants.
5.2. Fruit Trees Phytoplasmas

Apple proliferation (AP), Pear decline (PD) and European stone fruit yellows
(ESFY) are important fruit disease in Europe. They are associated with three
phytoplasmas belonging to the Apple proliferation group (16Sr X) (Lee et al., 2000).
Phylogenetic analyses revealed that the 16S rDNA sequences of the three
phytoplasmas are nearly identical (Seemüller & Schneider 2004), with the resulting
differences limited to 1.0–1.5% of nucleotide positions. Moreover, they have
biological and ecological similarities, are present in the same areas on plants
belonging to the same family (Rosaceae), and are transmitted by Psyllids.
However, on the basis of more accurate molecular, serological, vector
transmission and host-range experiments, it was demonstrated that the three
phytoplasmas are coherent but discrete taxa and can be distinguished at the putative
species level (Seemüller and Schneider, 2004). Thus for these species the names
“Candidatus Phytoplasma mali” (“Ca. P. mali”), “Candidatus Phytoplasma pyri”

(“Ca. P. pyri”) and “Candidatus Phytoplasma prunorum” (“Ca. P. prunorum”) were
proposed, respectively (Seemüller & Schneider 2004).
Given the importance of the disease associated with fruit tree phytoplasmas, it
was important to obtain information on their genomic organisation, in order to better
understand the host-pathogen molecular relathionship and, above all, the
pathogenicity. For this reason, physical maps of the chromosome of “Ca. P. mali”
and “Ca. P. prunorum” were constructed (Lauer & Seemüller 2000; Marcone &
Seemüller 2001), confirming a very similar genetic arrangement but also a certain
heterogeneity in phytoplasma populations.
5.2.1. Apple Proliferation

Apple proliferation (AP), associated to “Ca. P. mali”, is among the most
economically important plant disease in Europe, and is considered as a quarantine
disease in Europe and North America. For this reason, “Ca. P. mali” is a very
intensively studied phytoplasma. Several studies on diagnostic methods and
epidemiology were reported (Musetti et al., 2008). More recently, several genes were
identified and sequenced. A genetic variability of “Ca P. mali” has been reported,
and different strains were described (Jarausch et al., 2000b).
Apple is the main host of “Ca. P. mali” and almost all cultivars appear
susceptible, as well as wild and ornamental Malus. The most typical symptom is
witches’ broom, but also typical is the presence of smaller leaves, enlarged stipules,
leaf-rosette formations, virescent flowers, low quality fruits which appear pale in
colour or green, small and with a long, narrow petiole. The distribution of the
phytoplasma in the phloem tissues is not constant, and depends on the seasonal
photoperiod and temperatures, rendering its detection difficult or unreliable, during
certain periods of the year (EPPO 2006).
As described above, recovery can occur in apple plants affected by AP.
Symptoms remission can be transient or permanent, and it is influenced by host
genotype and environmental conditions (see paragraph 4). In recovered apple plants
phytoplasmas disappear from the crown, but they were found in the roots (Carraro
et al. 2004). The disease is spread in a persistent-propagative manner by psyllid
insect vectors: in particular, Cacopsylla picta (sin. C. costalis) and C. melanoneura
(Frisighelli et al., 2000; Tedeschi, Bosco & Alma, 2002). Both insect vectors
produce only one generation per year. In Spring, reimmigrant psyllas come back to
apple trees and lay eggs, from which a new generation hatches. This generation
leaves the apples to colonize secondary host plants (i.e. Coniferae), where they
overwinter as adults.
In nature, single transmission efficiency is quite low, but the diffusion of the
disease can be very severe and problematic because of the high number of active
insect vectors. At present, vector control and roguing remain the two basic ways of
combatting disease diffusion. In uninfected areas, control of the insect vectors by
precautionary insecticide treatments are advisable, particularly against adult
reimmigrant psyllas. In infected areas, when the epidemics are still at an early stage,
54 R. MUSETTI
it is strongly advisable to rogue the infected trees, even if they might recover and
produce regularly. In addition, contiguous trees must be eradicated, even if
asymptomatic. When epidemics of AP are already established in a given area, the
eradication of the orchard is strongly advisable.
5.2.2. European Stone Fruit Yellows

European stone fruit yellows (ESFY) is the common name of a group of
phytoplasma-related diseases, that occurr in different stone fruit species, such as
apricot chlorotic leaf roll, plum leptonecrosis, peach yellows and peach decline.
ESFY is particularly disseminated around the Mediterranean basin, causing very
serious problems in Spain, France, Italy and the Balkans, where the cultivation of
sensitive and susceptible Prunus species is widespread.
As reported above, the phytoplasma associated with ESFY (for which the name
“Candidatus Phytoplasma prunorum” has been proposed) belongs to the AP group,
being closely related to Ca. P. mali and Ca. P. pyri, but it is distinctly different from
other stone fruit phytoplasma diseases present in North America, such as X-disease
and peach leaf roll (Kison, Kirkpatrick & Seemüller, 1997). Different strains of Ca.
P. prunorum have been described, showing great differences in virulence (Kison &
Seemüller 2001). In particular, the most virulent strains are able to kill the host
plants very quickly, whereas the milder ones do not cause mortality, inducing
slightly reduced vigour.
Among Prunus species, the most susceptible to ESFY are P. armeniaca and P.
salicina (apricot and Japanese plum). In these plants the typical symptoms of the
disease, such as diffuse yellows, leaf roll (Fig. 3) followed by leaf reddening and
progressive necrosis, are very severe and can cause decline and eventual plant death.
Fruit production can be totally lost.
Figure 3. Symptoms induced on apricot by ESFY infection.

Other cultivated or wild species are highly tolerant to ESFY (Carraro et al.,
2002). In particular, P. avium demonstrated a high level of resistance (Jarausch et al.,
2000). The presence of wild plants is important for the epidemiology of the disease
because the pathogen can survive and spread, without the presence of susceptible
cultivated plants.
ESFY is spread by Cacopsylla pruni (Carraro et al., 1998b). The vector has an
European and Cental Asian distribution, and is strictly oliphagous on Prunus spp.
Similar to the vector of AP, the ESFY vector produces only one generation per year.
In Spring, reimmigrant psyllas return to plum trees and lay eggs, from which a new
generation hatches. In turn, this second generation overwinters as adults on
secondary host plants (probably Coniferae). As with other phytoplasma diseases,
ESFY is not curable and prevention is the only control method. In non-infected
areas, in the absence of the vector, the use of certified plant material is advisable and
this can be sufficient to control the disease spread. In areas with medium or high
infection pressure, treatments against C. pruni are necessary. Alternatively,
cultivation of tolerant species or plants with induced-resistance can be used (Morvan
et al., 1991).
5.2.3. Pear Decline

Pear Decline (PD) is a widespread disease present throughout Europe and North
America. The phytoplasma associated with PD (proposed as “Candidatus
Phytoplasma pyri”) belongs to the 16Sr X AP group (Lee et al., 1998). The disease
has been present in Europe for a long time (Refatti 1967), causing the death of
numerous pear plants (expecially Pyrus communis). Among cultivated pears the most
susceptible cultivars are: Decana del Comizio, William, Abate Fetel and Kaiser. On
the other hand, Conference seems to be quite tolerant.
The most typical symptoms occur in late summer, with the development of red
leaves but some cultivars may develop a yellow colour. Subsequently, the leaves
tend to become cupped or curled. A line of necrotic tissue in the bark may appear, at
the graft union between scion and rootstock.
PD may develop along two courses: the first is called “quick decline” where the
plant dies in a few weeks; the second is more common and is named “slow decline”,
with the plant declining slowly and death occurs in a few years.
The insect vectors responsible for the spread of PD are the pear psyllas
Cacopsylla pyricola L. and C. pyri Först (Carraro et al., 1998a). The first one
spreads the disease in North America and United Kingdom, whereas the second
species has been observed in Italy, France and Spain (Garcia-Chapa et al., 2005),
suggesting that the latter is probably the most important vector in the Mediterranean
area. Both vectors produce 3-6 generations per year, depending on the region, crop
and environmental conditions, and overwinter as adults on the same host. Cacopsylla
pyri is the most serious pest of pear trees in Europe and also an efficient vector of
phytoplasmas, so a great deal of effort has been made to control this vector.
The only method to control PD is to control the pear psyllas and maintain the
trees in good vigour (for example, reducing the stresses caused by inadequate
56 R. MUSETTI
irrigation or nutrient deficiency). Moreover, the use of tolerant rootstocks

(producing excellent crops), in areas exposed to the vectors, is advisable.
6. NEW APPROACHES AND PERSPECTIVES

As already mentionned, no curative methods are available against phytoplasma
diseases. In addition, among the most cultivated fruit crops and grapevine varieties,
not one is fully resistant. Management of phytoplasma-infected plants has mainly
focused on controlling the insect vectors and on roguing infected crops and weeds.
However, the actual concept of “management” implies the application of modern
measures compatible with the environment, as well as the cultural practices essential
for the crops and the economic thresholds.
The production of genetically engineered plants by introducing disease-
resistance genes into cultivated crops and so reducing the disease symptoms,
promises to control phytoplasma diseases.
Recently, studies on microorganisms as potential biocontrol agents against
vectors or plant resistance inducers, have been carried out, giving interesting results.
For example, some parasitoids that might be able to reduce the transmission
effectiveness of leafhoppers have been identified (Marzorati et al., 2006; Weintraub
& Beanland 2006). Lingua et al. (2002) demonstrated reduced symptom expression
associated with a reduction of nuclear senescence in phytoplasma-infected plants
treated with arbuscular mycorrhizal fungi. Fungal endophytes, antibiotic producers
or resistance inducers, have been isolated from recovered plants (Musetti et al.,
2005). Lherminier et al. (2003) reported the capacity of two elicitins to prevent
symptom expression in tobacco plants, infected with stolbur phytoplasmas.
Studies on the physiological bases of recovery and the metabolic pathways of
infected and recovery plants are also important to better understand resistance
phenomena (Musetti et al., 2004; 2005a; 2006). From a practical point of view, these
studies are proposed as a possible innovative opportunity to control phytoplasma
diseases.
ACKNOWLEDGEMENTS
Author is grateful to Prof. Ruggero Osler for critical reading and to Dr. Laurence
Cantrill for revision.
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4
SANTA OLGA CACCIOLA1 AND GAETANO MAGNANO DI
SAN LIO2
MANAGEMENT OF CITRUS DISEASES CAUSED

BY PHYTOPHTHORA SPP.
1
Dipartimento di Chimica Biologica, Chimica medica e Biologia
Molecolare, University of Catania,
Viale Andrea Doria 6, 95126 Catania, Italy
2
Dipartimento di Gestione dei Sistemi Agrari e Forestali, Faculty of
Agriculture, Mediterranean University of Reggio Calabria, 89122 Reggio
Calabria, Italy
Abstract. The complex of citrus diseases caused by Phytophthora spp. is reviewed, with reference to the
damages caused by Phytophtora root rot, gummosis and brown rot of fruits. Some aspects of the biology
and ecology of P. citrophthora and P. nicotianae are revised, like the inoculum dissemination, the fungus
reproduction and epidemiology. The symptomatic diagnosis of main diseases like foot rot or gummosis,
fibrous root rot, brown fruit rot and dieback of twigs and leaves, are reviewed. Biological and
instrumental diagnosis as well as routine laboratory tests are revised, for inoculum monitoring, sampling
and population dynamics procedures. Disease management methods based on interventions on the host-
plant, rootstock resistance, grafting, as well as nurseries sanitary practices are illustrated, together with
pruning, surgery, and cultural practices like soil preparation, fertilization, irrigation and soil management,
and weeds control. Chemical control methods are also reviewed, with reference to the use of systemic
fungicides for control of trunk gummosis, root rot and brown rot of fruits.
1. INTRODUCTION
Citrus are among the ten most important crops in terms of total fruit yield worldwide
(Table 1) and rank first in international fruit trade in terms of value. The term
“citrus” indicates a complex of species belonging to the sub-family Aurantioideae
(family Rutaceae) including the following genera: Citrus, Eremocitrus, Fortunella,
Microcitrus and Poncirus. More than seven million hectares are planted with
citrus throughout the world (Table 2). Although citrus are native to East Asia,
citriculture has expanded in tropical, subtropical and mediterranean climatic
regions (Table 3). Mediterranean countries are the leading producers for the
international fresh market. The all-inclusive term “Phytophthora root rot” indicates a
complex disease which is caused by several soil-borne species of Phytophthora and is
recognized as a major fungal disease of citrus almost universally (Boccas & Laville,
1978; Klotz, 1978; Gregory, 1983; Magnano di San Lio, 1994; Erwin & Ribeiro,
61
62 S. O. CACCIOLA AND G. MAGNANO DI SAN LIO
1996; El-Otmani; 2006; Sadowsky, 2006; Tuset, 2006). Phytophthora spp. attack
citrus plants at all stages and may infect all parts of the tree, including roots, stem,
branches, twigs, leaves and fruits. Root rot, foot rot (also known as “gummosis”,
“trunk gummosis” or “collar rot”), fruit brown rot, twig and leaf dieback (often
indicated collectively as “canopy blight”) and rot (better known as “damping off ”)
of seedlings, all caused by Phytophthora spp., may be considered different facies of
the same disease.
Table 1. Major crops worldwide (FAO, 2004).
Crops Yields (× 1000 tons)
Sugar cane 1,318,178

Mais 705,293
Wheat 624,093
Rice 608,496
Potato 330,518
Sojbean 206,410
Barley 155,115
Sweet potato 127,535
Tomato 124,112
Citrus 108,095
2. DAMAGES CAUSED BY PHYTOPHTORA ROOT ROT

Gummosis and root rot are the most serious facies of Phytophthora root rot. In
nurseries, gummosis can lead to the rapid death of young citrus trees, whereas on
adult trees the disease course is chronic. Usually a mature tree shows symptoms of
decline on the canopy, including leaf chlorosis, philloptosis, dieback of twigs, small
and poor colored fruit, offspring fruit production, twig dieback and withering of
leaves during periods of drought, if the infection affects more than 50% of the
circumference of the trunk.
Root rot is especially harmful if the plant is grafted on a susceptible rootstock.
Young trees are very susceptible. Seriously affected nursery-trees do not overcome
the crisis induced by transplant. The infections on bearing trees cause the decay of
the canopy, defoliation, leaf chlorosis and a reduction in the fruit size and
production. However, plants with a high percentage of infected roots may not show
symptoms. The tolerance to this facies of the disease depends on the capacity of the
plant to regenerate roots and to substitute the rotten ones (Graham, 1995). This
capacity is remarkably reduced if the soil is saturated with water. There are indirect
estimates of the damage caused by this particular aspect of the disease. According to
researches carried out in the United States, the damage in terms of yield losses
PHYTOPHTHORA MANAGEMENT ON CITRUS 63
caused by root rot is on average about 5%, while the damage caused by gummosis
was estimated to be on average about 1% (Menge & Nemec, 1997).
Table 2. Major citrus producers in the world (FAO, 2004).
Country hectares
China 1,476,679
Brasil 942,267
Nigeria 730,000
Mexico 523,503
U.S.A 430,080
Spain 296,950
India 264,500
Iran 232,500
Italy 168,507
Argentina 145,000
Egypt 143,883
World 7,295,135
Brown rot of fruit is a common preharvest decay of citrus fruit, which causes
the fruit to fall. The infection occurs with rain splash to lower hanging fruits.
Infected fruits picked during the incubation period of the disease can still infect
healthy fruits in storage. This disease causes occasionally severe damage when
heavy or lasting rainfall occurs before harvest.
The epidemic explosions of brown rot usually occur in areas where heavy
rainfall coincides with the early stages of fruit maturity as immature fruits are not
susceptible to the infection. Severe attacks have also been caused occasionally by
overhead sprinkler irrigation, due to the use of water contaminated by Phytophthora
propagules. Annual losses from brown rot vary greatly, even in the same site. As
much as 90% of the crop on an individual tree and up to 30% of the total production
of some orchards were estimated to be lost when the disease was noticed for the first
time in Florida (Knorr, 1956).
Severe damages caused by canopy blight have been occasionally observed in
the nursery and on potted ornamental citrus plants under greenhouse (Kuramoto,
1981; Magnano di San Lio, Tuttobene & Pennisi, 1986; Magnano di San Lio,
Pennisi & Tuttobene, 1986).
Seedling rot is a disease of citrus in nurseries and affects the seedlings just
before or just after they have emerged from soil. It has disastrous effects although
limited in 24 hours, and about 80% of the seedlings in a seedbed may be killed.
Table 3. Major citrus fruit producers in the world (FAO, 2004).
Country Yields (× 1000 tons)
Brasil 20,594
U.S.A 14,907
China 14,655
Mexico 6,475
Spain 6,206
India 4,750
Iran 3,825
Italy 3,493
Nigeria 3,250
Argentina 2,690
Egypt 2,562
Turkey 2,408
South Africa 1,850
Indonesia 1,600
Pakistan 1,585
Japan 1,470
Greece 1,227
Morocco 1,139
Thailand 1,116
World production 108,181
2.1. Causal Agents

The genus Phytophthora includes about 70 species. It belongs to the family
Pythiaceae, phylum Oomicota, kingdom Chromista or Stramenopila. At least ten
species of Phytophthora, including P. bohemeriae Sawada, P. cactorum (Leb. &
Cohn) Schröeter, P. cinnamomi Rands, P. citricola Sawada, P. citrophthora (R. E.
Smith & E. H. Smith) Leonian, P. hibernalis Carne, P. megasperma Drechsler, P.
nicotianae van Breda de Haan (= P. parasitica Dastur), P. palmivora (Butler)
Butler, and P. syringae Klebahn have been reported to attack citrus in the world.
However, the commonest species of Phytophthora in citrus orchards are P.
citrophthora and P. nicotianae. Other rarer Phytophthora species, such as P.
cactorum, P. citricola, P. hibernalis and P. syringae attack fallen fruits and
sporadically rootlets as well (Favaloro & Sammarco, 1973).
Phytophthora syringae and P. hibernalis, which have a lower optimal
temperature than other species, are found in citrus orchards during winter months.
Phytophthora palmivora is a very polyphagous species of tropical origin and

also attacks citrus orchards. It is very common in Florida (Zitko, Timmer & Sandler,
1991; Graham & Timmer, 2006). In Italy, although it has been found in olive trees,
ornamental plants and garden citrus trees, it did not spread to citrus orchards or to
commercial citrus nurseries (Magnano di San Lio et al., 2002).
2.2. Biology and Ecology
2.2.1. Dissemination and Reproduction

Phytophthora spp. produce various types of propagules: sporangia or conidiangia,
which can germinate directly, through a germ tube, or indirectly releasing zoospores,
motile biflagellate propagules, which lose the flagellum and encyst on contact with
the surface of the host and germinate; chlamydospores, resistant structures which
allow the pathogen to survive in unfavourable conditions; gametangia, called
respectively antheridium (male gametangium) and oogonium (female
gametangium); and oospores, which are formed after sexual reproduction and act
also as organs of preservation. Some species, including P. nicotianae and probably
P. citrophthora, are heterothallic, producing oospores only if the mycelium of the
two different mating types of sexual compatibility (A1 and A2) come into contact
with each other. Natural infections of all parts of citrus are most frequently caused
by zoospores and occasionally by direct germination of sporangia (Klotz & De
Wolfe, 1960).
2.3. Epidemiology
Both P. citrophthora and P. nicotianae are polyphagous, that is, they infect
numerous plant species. Phytophthora nicotianae is more active in warm conditions
than P. citrophthora (Table 4) and attacks mainly the rootlets. P. citrophthora is the
main causal agent of trunk gummosis and fruit brown rot. The primary source of
inoculum is the rhizosphere soil, where the pathogen survives in the roots in the
form of mycelium, chlamydospores and oospores. The infected rootlets and fruits
with brown rot infections are the sources of the secondary inoculum and, in fact,
sporangia are formed on their surfaces, whereas no sporangia are formed on the
gummy cankers at the foot of the trunk. As far as it is known, P. citrophthora does
not reproduce sexually and very probably P. nicotianae reproduces sexually only
occasionally, since in the majority of citrus orchards examined only one mating type
of mycelium is found.
Sporangia are produced on contact with air on the most superficial soil layers
and are transported on the fruits by rain, irrigation water and wind. They germinate
in water, and a single sporangium releases from 5 to 40 zoospores. Production and
germination of sporangia are influenced above all by temperature and soil water
potential. The zoospores are motile and can swim short distances by flagellar
movement or can be carried over longer distances by soil water. The zoospores are
attracted by root exudates and sweem towards roots and encyst upon contact. Cysts
then germinate and penetrate the cortex through wounds or directly. The zoospores
can affect any part of the plant, if it remains wet at least 18 hours. The trunk,
branches and roots are infected through lesions, but the zoospores germ tube can
penetrate fruits, leaves, shoots and green twigs directly even in absence of lesions.
Table 4. Cardinal temperatures (°C) for mycelium growth of citrus

isolates of Phytophthora citrophthora and P. nicotianae.
Minimum Optimum Maximum
P. citrophthora 5 25-28 35
P. nicotianae 5-10 28-30 35-38
Soils rich in calcium have a repressive effect on Phytophthora populations

(Campanella, Ippolito & Nigro, 2002), whereas the amounts of NaCl accumulated in
soil after using saline irrigation water may stimulate the production of sporangia.
NaCl may also damage the roots, increasing their susceptibility to infection (Blaker
& MacDonald, 1985). In general, host susceptibility is affected when roots are
stressed or damaged, and root exudates released by damaged or stressed roots attract
zoospores (Graham & Timmer, 2006).
Temperature is a major ecological factor affecting the seasonal fluctuations of
P. citrophthora and P. nicotianae and their distribution. In the Mediterranean region
P. nicotianae is not active in winter, while P. citrophthora is not inhibited by winter
temperatures (Timmer et al., 1989; Ippolito, De Cicco & Salerno, 1992).
Conversely, the activity of this latter species is dramatically reduced in hot summer
months with the exception of short periods following irrigation (Magnano di San Lio
et al., 1988; 1990).
Phytophthora nicotianae is more common in subtropical areas and causes foot
and root rot. Occasionally it attacks aerial parts of the tree causing brown rot of
fruits, but usually it does not infect far above the ground. Phytophthora citrophthora
causes brown rot outbreaks during fall and winter. In subtropical areas this species is
restricted to cooler weather sites and coastal areas (Feld, Menge & Pehrson, 1979).
However, as both species may grow and reproduce between 15 and 30 °C, the range
of soil temperature of most citrus orchards throughout the year, it appears unlikely
that summer soil temperatures may result sufficient to inhibit P. citrophthora.
There is evidence that Phytophthora inoculum fluctuations are strongly
influenced by the physiological conditions of the host-plant, which in turn are
correlated with temperature. Seasonal variations of citrus trees physiology, for
example, are a major factor determining the susceptibility of fibrous roots to the rot
incited by P. nicotianae (Lutz & Menge, 1991; Matheron & Matejka, 1993).
Summer activity of this species is directly correlated with both the production of
root exudates and the concentration of sugars in the roots, but it is inversely
correlated with starch concentration (Fig.1). Also the bark susceptibility to infection
by P. citrophthora in subtropical and mediterranean climates varies throughout the
year and is higher in spring and autumn and very low in winter and summer
(Matheron & Matejka, 1989; Adonia et al., 1992).
In subtropical and mediterranean areas, when soil temperature falls to about
12°C, citrus roots stop their growth. In these circumstances, P. nicotianae forms
chlamydospores and becomes inactive. The subsequent population increase of this
species occurs in spring, when soil temperatures rise again, and coincides with a
new flush of roots. In tropical regions, where roots grow almost all the year,
seasonal fluctuations of plants susceptibility to root rot infections are less evident.
Phytophthora palmivora is found in tropical areas but its epidemiology is more
similar to that of P. citrophthora. It attacks preferentially fibrous roots and fruit. The
fruit is susceptible to brown rot infections from the ripening phase.
Brown rot epidemics are more frequent in citrus orchards where trunk gummosis
is endemic. If the environmental conditions are favourable for infections, for instance
when there is heavy rainfall in the winter period, brown rot is associated with the
dieback of leaves and twigs. The incubation period of brown rot is 3 – 7 days,
according to the temperature (Schiffmann-Nadal & Cohen, 1966). Asymptomatic
infected fruits can still infect healthy fruits even after harvesting, during transportation
and storage.
2.4. Symptomatic Diagnosis
2.4.1. Foot Rot or Gummosis

The specific symptoms of this facies of the disease are the cankers and gummosis at
the base of the trunk (Fawcett, 1936). Gum oozes proceed from longitudinal cracks
of the bark around necrotic areas, which have a distinct water-soaked discoloration.
The dead bark turns soft and sloughs off the central cylinder below which a callous
is formed around the edges of the lesion. If the canker affects more than 50% of the
trunk circumference, the plant shows symptoms of decline in the canopy, chlorosis
of the veins and also midrib of the leaf, philloptosis, thinning of the canopy and
dieback of branches. Gum exudation can be seen on trees especially between the end
of spring and beginning of summer. The gum is water-soluble, but even if it is
washed away by the rain, the discolouration on the cortex is still visible. Since the
cankers are often formed below ground, it may be necessary to scrape away the soil
around the collar to see them and evaluate the severity of infection.
2.4.2. Fibrous Root Rot

This is the most difficult facies to diagnose by visual inspection, since similar
symptoms can be caused by different factors, like poor soil aeration and excessive
salt content in irrigation water (Klotz et al., 1958). The root cortex sloughs off
easily, leaving the stele bare, with the tip of the rootlet appearing thread-like. The
plant reacts to the infection by forming new rootlets. Adult plants may show no
symptoms even when there is a very high percentage of infected rootlets. The
symptoms of decay on the canopy appear when the plant is no longer capable of
producing new rootlets to substitute the rotten ones.
2.4.3. Brown Fruit Rot and Dieback of Twigs and Leaves

Infected fruit show a leathery brown rot with indistinct edges and have characteristic
rancid odour (Feld, Menge & Pehrson, 1979). If the humidity in the air is high,
white furry mould can be seen on the fruit surface. In environmental conditions
especially favourable to brown rot of fruits, symptoms can also be observed on
leaves and twigs. Infected leaves show dark brown oil-soaked necrotic spots with
indefinite margins, dry up and fall early. These necrotic lesions appear frequently on
the leaf tip. The infected twigs show gummosis, browning of the cortex, defoliation
and dessication.
2.5. Biological and Instrumental Diagnosis
2.5.1. Baits
In citrus orchards, the presence and quantity of Phytophthora inoculum in soil can
be determined empirically according to how frequently the ripe fruit left on the
ground for 3 – 7 days is infected. Ripe fruit of lemon and sweet orange can be used
as bait to capture P. citrophthora in the soil. Fragments of leaves from different
citrus cultivars are universal baits, i. e. they can be used to capture all Phytophthora
species living in citrus orchards. About ten grams of soil are incubated at ambient
temperature in the dark in a paper-glass filled with distilled water (soil : water ratio
1:6). After 4-6 days of incubation leaf pieces are picked up and observed at the
microscope for the presence of sporangia along the leaf cut edge. Another option is
to transfer the leaf fragments used as baits in Petri dishes on a selective isolation
medium and to identify the Phytophthora colonies grown from baits after 3-6 days
of incubation at 22-24 °C (Magnano di San Lio & Perrotta, 1982). However, to
identify the species or to determine the exact amount of inoculum, laboratory tests
are required.
2.5.2. Laboratory Analysis

In routine laboratory tests to obtain axenic cultures of Phytophthora from soil or
rotten tissues, isolation on selective media, such as BNPRAH or PARP (Erwin &
Ribeiro, 1996), is the most frequently used microbiological method. Similarly, serial
dilution of soil aliquots in Petri dishes on a selective medium is a very popular
method to determine the quantity of inoculum in soil. Diagnostic kits based on the
double-antibody-sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with
genus-specific polyclonal and monoclonal antibodies have been developed for
detection of Phytophthora spp. in roots and soil debris (Pscheidt et al., 1992;
Cacciola, Pennisi & Magnano di San Lio, 1995).
Although the ELISA method is highly sensitive and can detect the presence of
Phytophthora at lower population densities than dilution plating onto selective
media (Timmer et al., 1993), it has not been applied on a large scale for
Phytophthora detection in citrus orchard, probably because of the special laboratory
equipment which is needed to obtain quantitative data.
Molecular detection methods have been developed more recently, including
PCR with species-specific primers, nested-PCR with genus and species-specific
primers as well as real time-PCR (Ippolito, Schena & Nigro, 2002; Grote et al.,
2002; Ippolito et al., 2004). The molecular methods are very sensitive and rapid but
may be applied in specialised laboratories only.
2.5.3. Monitoring of Inoculum
2.5.3.1. Definition
Monitoring consists in periodically determining the quantity of inoculum of the
various species of Phytophthora present in the soil of the citrus orchard or in the
irrigation water. Quantitative methods are used, such as isolation from infected
organic material (roots, leaves, bark etc.) on selective media, insemination of the
substrate with a series of soil dilutions, the DSA-ELISA assay on infected roots or
molecular analysis using Real time-PCR of the DNA extracted from samples of
water, soil or organic material. Monitoring is useful, especially for the rational
timing and management of chemical treatments (Sandler et al., 1989; Matheron
Porchas & Matejka, 1997).
2.5.3.2. Critical Values of Inoculum Density

The quantity of inoculum determined with microbiological methods is generally
indicated as inoculum density (ID) and is expressed in terms of Colony Forming
Units (CFU) / g or cm3 of soil or water. The ID critical values in soil (also defined
threshold or danger levels) have been determined experimentally and can be used to
evaluate the effects of control options or as a guideline to program treatments. The
threshold intervention level in bearing orchards is 10-20 CFU/cm3 of soil (Magnano
di San Lio, Tuttobene & Perrotta, 1984; Menge, 1986; Timmer et al., 1988), but for
newly transplanted plants or for nursery stock it might be 3-5 CFU/cm3 (Magnano di
San Lio, Tuttobene & Perrotta, 1984; Magnano di San Lio et al., 2002).
The ID of P. nicotianae is directly related to the mass of fibrous roots, as
infected roots provide substrate for the propagules multiplication (Agostini, Timmer
& Castle, 1991; Sandler et al., 1989). Thus population levels of this species
generally diminish with soil depth and with distance from the tree. Highest
population densities of P. citrophthora (more than 100 CFU/cm3) have been found
in soil of citrus orchards during epidemic explosions of fruit brown rot (Magnano di
San Lio et al., 1988). Usually, however, in most citrus orchards ID values range
from 1 to 20 CFU/cm3.
Population levels show a seasonal pattern (Fig. 1) and may vary considerably
from year to year. It is also important to remind that Phytophthora populations in
soil are not uniform across the orchard. Studies on their horizontal spatial
distribution indicate either a random or an aggregate negative binomial pattern
(Magnano di San Lio, Reforgiato & Russo, 1987; Timmer et al., 1988; Magnano di
San Lio & Pennisi, 1994; Graham & Timmer, 2006). Because of this not uniform
spatial pattern of the inoculum, a great number of soil samples might be required to
detect lowest ID. Timmer et al. (1988) suggested that Elliot’s equation may be used
to calculate the number of soil samples theoretically needed to obtain a reliable
estimate of the ID of Phytophthora populations, fitting a negative binomial
distribution. They estimated that about 5-10 samples/hectare would be sufficient to
determine the mean ID in citrus orchards with moderate to high inoculum level.
2.5.3.3. Sampling
The criteria suggested in practice for collecting soil samples to determine routinely
ID are the following: the soil samples are taken from the rhizospere of the tree at a
depth of about 10-30 cm, under the tree canopy in the area soaked by irrigation
water, and they must contain rootlets. Each sample is obtained by mixing 20-40 sub-
samples taken from at least 4 trees over a surface of about 4 hectares, to give an
overall weight of 0.5-1 kg. It is advisable to analyse the sample within 24-48 hours
after its collection. If it must be kept for a longer period, it should be stored at room
temperature in a plastic bag, which should be left open to avoid water condensation.
Figure 1. Schematic representation of seasonal patterns of populations of Phytophthora

citrophthora and P. nicotianae in soil of citrus orchards in Mediterranean climate and
relationship between inoculum density, expressed as colony forming units (CFU) · gram of
soil-1, and physiological parameters of citrus trees.
2.5.3.4. Population Dynamics

The best period of the year to collect soil samples is determined by the seasonal
fluctuations of the quantity of inoculum of the two main Phytophthora species
present. Generally speaking, the ID values of P. citrophthora are highest in spring
and autumn, whereas those of P. nicotianae in summer months (Fig 1). In summer
P. citrophthora rarely can be isolated from soil, while P. nicotianae is dormant
during the winter months. As a rule, if it is not clear which Phytophthora species is
prevalent in the citrus orchard soil, the best time to take samples is between March
and November, 1-2 days after irrigation.
2.5.3.5. Molecular Methods

When applying molecular methods for the quantitative determination of the
inoculum, it has been shown that a direct correlation occurs between the ID values
determined by isolation on selective substrates and expressed in terms of CFU and
the quantitative analysis values of DNA determined by Real time-PCR (Ippolito
et al., 2004).
3. DISEASE MANAGEMENT
Disease management mainly relies on the deployment of some specific interventions

on the host-plant, including the selection and use of resistant rootstocks, grafting,
and further sanitary practices applied since the early plant growth phases in citrus
nurseries. Pruning and plant surgery may also be applied as routine practices.
Further phytosanitary practices include soil preparation, irrigation management,
fertilising, soil management and weeds control. Finally, chemical control is
considered, through the application of fungicides with different modes of action.
3.1. Interventions on the Host-Plant
3.1.1. Rootstock
One of the main factors on which incidence, intensity and spreading of the foot and
root rot infections depend is given by the rootstock degree of susceptibility.
Resistance to Phytophthora root rot, therefore, is one of the characteristics to be
taken into account when selecting a rootstock (Table 5).
Since the epidemic outbreaks of the 1860s in the Mediterranean area, the
control of Phytophthora foot and root rot has been mainly based on the use of
resistant rootstocks (Magnano di San Lio, 1994; de Franqueville, 2002). In Japan,
for example, foot rot is considered a minor disease thanks to the widespread use of
trifoliate orange, Poncirus trifoliata (L.) Raf., as rootstock (Kuramoto, 1981).
Although the use of resistant plants is one of the best and most practical means
to control Phytophthora root rot, some rootstocks resistant to Phytophthora are
susceptible to other diseases or are horticulturally unsatisfactory (Ferguson,
Sakovich & Roose, 1990). The rootstocks resistant to P. citrophthora infections are
also resistant to P. nicotianae. However, it is not always true that a rootstock

resistant to foot rot is also resistant to root rot. For example, sour orange (C.
aurantium L.) and “Carrizo” citrange (C. sinensis Osbeck × P. trifoliata [L.] Raf.)
are tolerant to foot rot but are susceptible to root rot (Graham & Timmer, 2006).
Trifoliate orange is resistant to both foot and root rot.
3.1.2. Grafting
The bud union must be enough far from ground level (above or at least 40 cm) to
prevent the Phytophthora inoculum present in soil from reaching the scion through
water splashing (Whiteside, 1972). Most of the citrus species and cultivars used as
scions, in fact, are susceptible to Phytophthora infections. Grafting with highly
susceptible species or cultivars such as clementines or nucellar clones of sweet
orange may reduce rootstock resistance (Boccas & Laville, 1978; Laville, 1984;
Feichtenberger et al., 1994; Ippolito et al., 1994; Ippolito, Nigro & Lima, 1997).
3.1.3. Sanitary Practices in Nurseries

The inoculum found in bearing orchards usually comes from the nursery. Ideally,
when a new citrus orchard is planted, the plantlets must be free of Phytophthora
infections. This is reccomended in countries that have introduced certification
schemes for nursery citrus plants. The plants with symptoms of gummosis on the
trunk must be eliminated before planting. If a high percentage of plants in a nursery
show symptoms of gummosis or the mean value of Phytophthora ID in the
rhizosphere soil is above 3-5 CFU/cm3, it would be advisable to discard the whole
lot. Management of seedling damping off is based on prevention by using seeds
extracted from healthy fruit and dressed with fungicides, then sowing them in
sterilised soil and irrigating with non-contaminated water. Other sanitary
recommendetions in citrus nurseries can be summarised as follows:
- soil fumigation with i.e. Vapam or sterilisation with steam, before planting;
- limit as far as possible the transit of people or vehicles in the nursery;
- grow plants in separated containers;
- keep the containers above ground level on benches or gravel beds
- avoid resting containers on water-proof plastic sheets, tarmac or cement;
- examine the plants periodically and eliminate those with symptoms of
gummosis to prevent the disease from spreading;
- do at least one quantitative determination of the Phytophthora inoculum
density in the soil of the containers between April and November;
- do not excede with irrigation;
- select a nursery site at some distance from commercial citrus orchards;
- avoid using machinery and tools previously used in other citrus orchards.
3.1.4. Pruning
Pruning modifies the architecture of a tree and may have an effect on the diseases
caused by Phytophthora. For example, the removal or the thinning of the lower
branches can create an unfavourable habitat for trunk gummosis infections and
reduce the risk of infections of fruit brown rot. Drastic pruning or topworking of
plants with symptoms of decline due to foot or root rot reduce the volume of the
canopy and prevent the tree from collapsing. Moreover, it helps the tree to recover,
given the predisposing causes of infection are removed, and will result in a more
efficient management if complemented by chemical treatments.
3.1.5. Surgery
Surgical intervention, practised in the past before systemic fungicides were
introduced to help infected plants recover from foot gummosis (Klotz, 1978), is now
almost obsolete. This is indeed a laborious and time consuming practice. It consists
in carving out the rotten bark. There must be a clean cut to help the scar to cicatrize
quickly and it is not necessary to penetrate too deeply into the woody cylinder. The
lesion can be disinfected with copper-based products, such as the Bordeaux mixture,
copper oxychlorides or mixtures of systemic copper-based fungicides. An alternative
method could be to cauterise the gummy canker with a flame, without removing the
bark (an ordinary blowtorch can be used).
3.2. Cultural Practices

3.2.1. Soil Preparation
An accurate preparation of the planting site, including 80-100 cm deep ditch before
the planting, underground drainage and a superficial soil levelling to allow rainwater
to run off and drain away, prevent stagnation and consequent water saturation of the
soil. Water saturation is one of the main predisposing factors for Phytophthora
infections. When a site has to be replanted, a period of 6-12 months fallowing will
effectively reduce Phytophthora populations as well as other soilborne pests such as
citrus nematode. The young trees must be planted at the same depth as in the nursery
to avoid burying the collar. Putting soil in raised beds (also named mounding or
“meseta” in Spanish), to avoid burying the collar and soil waterlogging under the
tree canopy where the root density is higher, is an example of prevention based on
soil management. This type of soil preparation is especially used and popular where
citrus orchards are planted in heavy soils (El-Otmani, 2006; Schillaci et al., 2006).
3.2.2. Irrigation Management

A fundamental part of the life cycle of P. citropthora and P. nicotianae is spent in
water. The sporangia release zoospores if the soil or the atmosphere is saturated with
water. The released zoospores reach their targets (i.e. the roots, trunk, fruit, branches
or leaves) through water. Finally, an essential condition for the infection to take
place occurs when the host surface remains moist for some hours (at least 18),
giving enough time for the zoospores to germinate and for the germ tube to
penetrate. Another aspect to consider is that hypoxia, which is a consequence of the
soil water saturation, increases the susceptibility of citrus trees roots to
Phytophthora infections and inhibits the growth of new roots.
From this epidemiological knowledge it is convenient to follow some simple
general principles for rational irrigation management:
a) use water not contaminated by Phytophthora;
b) do not wet the trunk;
c) avoid flooding the soil.
Irrigation methods that wet the trunk favour gummosis infections. When one of
these methods is used, it is preferable to irrigate during the day and for short periods,
in order to allow water to evaporate and reduce the time the bark remains wet. As a
root rot preventive practice, the time intervals between irrigations can be extended
(Ohr & Menge, 2006) to reduce the water potential of the upper layers of soil below
the lowest values for P. citrophthora and P. nicotianae activity (Fig. 2). By this
way, the ID in the soil layers with the higher concentration of roots is reduced.
However, the practical application of this concept is difficult. In a study aimed at
determining the influence of different irrigation regimes on Phytophthora rot of
feeder roots, it was shown that in the presence of Phytophthora spp. larger plants
with healthier roots were obtained with frequent irrigation scheduled on the basis of
tensiometer readings, thus suggesting that the practice of drying out orchards soils to
reduce root rot problems is unnecessary, unless excess water was added to soil
(Stolzy, 1959). In the case of irrigation water, it is important to avoid premature
irrigations in spring, when roots are inactive. A recommended practice would be
irrigations of shorter duration with the frequency adjusted on instrument readings
(Ohr & Menge, 2006).
Generally speaking, the use of localised irrigation methods, such as drippers,
makes the plants more vulnerable to root rot. In fact, the ID of P. nicotianae is
directly correlated to the root density which, in arid or semi-arid environments, is
inversely proportional to the volume of soil wetted by irrigation water. In citrus
orchards irrigated in this way, constant monitoring of the water status using
tensiometers is recommended to avoid soil saturation. The effect of various methods
of irrigation on soil populations of Phytophthora has also been investigated by many
authors (Magnano di San Lio et al., 1988; Feld, Menge & Stolzy, 1990; Ippolito,
Lima & Nigro, 1992).
3.2.3. Fertilising
In citrus orchards with problems of root rot, it is better to apply nitrogen in nitrate
rather than ammonium form. The ammonium nitrogen, in fact, is rapidly
metabolised to asparagine and glutamine. These amino acids provide ideal
nourishment for P. nicotianae and attract zoospores (Menge & Nemec, 1997).
Figure 2. Effect of soil water status on the epidemiology of Phytophthora

citrophthora and P. nicotianae.
3.2.4. Soil Management and Weeds Control

Removing the soil around the collar creates unfavourable conditions for gummosis
infections, since it prevents the bark at the foot of the trunk from remaining moist
for too long, and helps the cankers to heal. Vice versa, the development of weeds
around the collar creates a favourable habitat for gummosis infections, since it
prevents the quick drying of the bark.
The use of herbicides is a way of removing this problem and makes periodic
inspections to detect gummosis infections and their treatment easier. The use of
herbicides on the row, moreover, reduces the risk of wounds on the trunk, which
provide entry points for infection. The lesions, before healing, are prone to
infections for about two weeks (Graham & Timmer, 2006).
Mechanical tilling of the soil near the trunk leads to burying the base of these
trunk, causes lesions and therefore aids collar gummosis infections. Deep tilling aids
root rot since it damages the roots, making them susceptible to infections. Grass
growing between the rows during the winter months reduces the risk of epidemic
explosions of brown rot since it softens the impact of the rain on soil. It also
prevents the inoculum present in soil from coming into contact with fruits and
leaves, trough water splashing.
3.3. Chemical Control
3.3.1. Systemic Fungicides

Control by chemical means is a widespread practice, especially since very effective
systemic fungicides like metalaxyl and Al ethyl-phosphyte (or fosetyl-Al) were
made available (Farih et al., 1981; Davis, 1982; Timmer & Castle, 1985). More
recently, metalaxyl was substituted by its enantiomer metalaxyl-M (or mefenoxam)
which is effective at a lower dosage. Another derivate of phosphorous acid,
potassium phosphonate, which is on the market as fertiliser, acts in the same way as
Al ethylphosphite (Walker, 1988; Adonia et al., 1992; Tuset, Lapena & Garcia-
Mina, 2003). On the whole, these fungicides are effective against both P.
citrophthora and P. nicotianae.
However, there are slight differences in their range of action: fosetyl-Al is
slightly more effective against P. citrophthora, whereas mefenoxam is more
effective against P. nicotianae. The derivates of phosporous acid are translocated up
and down in the plant so they can be applied to roots, trunk or leaves. Their
application is recommended in a preventive way, to trees that are still apparently
healthy or slightly infected and during periods of plant intensive physiological
activity. Mefenoxam only moves upwards and must be applied to the ground or to
the bark to be effective. Both groups of fungicides will remain active in the plant
tissues for 3-4 months (Matheron & Matejka, 1988). Mefenoxam has a toxic action
directly on mycelium growth and on the sporulation of the two species of
Phytophthora. The activity of the phosphorous acid derivates, on the other hand,
depends mainly on their capacity to trigger off the defence mechanisms of the plants
based on the production of phytoalexins and, to a lesser degree, on inhibiting the
development of the pathogen (Afek & Sztejnberg, 1988).
Recently, a new systemic fungicide, dimetomorph, applied as trunk paint at high
concentrations proved to be as effective as fosetyl-Al and mefenoxam in suppressing
canker development on citrus bark, after inoculation with P. citrophthora and P.
nicotianae (Matheron & Porchas, 2002). Other systemic fungicides active against
Oomycetes are being testing and evaluating for a possible use against Phytophthora
diseases of citrus. Some of these compounds appear promising and showed the ability
to inhibit gummosis when applied as foliar spray on citrus plants artificially inoculated
with P. citrophthora at various time intervals after the treatment (Fig. 3). Moreover,
like fosetyl-Al and mefenoxam, these new fungicides showed a long-lasting preventive
activity (Cacciola et al., 2007). Figure 3 e.g. shows that fosetyl-Al, applied as reference
product, and product A (new a. i.) were systemic and showed a long-lasting preventive
activity. Foliar spray with these two fungicides 49, 35, 21 and 7 days as well as 24
hours before inoculation inhibited canker development on twigs as well as on the basal
part of the stem. Product B (a new dipeptide fungicide) was less effective and
persistent. Inhibition of canker on scion twigs and basal stem of the rootstock was
significant only in trees treated 24 hours before inoculation.
Figure 3. Effect of three systemic fungicides, applied as foliar spray (175 g a.i. ·100 liter -1
H2O) at various time intervals before inoculation with Phytophthora citrophthora, on the
development of cankers on sweet orange trees grafted on sour orange. A) Effect of
treatments on the length of cankers on the twigs (sweet orange), 21 days after
inoculation. B) Effect of treatments on cankers size on the basal portion of the rootstock
stem (sour orange), 70 days after inoculation (mean of 8 replicates ± SE).
3.3.1.1. Trunk Gummosis

To control this facies of the disease, mefenoxam can be applied to the plant through
trunk painting or through the ground. The phosphorous acid derivates are applied via
the leaves. They are effective also as trunk paints. Painting and spraying the trunk
with high concentrations of these fungicides (6% a.i. of mefenoxam and 10% a. i. of
fosetyl-Al) also help the plant to recover. The treatment must be started at the first
sign of symptoms and must be repeated after 3-4 months. If more than 50% of the
tree circumference is affected by gummosis, then the treatment is no longer effective
and it is better to substitute the plant. Painting and spraying of the trunk can also be
carried out using copper-based products, but this has only a preventative effect,
since these products do not penetrate the bark. Painting is impractical if trunk
infection is under the soil level.
3.3.1.2. Root Rot

To control this facies of the disease, both mefenoxam and fosetyl-Al can be used in
the ground. However, fosetyl-Al and the other phosphorous acid derivates are more
effective when applied as foliar spray. In young plants the treatment must be applied
routinely for the first 2 years since the young citrus seedlings are highly susceptible
to root rot. In bearing citrus orchards the treatment should be applied when at least
one of the following premises is met:
- the trees are grafted on a susceptible rootstock;
- the citrus orchard is on a site where root rot is chronically present because
of environmental conditions which cannot be modified;
- the ID values of Phytophthora in the rhizosphere soil reach a critical level
around 15-20 CFU / g or cm3 of soil (lower values can also be considered
critical if the Phytophthora population is made up mainly of P.
citrophthora, or if the rootstock or scion - rootstock combination is highly
susceptible).
It is advisable to apply mefenoxam through the irrigation system, if localised .
To avoid being washed away, the fungicide must be distributed at the end of the
irrigation cycle. In some soils mefenoxam may be degraded quickly by the bacterial
population in the soil (14-28 days) (Bailey & Coffey, 1985). However, if the product
is applied through an irrigation system, it may be absorbed in 2-9 days. Another way
of safeguarding against biodegradation in soil is to alternate applications with foliar
treatment of phosphorous acid derivates, in order to reduce selective pressure on the
microbial populations in the ground. This alternation of active principles also helps
to prevent phenomena of resistance to mefenoxam in the Phytophthora populations.
Treatments against foot and root rot using systemic fungicides protect the fruit
from brown rot infections in both the pre- and post-harvest phases. The treatments
must be timed according to the population dynamics of the pathogenic agents and
the physiological state of the plant.
In the case of root rot caused by P. nicotianae the most suitable period for
treatment is immediately before roots begin to develop, when the first spring growth
flush is three quarters of maximum. The treatment should be repeated in summer. If
root infections are caused by P. citrophthora and are associated with foot rot, the
applications should also be repeated in autumn before winter rains and plant
dormancy or by the end of winter, a few weeks before the spring foliage flush. Even
in this case a second application may be necessary in summer. Since both

mefenoxam and phosphorous acid derivates are systemic, applications must be
timed well in order to obtain a high concentration of fungicide in the trunk bark and
in the roots when the plants are most susceptible to infection.
3.3.1.3. Brown Rot of Fruit

The risk of epidemic explosions of brown rot of fruit is greater in citrus orchards
where there is a high incidence of foot and root rot caused by P. citrophthora. To
control this facies of the disease in the field, the canopy is preventatively treated
with phosphorous acid derivates or copper-based products. Some commercial
formulations of copper fungicides are not coloured purposely in order not to stain
the fruit close to harvest. The most suitable time for treatment is before the winter
rains, generally by the first week of November and if the winter is especially wet, a
second application in January-February is recommended.
4. CONCLUSIONS
Research on the ecology and epidemiology of P. citrophthora and P. nicotianae has
provided a corpus of knowledge and data which has been essential for the
development of rational strategies of integrated management of Phytophthora root
rot in citrus orchards and nurseries. These strategies are based on concepts, such as
inoculum density, threshold levels, host susceptibility and pathogen population
dynamics, as well as on general principles, such as use of genetic resistance of the
host, monitoring of inoculun, reduction of inoculum potential, timing of treatments,
sanitation measures, management of cultural practices to obtain an environment less
favourable to the pathogen and to reduce the disease pressure, induction of
resistance and eradication of infections by chemicals.
The efficacy of fosetyl-Al and mefenoxam has relaunched chemical control as
essential part of a rational and effective management strategy of this complex
disease. Proper timing and mode of application of these fungicides based on the
knowledge of the type of fungicide activity, the dynamics of pathogen populations
and the seasonal fluctuations of host susceptibility can help in reducing the
environmental impact of chemicals. The introduction in the scenario of new
systemic active ingredients adds flexibility to the chemical control and can help in
reducing the potential rik of fungicide resistance in the patogen populations.
The substitution of a resistant rootstock such as sour orange, due to further
spread of Citrus Tristeza Virus in the Mediterranean area and the diffusion of
cultivars, such as clementine and nucellar clones of sweet orange (Laville 1984,
Ippolito, Nigro & Lima, 1997), which induce susceptibility even in a tolerant
rootstock, might encourage the use of fungicides as routine control methods.
However, to be effective chemical control of Phytophthora root rot must be
complemented by cultural practices in order to make the environment less
favourable for infections and to reduce the disease pressure.
Genetic resistance of the rootstock appeared to be the most effective means to
control Phytophthora gummosis since the devastating epidemics of the second half
of the 19th century. There is evidence that in the Phytophthora-citrus pathosystem

both olygogenic and polygenic types of resistance are involved (Laville, 1975)
suggesting that it is advisable to select new resistant rootstocks from hybrid
populations obtained by interspecific crosses (de Franqueville, 2001). Phytophthora
root rot of citrus, like other tree diseases, is characterised by a high potential
variability of the pathogen, a low variability of the host and a strong selection
pressure on pathogen populations. Using a polygenic horizontal resistance ensures
durability and efficacy in different geographic areas.
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5
TULLIO TURCHETTI 1 AND GIORGIO MARESI 2
BIOLOGICAL CONTROL AND MANAGEMENT

OF CHESTNUT DISEASES
1
Istituto per la Protezione delle Piante, CNR
Via Madonna del Piano, 50019 Sesto Fiorentino, (FI) Italy
2
IASMA Research Center, Natural Resources Department,
Via E. Mach 1, 38010 San Michele all’Adige (TN), Italy
Abstract. Chestnut blight and ink diseases caused, respectively, by Cryphonectria parasitica and
Phytophthora cambivora and P. cinnamomi, are revised. The main strategies for efficient biological
control and management are discussed, considering their appearence, symptomatology, epidemics and the
actual situation. The types of cankers of chestnut blight are shown, as well as the characters of the
different types of infection caused by C. parasitica. The evolution of the diseases and the spread and
effectiveness of hypovirulence traits are revised, considering the morphology, physiology, presence and
transmission of dsRNA. Chestnut resistance, and the role of environmental and other ecological factors in
ink disease, including the action of the soil microflora, are discussed. The role of silviculture and the
evaluation of biological control strategies for blight and ink disease management are also revised. The
improvement of the management of chestnut disease needs a better understanding of the ecological
dynamic of chestnut ecosystems. An holistic approach including all the factors involved in the chestnut
trees ecology is proposed in planning the management of such ecosystems and in undertaking the best
measures of conservation and improvement.
1. INTRODUCTION
Chestnut. Castanea sativa Mill., can be considered as a ‘multipurpose’ tree because
of its ability to satisfy multiple and changing demands from society, and to carry out
various, always useful roles, in accordance with the location and time. Chestnut
stands and orchards constitute, indeed, agro-forest ecosystems of great importance.
They are highly topical, thanks to their productive aspects, hydro-geological
defence, and ecological and biodiversity functions.
In the European and Mediterranean areas, chestnut has been characterizing the
mountain landscape, as the mountain societies, for thousands of years. What can be
defined as a ‘chestnut civilization’ has been in existence since medieval times, and
probably also during the Roman era. This started from the key role of chestnuts as a
source of food, and influenced all aspects of life, particularly in the mountain
(Gabrielli, 1994; Arnaud et al., 1997; Conedera et al., 2004). The importance of
chestnut ecosystems decreased as a consequence of a long period of crisis that began
85
86 T. TURCHETTI AND G. MARESI
in 50’s, when cultivation became restricted for social, economic and phytosanitary
factors. However, chestnut trees have remained an essential feature of mountain
areas.
Orchards and stands constitute a major component of the rural landscape because
of their distinguishing and dominant characteristics, particularly as far as the
specificity and typicality of the spatial organization are concerned, but also due to
the presence of a significant biodiversity and to signs of the material culture.
Nowadays, as indispensable suppliers of food, wood and other products, these
anthropogenic ecosystems are able to satisfy an increasing demand for genuine
products, as well as the need of life suitability for mountain people. These new
aspects are yielding a renewed economic interest in the cultivation of chestnut trees,
and are enhancing the recovery of several orchards. In almost all areas of Europe,
chestnut cultivation is once again playing an important role in mountain economy
and development.
Asia is another important continent for the production of chestnut fruits: South
Korea and China are the main producer countries. Chestnut trees (C. mollissima
Blume and C. crenata Sieb. et Zucc.) grow in Korea, and improvements in their
cultivation have been carried out during the past 30 years. Orchards of Chinese
chestnut trees, including chestnut stands, are cultivated over 200,000 hectares
(Bonous, 2002). Castanea mollissima, which has been cultivated for centuries
(about 5000 years), is an important source of food for populations living in the
mountains. Recently, an increased interest for chestnut fruits has determined an
intense development of this cultivation, and many orchards have been planted.
Japan is another Asian Country involved in chestnut cultivation, based on the
Japanese specie of chestnut (C. crenata). Its cultivation is distributed throughout the
country, especially in the southern part of Japan, and the production is characterized
by large-size, well appreciated fruits. As in Europe, also in Japan a decrease
occurred in cultivation starting in 80’s, and large quantities of chestnut fruits are
now imported from Korea and China.
The American chestnut (C. dentata Bork.) was one of the most important forest
species in north-east America for the production of wood and fruit, with an
incomparable ecological role. It had an extensive area of distribution, and was the
dominant specie in many American forests. American chestnut trees were the giants
of the species, and reached very large dimensions (35 m in height and more than 3 m
in diameter). Chestnut trees disappeared from American forests due to the spread
and activity of the fungus Cryphonectria parasitica (Murr.) Barr, which was
imported from China and is responsible for the chestnut blight pandemic.
At present, programs based on the biological control of blight and on intra-
specific breeding are attempting to improve the restoration of the American chestnut
for silvicultural, traditional and landscape purposes (Griffin, 2000).
This situation is symptomatic of the effects caused by constraint factors such as
diseases. Control means for the main parasites, such as C. parasitica, Phytophthora
cambivora (Petri, Buism.) and P. cinnamomi (Rand.), agents of chestnut blight and
ink disease, respectively, are necessary. Moreover, an implementation of biological
IPM OF CHESTNUT DISEASES 87
control is displaying encouraging prospects in the management of chestnut stands

and orchards, thanks to its ability to limit the severe damage caused by these fungi.
With the contribution of human beings, chestnut trees can still concur in the
improvement of life sustainability, with their beneficial effects on the atmosphere,
landscape and hydro geological defence. Furthermore, all the associated economic
activities can be turned into yield, employment, and incentives for counteracting the
migration of mountain populations (Adua, 1998).
2. DISEASES APPEARENCE, EPIDEMIC AND ACTUAL SITUATION

There are two main fungal diseases in chestnut trees: blight and ink disease. Both of
these pathologies have a severe impact, and their evolution fits a model of pandemic
dynamics.
2.1. Chestnut Blight

In America, blight epidemic has shown the real potential of a plant disease for
making dramatic changes, not only in productive sectors, but also in the ecology of a
whole geographic area. The first blight recorded in the history of the pathology was
in 1904, at the Bronx Zoological Park, in New York City. Since then, the fungal
agent has spread quickly amid the principal chestnut forests of the Allegheny
mountains, invading the entire natural range of C. dentata. Forty years later, the
fungus had killed about 3.5 billion American chestnut trees. The most important
forest tree in eastern North America was hence reduced to insignificance. No
comparable devastation of a species exists in history, as American chestnut trees
were reduced to small understory seedlings and stump sprouts (Griffin, 1986).
The blight fungus was probably introduced into North America by Japanese and
Chinese chestnut trees. Cankers found on imported Japanese and Chinese chestnut
trees indicated the Orient as being their origin, and this was confirmed by the
subsequent discovery of the parasite in China and Japan (Shear & Stevens, 1913).
The endemic presence of blight on indigenous chestnut species was discovered in
these two countries by Meyer in 1912 and 1913 (Shear, Tiller & Stevens, 1917). In
Korea, the disease was first reported in 1925 (Lee, Park & Lee, 2004).
In Europe, 34 years after the discovery of the disease in North America, the
chestnut blight fungus was located in European chestnut trees near Genoa, Italy, in
1938 (Biraghi, 1946). New foci were then found in two other chestnut areas: Udine
and Avellino, in 1940 and 1943, respectively (Biraghi, 1950). The first spread of the
disease near harbours suggested an human role, contributing to vectoring this
disease. From these different disease centres, C. parasitica spread throughout the
whole country in about 30 years.
From Italy, the blight spread to other European countries. In France, the disease
was discovered in 1956 (Darpoux, Ridé, & Bondoux, 1957). The parasite was found
on C. crenata var. Tamba in Spain in 1947 (Biraghi, 1947), even if symptoms had
been observed also on Japanese diseased seedlings, presumably imported from
France as early as 1940. Blight was recorded on European chestnut trees only in
1978 (De Ana Magan, 1984; Muñoz & Cobos, 1991).
From 1950 to 1975, the blight spread into Switzerland, Austria, the former
Yugoslavia, Greece and Albania. In southern Russia, infections were noted in the
1950s in the Caucasus mountains (Pridnya, Cherpakov & Paillet, 1996).
Cryphonectria parasitica was discovered in Hungary, near Zalaerszeg, in 1969
(Körtvély, 1970). The disease was then recorded in Turkey in 1967 (Delen, 1975).
In the following years, blight attacks were observed also in Slovakia (1976),
Portugal (1989) and Germany (1992) (Heiniger & Rigling, 1994).
Some scattered chestnut stands in Spain, northern Europe and Great Britain were
to remain blight-free. Griffin (1986) reported the presence of large C. sativa trees
without any blight symptoms on the island of Jersey. Despite these particular
situations, blight is currently present and spreading over the entire European
chestnut range.
The presence of blight is still acting as a constraining factor for the American
chestnut. In America, chestnut trees lived in natural stands where stump vitality is
decreasing for the blight pressure, which is able to kill most of re-sprouts, regularly.
Only a few trees or stumps sprouts show evidence of partial blight resistance, which
also permits the presence of artificially-induced non-lethal infections.
In oriental chestnut trees, the presence of blight can produce occasional damage
in natural stands. The increase in plantations has created different situations in
which chestnut trees may result more susceptible to blight attacks. Moreover, the
effects could be staggering as far as the production of chestnuts is concerned, with
serious economic consequences.
The role and evolution of C. parasitica in Europe were completely different. In
an initial period of high mortality, many chestnut stands and orchards were badly
damaged. A spontaneous re-growth of most of the affected trees was then noted, and
stands and orchards have been recovering now, for some fifty years.
The presence of non-lethal blight infection has been recorded in Italian stand and
orchards since 1950 (Biraghi, 1950). These infections were able to encircle, but not
kill, infected branches and stems and made it possible for the host to react
vigorously. Non-lethal cankers were compared with infections observed in oriental
chestnut species, such as C. crenata. This occurrence was initially explained as
being symptomatic of a tree acquired resistance to the parasite attacks. Further
studies emphasized the presence of atypical strains of C. parasitica (Grente, 1961;
1965; Grente & Sauret, 1969a; 1969b; Bonifacio & Turchetti, 1973). Sporulation
and coloration in culture were less than in normal isolates, and the reduced virulence
of these atypical strains was confirmed by artificial inoculations. This unusual
development of the disease was defined as “Hypovirulence”. It guaranteed the
survival of most of the trees after the first impressive damage, and showed a
widespread distribution in all the chestnut woods affected by blight.
Cryphonectria parasitica was found on other hosts both in North America and
Europe. In the USA, the fungus usually attacks Castanea pumila (Chinkapin) and
several Quercus species, especially Q. virginiana. In Europe, three species of
Quercus are affected (Q. pubescens, Q. ilex and Q. petrea). In Italy, Ostryia
carpinifolia has also been reported with infections, such as Alnus cordata (Luisi &
Laviola, 1977; Turchetti, Maresi, & Santagada, 1991). In general, other hosts
appeared to be infected in areas where normal infection and severe damages are
prevalent on chestnut trees.
2.2. Ink Disease

Ink disease mainly affects European chestnut stands and orchards. It was widespread
in the past on all continents, as a result of international trade. It was imported into
Europe at the beginning of the 18th century. However, the age of introduction of the
pathogenic agent in Europe is not known, while the origin is probably Asia.
Ink disease was probably first introduced in the Azores, but after a few years it
was present also in various European countries. In a first phase, the disease was
discovered in the Iberian peninsula (Portugal and Spain): in Spain, symptomatic
chestnut trees were detected in 1726, as referred by Petri (1918). In Italy, it was
observed by Selva (1845) in Piedmont, where the disease was most prominent, and
was described by the name of ‘ink disease’ for the first time by Puccinelli (1859). In
1876, it was observed in France in the region of the Pyrenées and in some sites in
the Cévennes (Allain, 1935). Germany and England were struck by ‘ink disease’ at a
later date.
The disease aetiology remained unknown for a very long time, in spite of an
extensive amount of research that was carried out mostly in Italy and France. In
1870, Celi and Gibelli were appointed by the Italian Ministry of the Agriculture to
find out the causes of the disease. Gibelli (1876) stated that it was contagious and
that it was caused by a blackish mycelium capable of attacking the mycorrhizae
Diplodia castaneae var. radicicola and Torula exitiosa. In France, De Seynes and
Planchon began research projects in parallel with Gibelli in Italy. Planchon (1879)
attributed the disease to the parasitism of a species of Rhizoctonia and Agaricus
melleus. On the contrary, De Seynes (1879) failed to confirm the presence of
Diplodia strains, but frequently obtained strains of Torula. Delacroix (1879),
instead, interpreted the disease as the result of a nutritive deficiency in soil, just as
Gibelli had suggested (1879). The latter considered the absence of humus, in the
poor soil favourable to the ‘ink disease’, as a predisposing factor. Cornu (1881)
regarded the disease as a consequence of the severe winters of 1870 –1871 and 1879
– 1880. Malinvaud (1898) ascribed the disease to Pseudocommis vitis, the causal
agent of darkening in grapes. Crié and Ducomet noted the bacterial origin of ‘ink
disease’ in 1900 (Ducomet, 1913). Prunet (1900) excluded the influence of
mychorrizae and soil organic matter, and verified the disease contagious ability.
Mangin (1903) distinguished two different diseases: one of physiological origin,
due to a soil exhaustion that had an influence on the plant, the other being caused by
‘cryptograms’ that attack only the roots. Mangin turned his attention to mychorrizae,
and suggested that their destruction was caused by a fungus known as Mycelophagus
castaneae, similar to an oomycete. Henry (1909) accepted the hypothesis suggested
by Delacroix and Camara Pestana, which was based on the absence of the processes
of nitrification and an air insufficiency in soil. Briosi and Farneti (1909) asserted
that the root rot was a secondary phenomenon: the disease started at the epigeal part
of the chestnut tree on the branches and twigs, and subsequently spread along the
stem until it affected the root system. The agent was Corynenum perniciosum, while
the ascogenous form was Melanconis perniciosa. Griffon and Maublanc (1910)
resumed their studies on Coryneum, and identified the Coryneum kunzei var.
castaneae Sacc. with the ascogenous form Melanconis modonia. They did not,
however, confirm the ability of this pathogen to cause ink disease. Documet (1913)
ascribed the destruction of mychorrizae in plants with symptoms of the disease to
bacteria.
Finally, through his research work, Petri (1917a; 1917b) succeeded in obtaining
the isolation of a mycelium different from Coryneum from the cambium of a
chestnut tree affected by the disease. He named it Blepharospora cambivora, in
confirmation of its patogenicity (Petri, 1925). Buisman (1927) revised the
Phytophthora genus, and re-named this pathogenic agent Phytophthora cambivora
(Petri) Buisman (Waterhouse & Waterston, 1966). The other agent of ‘ink disease’,
Phytophthora cinnamomi Rand, which was studied in America and Europe, was
attributed to the same genus.
After a long period of quiescence, an upsurge of ink disease in chestnut trees has
recently occurred in various parts of Europe (Turchetti, 1986; Turchetti & Parrini,
1993; Abreu, 1996, Anselmi et al., 1996; 1999). The appearance of these new foci
seriously threaten the recovering project of chestnut stands, and in some cases
frustrate chestnut growers, because of the destruction of recovered trees.
3. SYMPTOMATOLOGY
3.1. Chestnut Blight and Types of Cankers

Cryphonectria parasitica is a wound parasite that is able to infect sprouts, branches
and stems of Castanea species. The disease is easily detectable during the early
stages of the infective processes, when it appears as an initial undifferentiated
infection in the young chestnut bark. During this attack phase, the bark superficial
tissues develop rust-red areas with irregular raised edges. An increasingly reddish
spot is also evident. The fungus grows in the bark tissues, producing characteristic
yellow mycelial fans. The discriminating factor for the effect of the infection is the
ability of the fungus to cause the death of the cambium.
The most pronounced symptom of the disease in chestnut trees is an occasional
‘flag’, a dead branch with wilted yellow or brown leaves. A girdling canker is
usually found on the branch, below the discoloured foliage. If the canker occurs on
branches or sprouts of stumps, the entire upper part may be killed outright.
However, new sprouts readily develop on the branch or stem below the canker, and
produce ‘witches’ brooms’ from the dormant buds. In these infections, which are
referred to as normal, the fungus spores or conidia develop mycelia upon
germination, that rapidly penetrate the inner bark and cambial layers.
It should be noted that the parasite acts as a necrophyte, killing the cells before it
colonizes them. After the fungus has grown under the outer bark for some time, the
tissues appear reddish, completely sunken and with several cracks. Minute pustules
erupt in these fissures or through the surface bark. On the cankered bark they are
yellow, orange, or reddish-brown, about the size of a pinhead. These fruiting

pustules are of two kinds. The first one is formed by pycnidia, which produce tiny,
one-celled, sticky conidispores that ooze out in long twisted tendrils (cirrhi) during
moist weather. These conidispores are disseminated by birds, by crawling of flying
insects, or by splashing rain. The other kind is given by perithecia, which produce
slightly larger, two-celled ascospores that are shot into the air through a small
opening at the top of the pustule. Ascospores are windborne, and may be carried for
miles. They are, however, relatively short-lived.
In addition to the wilting foliage, epicormic shoots, cankers and fruiting pustules
associated with the disease, mycelial fans can readily be seen when the overlying
bark is removed, at the edge of an active canker. On large branches or stems with
thick fissured bark, the disease is difficult to detect until longitudinal splits appear,
or fruiting pustules develop in the bark fissures.
It should be noted that roots are not infected and that stumps can produce sprouts
again. These sprouts, in turn, become blighted, and the repetition of infections leads
to the death of the entire stump.
In other instances, cankers have encircled the branches and stems. However, they
have a swollen appearance, indicating that the fungus has not infected the cambial
tissues. These infections are defined as abnormal cankers, which do not kill the
infected parts of the trees. Mycelia are not able to colonize the inner layers of the
bark, and so the cambium is able to react, producing new tissues. The survival of
bark tissues leaves the encircled stem alive, while the appearance of the canker is
swollen, with more or less pronounced swellings. The mycelia colonize the
superficial layers of the bark in which cream-coloured fans are recognizable.
Reddish-orange areas are detectable on the infected surface, and a few orange or
reddish-brown pycnidia are produced. Perithecia are not commonly observed in
these infections.
Some of these cankers colonize wide surfaces of stems or branches. No wilting
appears on the upper part of the crown, and epicormic shoots are not produced
below the infection. Plants survive and grow without any effect on either the
production of wood or the yield of fruit.
This type of abnormal canker is known as a healing canker. As a rule, in a few
years this attack evolves into a completely healed canker, in which the stem appears
to be colonized over a large vertical portion of the stem or branch, the bark is
slightly cracked, and looks rough and dark in colour. No evidence of stromata is
observed during this stage, but sometimes mycelia are still alive under the black
bark, and are able to grow.
Intermediate characteristics between those of the normal and abnormal cankers
are found in other kinds of cankers, and these are known as intermediate cankers.
This type of canker starts as a normal virulent infection that kills the inner bark and
exposes the sapwood with abundant mycelial mats of the fungus. Then, however,
vigorous wound cork barriers arise in the reactive swollen zone, surrounding the
dead area. Epicormic shoots occur normally below the canker, together with
fructifications of the fungus in the central zone, while the upper part of the infected
stem still remains alive. Even if it started as virulent, this type of infection loses its
virulence, probably because of the subsequent growth of hypovirulent strains, and
yields a healing or healed canker, in the majority of vigorous branches or stems

(Turchetti & Maresi, 2000).
The fungus may continue to survive in the bark on logs or sprouts and branches
and to produce many pycnidia and perithecia, both in normal and abnormal cankers
(Diller, 1965; Prospero et al., 2006). In normal infections, mycelia are able to
colonize large parts of dead tissues.Table 1 summarizes the canker classifications
adopted for the Italian and European stands (Turchetti & Maresi, 2000; Turchetti
et al., 2008).
Cryphonectria parasitica produces cankers in Quercus trees that are similar to
the ones visible on chestnut trees. Infections on branches and young stems resemble
swellings caused by the reaction of the infected tissues to invasive parasites.
Longitudinal splits appear on the bark, and stromata develop in the bark fissures.
Epicormic shoots are rarely observed, and the mycelial development is notable for
the presence of evident fans.
Similar symptoms are described in other hosts, such as Alnus cordata and Ostrya
carpinifolia. It has been reported, however, that chestnut blight fungus causes little
damage to these host species.
Figure 1. Healing canker.

Table 1. Characters of the different types of infection caused by Cryphonectria parasitica

(from Turchetti, Ferretti and Maresi, 2008; modified).
Canker type Symptoms
Death of above stem

Encircled and killed stem
Normal Virulent Depression of reddish infected bark
Mycelia fans in the bark
Abundant stromata presence
Epicormic shoots under affected area
Survival of affected stem

Encircled stem
Healing Swelling of reddish infected bark
Mycelia present in the superficial tissues of bark
Reduced stromata presence
Strong cambium reaction
Absence of epicormic shoots

Encircled stem
Healed or swollen Swollen and dark bark
Mycelia with reduced vitality
Absence of stromata
Strong cambium reaction

Encircled stem
Intermediate Swelling of the bark but with exposed wood
Mycelia fan present in the superficial tissues of bark
Reduced stomata production
Strong cambium reaction with callus
Presence of epicormic shoots

Initial and undifferentiated Stem not encircled
infection Reddish affected bark
Mycelia fans present in the superficial tissues of bark
Reduced stomata production
3.2. Ink Disease

The crown of chestnut trees affected by P. cambivora becomes transparent, unlike
what happens in healthy trees or in those infected by blight. Leaves lose their
brilliant green and become glaucous, then often yellowish, their growth is arrested,
and branch growth is reduced, so that branches appear shortened. Sometimes, there
is a precocious leaf fall (in August), leading to a thinning of the canopy. Chestnut
husks contain fruits that are smaller than normal and are concentrated at the top of
the crown, all at the same high level.
The roots become soft, spongy and brittle, exhibiting deep purple or almost black
areas. A blue-black inky substance exudes from these, that stains the nearby soil,
and this is what gives the disease its popular name.
Infected or dead areas that are slightly sunken and have small cracks appear at
the base of the stem on young tree. At a later stage, an elongated sunken area with a
distinct edge forms near the tree base. When the bark is removed from the base of
the trunk or from big roots, brown necrotic areas are observable at the cambium
level. These areas, which spread from the roots up to the stem, are shaped like an
acute-angle triangle: they are called ‘flame blots’. Sometimes, these typical blots do
not appear, because the young roots infection kills the tree before the blots have time
to develop (Biraghi, 1953a). Shoots arising from the collar desiccate early.
At an advanced stage, the trees are encircled at the collar, many branches begin
to wither, and then death of the entire tree results from the infection. Parasite
development in the host is usually very fast, and the death of the tree follows within
a year of infection. Sometimes, however, the infection progresses slower. In that
case, the trees generally die by the end of the second year. This difference in the
infection outcome may be due to the condition of the roots, since vigorous roots take
longer to be colonised than weak ones, suffering from i.e. environmental stress.
Symptoms of P. cambivora often resemble those caused by other root rot or collar
rot pathogens. Decayed bark at the base of the trunk is a symptom of collar rot, which
often starts at several points. As the infection progresses, it colonizes the cambium and
cortical parenchyma of the host around the trunk, until the lower part is entirely
encircled. Ink-infected roots become brown, brittle and necrotic, in contrast with the
soft rot typical of other root rot agents. Inhibition of the root system and necrosis of the
lateral roots and taproot, which are also caused by other Phytophthora species, can
influence the vegetative condition of the tree. Some vigorous trees cope with this root
reduction without appreciable crown symptoms, even though their water relations and
nutrition uptake are affected. Consequently, the infection is difficult to detect in
seedlings of walnut and other tree species after transplanting. Moreover, infected adult
trees exposed to nutritional stress and unfavourable environmental conditions may
exhibit a slow decline (Vettraino et al., 2003).
Phytophthora cambivora is difficult to isolate from dead trees, even if the tree
has died only recently. Mycelium in dead host tissues is not permanent, so the
fungus is very difficult to isolate from dead stems or trunks. It is hence advisable to
inspect the trees for P. cambivora when they are symptomatic, but still green and
alive.
Collar rot is symptomatic of the disease, but it may also be due to many other
factors, such as mechanical wounding, cracking, insect attack and other injurious
agents. A pathogenic effect similar to that of ink disease from P. cambivora is
infrequently produced by P. cactorum and P. citricola in chestnut trees (Biocca et al.,
1993). In view of this, it is difficult to identify P. cambivora positively without
an isolation test. Infected areas on the stem, initiating from the collar, are visible as
slight depressions in the bark. One useful way to detect the fungus is to remove the
bark with a blade: if the tree is infected, this will reveal the dark-brown, flame-
shaped lesions typical of P. cambivora underneath.
Many lateral roots have to be destroyed before infected trees show above-ground
symptoms, and this fact must be taken into account when making a diagnosis.
Symptoms and dieback occur in single plants or in groups of trees growing both in
humid places or in the lower valleys, and also on mountain slopes or crests.
Attacks of the diseases are induced by mild winters, and a succession of dry and
wet spells favours P. cambivora infections. Winters that are drier and warmer than
usual allow the trees to undergo a water stress during spring growth, which is a
period that is very favourable to ink disease infection.
The disease has been observed in chestnut orchards and abandoned stands where
chestnut trees were still growing in competition with other invasive trees species
(Turchetti & Maresi, 2003).
All these symptoms and characteristics are common to P. cambivora and P.
cinnamomi. Therefore, isolations need to be made to enable secure identification. In
Italy, however, P. cinnamomi is not commonly recovered from forests. Thus, any
symptoms found are almost certainly those of P. cambivora. Phytophthora
cinnamomi was recovered once, however, in a chestnut coppice in the Lazio Region,
but it was probably introduced by infected seedlings proceeding from a nursery
(Cristinzio, 1986). Phytophthora cinnamomi has also been detected in chestnut
seedlings in nurseries (Turchetti & Parrini, 1993), as well as in walnut nurseries and
plantations. It was probably introduced into these plantations along with infected
seedlings being planted for reforestation on former agricultural lands. This
threatening parasite may now be starting to spread, therefore, throughout Italy
(Belisario, Cacciola & Magnano di San Lio, 1997; Belisario, Maccaroni &
Vettraino, 2001; Belisario et al., 2002; Vettraino et al., 2003).
Figure 2. Initial symptoms of ink disease in a recovered stand.

4. EVOLUTION OF THE DISEASES
4.1. Spread and Effectiveness of Hypovirulence

The natural spread of hypovirulence is a main factor for the biological control of
blight in Italy and others European Countries, as well as in certain locations in the
United States (Michigan, Connecticut and Virginia). It appears to be related to the
natural re-growth of chestnut stands and to the survival of the trees. Initial
observations and investigations on healing and healed cankers were carried out in
the past in Italy and France (Biraghi, 1950; Grente, 1965; Grente & Sauret 1969a;
1969b; Bonifacio & Turchetti, 1973). Subsequently, hypovirulence was detected in
other European countries, and it is now possible to affirm that almost the entire
natural range of C. sativa is involved in this phenomenon (Table 2).
It should be emphasised that hypovirulence spreads generally in the absence of
any sort of silvicultural intervention, and without any artificial treatment for
biological control. This points up the great efficacy of the natural spread of
hypovirulence, which until now has been shown to be rapid and more efficient on
the European chestnut than all the artificial interventions, as reported by Robin,
Soutrenon and Rigling (2002) and Milgroom and Cortesi (2004). The main result is
a severe reduction in the trees mortality level.
Table 2. Detection of hypovirulence in some European countries (from
Heiniger & Rigling, 1994, updated with data collected by the authors).
Country FRCp* HC HS dsRNA

Albania 1967 1997 1998 +
Austria 1970 1993 1993 +
Bosnia Herzegovina 1961 1980 1980 +
Croatia 1950 1978 1981 +
Czech republic 2002 - -
France 1956 1964 1964 +
Germany 1992 1993 1993 +
Greece 1964 1975 1984 +
Hungary 1965 1995 1996 +
Italy 1938 1951 1964 +
Macedonia 1974 1995 1995
Portugal 1989 ND 2004 ND
Romania 1984 - - +
Slovakia 1976 ND ND ND
Slovenia 1950 1995 1999 +
Spain 1947 1992 1992 +
Switzerland 1948 1975 1975 +
Turkey 1967 1998 1998 +
*
FRCp = First record of C. parasitica; HC = Healing cankers; HS = Hypovirulent strains;
+ = Positive; - = Negative; ND = Not determined.
Chestnut stands were examined in the Cévennes region of France showing a 16%
average mortality, with a range between 9% and 29%. The mortality was higher in
only a few plots, due to the influence of environmental factors, such as drought
(Turchetti, 1994). In Spain, 8 stands visited in the Bierzo region (Castilla – Leon)
showed a percentage of sprouts ranging from 6% to 17%, with an average of 11%
dead because of blight. In several chestnut stands located in Slovakia, a mortality
level ranging between 3% and 21%, with an average of 11%, was determined.
The Italian situation, in particular, shows a reduced mortality rate almost
everywhere, with the survival of most infected plants. Field investigations on
unmanaged coppices have found a large, but variable, presence of chestnut blight in
the plots visited, with blighted sprouts ranging from 11% to 91% (Table 3). Limited
mortality levels were recorded in all stands surveyed. The sprouts that died due to
blight ranged from 2% and 21%, with an average of 11% (Turchetti & Maresi, 1990;
Leonardi et al., 1995). The highest mortality rate observed in Sicily was linked to
the effect of other disturbances (Leonardi et al., 1995). In Southern Tuscany,
Amorini et al. (2001) recorded 13% of blight-killed sprouts in coppices in 1994,
while the mortality due to blight in three different provinces of Lombardy (Sondrio,
Varese and Brescia) was 11% in 16872 sprouts and 826 grafted trees examined
(Davini et al., 1998).
Table 3. Incidence of Cryphonectria parasitica in several Italians stands and coppices.
Regions and Year Surveyed Total Healthy Live and

province plots surveyed sprouts blighted dead (%) due to
sprouts (%) sprouts
other
(%) blight
causes
Tuscany (Pistoia) 1981 7 1314 59 22 12 7
Tuscany (Florence) 1990 4 777 45 47 6 2
Marche (Ascoli
1992 12 2217 23 44 15 18
Piceno)
Sardinia (Nuoro) 1992 51 5768 76 9 2 13
Piedmont (Turin) 1993 2 274 38 42 13 7
Campania Salerno) 1994 6 2098 60 21 5 14
Tuscany (Grosseto,
1994 15 3625 29 55 15 1
Siena)
Lombardy
(Sondrio, Brescia, 1993 116 16872 32 32 11 21
Varese)
Sicily (Catania) 1994 4 1669 9 70 21 -
Tuscany (Lucca) 1997 7 1250 22 61 9 8
During these investigations, carried out from 1991 to 1993, the mortality rate due
to other causes (competition, damage by wild game, wildfire, etc.) was higher than
blight-caused mortality, reaching 21% of the examined trees. Mortality factors other
than blight were common in all plots, causing severe dieback in Sardinia and
Campania (Table 3). Similar results were obtained in some coppices in Switzerland,
where blight killed 15% of sprouts. An additional 20% of thin sprouts were killed by
other factors, including competition between stems (Bissiger et al., 1997). In France
and Spain, similar results were obtained, with 13% and 12% of sprouts killed by
factors other than blight.
A clear relationship between blight dieback and sprout diameter was reported by
Davini et al. (1998) and by Amorini et al. (2001), and confirmed by the observations
carried out by Turchetti and Marinelli (1980). Mortality due to normal infection was
concentrated on smaller diameters, while subjects with larger diameters always
showed a reduced level of dieback. In Switzerland, investigations carried out on two
coppices by Bissiger et al. (1997) reported both a reduced blight-caused mortality
and the influence of the tree diameter.
Figure 3. Chestnut coppices with a clear predominance of healed and healing infections.
Blight mortality seems to be related to sprouts vitality, and so it can be

influenced by its social position: recent investigations carried out in chestnut
coppices in Tuscany have confirmed this statement, showing a statistical
relationship between normal infections, death due to blight, and sprout social
conditions (Amorini et al., 2001; Turchetti et al., 2008).
In the face of the low mortality level, blight infections involved most of the trees
surveyed, with a clear prevalence of abnormal infections. This large predominance
of healing and healed infections has beens found to be maintained over time.
Turchetti et al. (2008) have evaluated the clear prevalence of healing and healed
cankers in a completely natural environment. Abnormal infections increased over
the fifteen years of observations, and ended up with more than 80% of the total
attacks recorded. Moreover, during the survey period, almost all the new
undifferentiated infections developed into abnormal ones. These data are consistent
with those of other investigations carried out both in Italy (Davini et al., 1998) and
in Switzerland (Bissiger et al., 1997), emphasising the great effectiveness of
hypovirulence.
In the natural range of American chestnut trees, hypovirulent cankers and strains
were found in Virginia (Griffin et al., 1983) and Michigan (Elliston, 1978).
Hypovirulence was found to be associated with surviving trees also in Ontario
(Melzer & Boland, 1999). In contrast to these highly-localised situations, large
amounts of virulent inoculum were still prevalent in the Appalachian forests
(Griffin, 1986).
4.2. Morphology, Physiology and ds-RNA Presence and Transmission

As indicated by Grente and Sauret (1969b), Bonifacio and Turchetti (1973), and
Turchetti (1978), the isolates were defined as: white (hypovirulent), with white
mycelium and few and large pycnidia; normal, or virulent, when they had cream-
coloured mycelium with abundant orange pycnidia, often scattered within concentric
rings and spore tendril production; intermediate, when they developed whitish-
cream mycelium with pycnidia distributed over the entire colony; red-orange
pigmented, with many abnormal small pycnidia. While only the red-orange
pigmented type showed - fairly infrequently - a relative stability in its morphological
characteristics, a certain morphological variability and instability was observed in
the other types. This could be a problem for an accurate classification. It is necessary
to consider that massive populations of the parasite spread in natural conditions, and
that the isolates proceed from infected tissues. Instead, the monosporic strains are
selected in the laboratory, and are studied for specific research purposes.
Cryphonectria parasitica is characterised by a complex physiological activity,
and phytotoxic metabolites are produced. Some of these are characterised and
defined as: diaporthin, skirin, rugolosin, orthosprin and cryphonectric acid (Shibata
et al., 1955; 1956); Gaumann & Naef-Roth, 1957; Bonifacio & Turchetti, 1973;
Sparapano, Mairota & Lerario, 1989; Arnone et al., 2002). Several extracellular
enzymes are produced by C. parasitica in culture media: rennin, laccase, phenol
oxidase, tannase, cutinase, protease, cellulase and polygalacturonase, and some of
these are associated with the pathogenicity of the fungus (Sardinas, 1968; Whitaker,
1970; McCarroll & Thor, 1985; Rigling, Heiniger & Hohl, 1989; Varley, Podila &
Hiremash, 1992; Gao & Shain, 1994; 1995a; Farias, Elkins & Griffin, 1992). Oxalic
acid, which is another product of the chestnut blight fungus, is related to the
virulence of isolates (Havier & Anagnostakis, 1983; 1985; Vannini et al., 1993).
Differences in the production of the cited enzymes were observed between the
different fungus isolates, especially between hypovirulent and normal strains.
Physiological and morphological differences between hypovirulent and normal
strains are supposed to be due to a cytoplasmatic determinant (Grente & Sauret,
1978) and closely related to the presence of the dsRNA hypovirus in the cytoplasm
(Morris & Dodds, 1979; Hillman et al., 1995). Further investigations on the
molecular properties of the virus-like agents associated with hypovirulence have
been carried out, and more is now known about viruses in C. parasitica than in any
other fungus (Milgroom & Cortesi, 2004). Different virus species have been
characterised: four hypoviruses, two Mycoreovirus and one Mitovirus. DsRNA is
able to spread in cultures, and can be transmitted in varying proportions in conidia,
but not in ascospores (Van Alfen et al., 1975; Turchetti & Maresi, 1991).
Transmission in mycelia occurs via hyphal anastomosis. The ability of isolates to
produce viable hyphal anastomosis is linked to the vegetative compatibility (v-c)
system, which is controlled by at least 5-7 v-c loci (Anagnostakis, 1988; Heiniger &
Rigling, 1994; Cortesi & Milgroom, 1998). When the alleles present are identical in
all v-c loci, the strains are compatible and anastomosis occurs, making cytoplasmic
transfers possible. When no correspondence between alleles occurs, strains are
incompatible, reducing potential hypovirus transmission.
V-c groups in the different populations of the fungus are identified by pairing
previously-determined unknown isolates and v-c testers in laboratory assays. In
Europe, only 31 out of 64 potential v-c groups have been identified in laboratory
tests until now, with differences between the examined populations (Milgroom &
Cortesi, 2004). On the contrary, it has been suggested that the widespread presence
of different v-c groups is the main reason for the poor natural dissemination of
hypovirulence in the USA (Anagnostakis, 1988; Heiniger & Rigling, 1994; Cortesi
& Milgroom, 1998). Almost all studies on C. parasitica population and
hypovirulence have been based on v-c determination, and the genetic mechanism
involved has been dealt with in numerous scientific papers (Day et al., 1977;
Anagnostakis, 1983; 1988; Cortesi, Milgroom & Bisiach, 1996; Cortesi, Rigling &
Heiniger, 1998; Cortesi et al., 2001; Cortesi & Milgroom, 1998; Garbelotto,
Frigimelica & Mutto-Accordi, 1992; Milgroom & Cortesi, 1999; Milgroom, 1996;
Sotirovski et al, 2004; Liu & Milgroom, 2007; Robin, Anziani & Cortesi, 2000).
In any case, recent studies (Carbone et al., 2004) have suggested no evidence for
a restriction of viral transmission between different v-c groups in two natural Italian
populations (Milgoom & Cortesi, 2004). The whole v-c system seems to be overrun
by real transmission rates, with clear disagreement as regards theoretical
probabilities.
Field tests had already suggested a better transmission of hypovirus in the
field (Double, 1982), as some lack of biological control of a few v-c groups
population with compatible hypovirulent strains was recorded in Wisconsin
(Cummings-Carlson et al., 1998; Jarosz, Dahir & Double, 2002). The data therefore
suggest that the real importance of vegetative compatibility in the natural spread of
hypovirus has not yet been clearly defined, and that it may have been overestimated
(Carbone et al., 2004; Milgroom & Cortesi, 2004). Within this context, the effective
role of v-c compatibility in explaining or predicting the success or failure of
hypovirulence is disputable.
4.3. Mixed Inoculum

White isolates showing great compatibility and conversion range have been
identified in the natural populations of the fungus (Maresi et al., 1995) as like as
numerous intermediate strains compatible with more than one v-c groups. These can
all act like bridge strains, thus allowing transmission between different compatibility
groups (Bazzigher, Kanzler & Kobler, 1981; Turchetti et al., 2008). Intermediate
strains have often been overlooked in populations of C. parasitica. However, they
are a consistent part of fungus population, having been obtained from healing
cankers in areas characterised by the dominance of hypovirulence (Grente & Sauret,
1969; Bonifacio & Turchetti, 1973; Davini et al., 1998; Garbelotto, Frigimelica &
Mutto-Accordi, 1992; Turchetti et al., 2008). The presence of dsRNA in
intermediate isolates has been observed in several cases (Turchetti & Maresi, 2006;
Hogan & Griffin, 2008).
Table 4. Inocula obtained from pycnidia produced on cankers collected in

two different Italians Regions
Type of canker Type of isolate

Region Site
Normal Intermediate White
Healing Normal
(%) (%) (%)
Tuscany Pomino × 35 45 20
Tuscany Pomino × 100 - -
Tuscany Montecuccoli × 2 81 17
Tuscany Montecuccoli 100 - -
Tuscany S. Donato × 73 8 9
Tuscany S. Donato × 100 - -
Campania Mercogliano × 8 22 70
Campania Mercogliano × 100
Campania Stio × 34 54 12
Campania Stio × 100 - -
Campania Agropoli × 12 81 7
Campania Agropoli × 100
The presence of a mixed inoculum with different percentages of white, normal

and intermediate isolates in healing cankers was detected in situations of
hypovirulence established both in America (Hogan & Griffin, 2002; Hogan &
Griffin, 2008) and Italy (Bonifacio & Turchetti, 1973; Turchetti, Ferretti & Maresi,
2008). Table 4 shows the percentages of different isolates obtained from stromata
collected on natural cankers in two Italian regions: white, intermediate and normal
isolates are present with different values in healing cankers. Occasionally, also
pigmented strains were obtained from inocula assays.
It should be noted that inocula composed as natural ones were also obtained by
using four different hypovirulent strains in artificially-combined inoculations
(Turchetti & Maresi, 1991). What is reported above suggests that hypovirulence
results from the action and interaction of different morphological strains (white,
intermediate, normal and pigmented), all originating from the natural circulating
inocula, harbouring (or not) dsRNA and with different virulence rates. This mixed
inoculum is able to produce different mycelia which, in growing together, cause
healing cankers, that are subsequently healed (Griffin, 1999; Turchetti & Maresi,
1988; 2006).
The mixed inoculum observed in natural cankers seems to exceed the variability
based on the v-c groups detected, thus enabling both transmission of the hypovirus
and the development of non-lethal infections. This inoculum and the effects on
predominance of hypovirulence seem to be stable over the years, even if new v-c
groups may be produced and observed (Turchetti, Ferretti & Maresi, 2008). Thus,
the mechanism of virus transmission and the establishing of hypovirulence seem to
be very complex: a more effective and holistic approach is desirable in order to
increase our knowledge on this important phenomenon.
4.4. Chestnut Resistance

The effect of a mixed inoculum is obviously mediated by host resistance. The C.
sativa resistance has already been suggested as a main factor in the spread of
hypovirulence in Italy and Europe (Biraghi, 1953b; Bazzigher, 1981; Bazzigher &
Miller, 1991). In Europe and North America much research and numerous
investigations have been dedicated to detecting resistance in chestnut species.
Breeding programs have been carried out and increased in order to select resistant
clones and to introduce resistance, so as to obtain hybrids between the different
species of chestnut, especially between Asian and American or European chestnut
varieties. European chestnut appears to be somewhat more blight-resistant than
American chestnut, but even less so than Asian species (Griffin, 1986).
Castanea crenata and, especially, C. mollissima which have the highest level of
resistance, have been used extensively in breeding programs. A few surviving
American chestnut trees also proved to be resistant to blight, harbouring superficial
non-lethal cankers (Griffin, 1986). In Virginia, some of these trees were grafted and
then inoculated with hypovirulent blight strains. Within this integrated management
systems, host resistance and the spread of hypovirulent strains, such as hardwood
competition control, have been closely associated with blight control (Robbins &
Griffin, 1999).
Until now, most of the breeding work has been done by American researchers
with the aim of saving and restoring American chestnut trees. Two strategies have
been pursued, the first of which was aimed at introducing the Asian resistance gene
in the American genotypes. The second sought to recover natural resistance in the
surviving trees. In this option, resistant chestnut trees were created by means of a
hybridisation between resistant American chestnuts from many locations, in an
attempt to improve the low levels of blight resistance and to make an all-American
chestnut tree that can compete in the forest (Griffin et al., 1983). After several years
of work, encouraging results were obtained and some trees grafted with resistant
scions showed a good capacity to support the presence of hypovirulence (Dierauf
et al., 1997).
As for C. sativa, Bazzigher and Miller (1991) assert that, after thirty years of
selection for resistance, only subtle differences between blight-resistant and blight
susceptible chestnut trees have been recorded. There is a continuous gradient from
very susceptible to very resistant, but complete immunity has never been detected.
Environmental condition such as physiological status have a great influence on
canker severity, and so it is really extremely difficult to evaluate genetic resistance.
During the 1980s in France, hybridization and selection work carried out by
INRA researchers yielded different resistant hybrids C. crenata × C. sativa. These
trees perform well against canker, even if they all are still susceptible to the disease.
However, their productions have a low level of quality, due to the flavor of the
fruits.
4.5. Environmental Factors

Site conditions and related environmental constraints greatly influence blight
disease. In Chinese and Japanese chestnut trees, the presence of damage due to
disease infections is closely related to environmental factors that are not optimal for
these species.
In Europe and, in particular in Italy, blight recrudescence has been recorded after
severe periods of drought (Davini et al., 1998). Drought seems, in fact, to be one of
the most important constraint factors for C. sativa. Furthermore, as reported by
Barthold et al. (2004), dieback of trees, which only sometimes is also related to the
appearance of blight, follows prolonged periods of low or absent rainfall, as in 2003.
Heavy hailstorms, which produce wounds on small branches, can act locally to
induce the insurgence of new blight damage on the crown: the fungus can easily
colonise small branch tissues that are badly mechanically damaged by hail. Wildfire
can also weaken trees and branches, increasing the appearance of blight through the
presence of a large number of dead sprouts or branches (Leonardi et al., 1995). It is
important to note that these perturbations are capable of producing transitory
outbreaks of blight damage. However, in general, or at least in Italian chestnut
woods, the predominance of hypovirulence does not seem to be unbalanced. After
one or two years, the damage returns to a lower level.
Intra- and inter-specific competition plays a fundamental role in weakening

sprouts or trees. In Italian stands where hypovirulence is predominant, the mortality
caused by blight is reduced and it is limited to subjects already suffering from inter-
and intra-specific competition (Turchetti et al., 2008). The mortality caused by
blight is flanked or overlapped by the mortality linked to the evolutionary dynamics
of the stands. Perhaps it contributes to accelerating it, but it does not succeed in
distorting it. Several studies (e.g. Pividori, 1997) have demonstrated the strong
competition and natural thinning that occurs between both stumps and sprouts in
chestnut coppices of the same age, particularly during the youthful stages.
As for American chestnut trees, both competition and winter frost have been
reported as factors capable of influencing the worst effects of blight infections
(Griffin, 2000). Hardwood competition has been identified as a critical factor, not
only for blight, but also for the survival of stumps after blight damage. Xeric and
intermediate site sprouts re-grow quite vigorously after stem killing for blight, while
the high level of competition on mesic sites, sometimes added to browse damage,
results in the death of sprouts and rootstocks. Therefore, a chestnut tree is not able to
survive in these more favourable sites if competition is not controlled by forest
management. Controlling hardwood competition resulted in high survival rates in
American chestnut and in the natural development of superficial cankers associated
with hypovirulent strains (Griffin et al., 1991; Griffin, Khan & Griffin, 1993).
Physiological stress due to low temperatures greatly influences the response of
C. dentata to blight at high altitudes and in frost pockets. Moreover, drought stress has
also been reported as a constraint factor in American chestnut survival against blight
attacks (Gao & Shain, 1995b).
4.6. Ecological Factors in Ink Disease

Phytophthora cambivora and P. cinnamomi occur worldwide in soils of natural
forests, agricultural fields and orchards. Stands of Eucalyptus sp. are a favourite
habitat for P. cambivora and P. cinnamomi in Australia. In these areas they are
commonly recovered from soils, together with other Phytophthora spp., but no
pathogenic effects are observed on the trees (Gerretson-Cornell, 1977; 1978).
In Europe, P. cambivora and P. cinnamomi are widespread in soils. In Italy,
however, only P. cambivora infects the soils of chestnut stands and it is the main
agent of chestnut ink disease (Petri, 1918; Biraghi, 1953a; Turchetti, 1986; Anselmi
et al., 1996).
The causal agent of chestnut ink disease is mainly P. cinnamomi. It accounts for
the majority of disease problems in chestnut trees in Portugal, limiting the yield in a
large number of stands and impeding the establishment of trees in new areas. A
survey was carried out on 32 chestnut stands in the Padrela Mountains of Northern
Portugal in order to investigate the relationship between the occurrence of ink
disease, edaphic factors, and management practices. Results showed that the main
factors affecting disease were soil compaction (COMP), soil organic matter level
(OM), and manuring practices (MA). A logistical model containing the soil variable
COMP and the interaction term OM · MA correctly predicted the health status of
stands with 94% accuracy (30 of the 32 stands studied) (Fonseca, Abreu, &
Parresol, 2004).
During a three-year study in Portugal, quarterly assessments were made of air
temperature, air humidity, wind speed, solar radiation, soil water content and soil
temperature (at a depth of 25 cm). Soil and climatic parameters were found to be
more stressful in stands with a Southern orientation: i.e. the soil temperature was
higher and the soil was drier, while temperature and wind speed were higher and air
humidity was lower. Thus, the severity of the disease was greater on south-facing
stands (Martins, Oliveira & Abreu, 1999).
Moisture, which includes rainfall, dew deposition and irrigation, is the main
environmental factor favouring the pathogen. In general, shallow soils are
favourable to P. cambivora and P. cinnamomi and the disease. In soils of this type,
which can be found on all continents, plant roots are produced in a high
concentration and become infected more rapidly, thus leading to a larger pathogen
population. In shallow soils, the effects of drought are more marked: both the soil
and the roots dry out more quickly, and in hosts with fine roots, water stress is more
likely to cause infection and death.
Shallow soils may have an underlying impervious clay layer or a rock base,
impeding drainage. Thus, the soil rapidly becomes wet and saturated with rainwater
in which zoospores are dispersed, and the roots are also predisposed to disease, due
to the resulting anaerobic soil condition. Recovery of the fungal pathogen is more
frequent in shallow soils: soils with low fertility and low mineral nutrient levels,
particularly phosphorus, seem to favour infection. Furthermore, sites facing south
show a higher occurrence of P. cinnamomi, which is also more frequent on slopes
and valleys than on hilltops (Moreira & Martins, 2005).
Phytophthora cinnamomi has never been detected in sites characterised by soil
pH below 5.4, or minimum temperatures below 1.4 °C, and maximum temperature
above 28 °C. Phytophthora cambivora may be found colonising different soil types
in Italy, but often the surrounding chestnut trees are not diseased. These results
provide useful information for modelling the probability of ink disease, crown
decline, and associated Phytophthora species, in chestnut groves in global climatic
change scenarios (Vettraino et al., 2004).
Data from chestnut groves exhibiting different degrees of infection were
submitted to Principal Component Analysis (PCA), in order to investigate the
relationship between the severity of ink disease and site characteristics, soil
properties and cropping practices. The relationship between the severity of ink
disease and concentrations of plant nutrients was also submitted to PCA. The
importance of soil fertility parameters, soil organic matter, and effective soil depth
in improving the health conditions of chestnuts was pointed up, while radiation, the
frequency of tillage and imbalanced mineral fertilization were found to contribute to
the severity of ink disease. The relative position of a chestnut grove, i.e. on the
upper or the lower slope, affects the hydrological conditions and rooting depth of
chestnuts. Results showed that it is not possible to identify just one single factor as
being responsible for the development of chestnut ink disease. Factors that debilitate
trees and reduce their capacity to recover from damage, such as restrictions to root
expansion, poor soil fertility, low aeration and soil disturbance by tillage, are
associated with the occurrence of ink disease (Portela et al., 1998).
Phytophthora cambivora and P. cinnamomi, like other species of Phytophthora,
can cause genetic erosion in different species and cultivars of fruit trees, but the use
of resistant rootstocks can overcome this risk. The impact of P. cambivora on
chestnut and beech forests is significant, because its presence undermines the
stability and evolution of these ecosystems at risk. Trees on mountain slopes or
ridges that died from ink disease can compromise the stability of soils by leaving
them exposed to erosion from runoff rainwater.
In some stands in Italy, P. cambivora infection led to the natural replacement of
chestnut trees: dead chestnut stands have gradually been invaded by the more
resistant oaks (Quercus pubescens), leading to the formation of new oak woods
(Turchetti, 1986). Chestnut is also the main ectomycorrhizal host of most of these
stands, and the mushroom population suffers when it disappears. Phytophthora
cambivora, like other Phytophthora species, colonises anaerobic soils that are
unfavourable to other fungi, including Phytophthora antagonists, which greatly
decrease in these soils.
4.7. Soil Microflora Action

It is difficult to study the influence of soil microorganisms on ink disease agents
because of the complexity of this microhabitat. Only a few studies on this subject
have been carried out for P. cambivora, while more knowledge is available for P.
cinnamomi. There is much evidence to demonstrate that most Phytophthora spp. are
relatively poor saprophytes and do not grow well in competition with other micro-
organism in soil.
In general, the competitive saprophytic ability of Phytophthora spp. is extremely
low, and rapid lysis of mycelium occurs in natural soils. Several genera of bacteria,
actinomycetes, fungi, amoebas, as well as nematodes and mites, have been known to
parasitise and to lyse propagules of Phytophthora spp. in soil (Erwin & Ribeiro,
1996). Their survival varies inversely with the number of micro-organism present.
Although oospores are long-lived in soil, they are parasitised by various
microorganisms. More than a dozen species are capable of infecting oospores (Sneh
Humble & Lockwood, 1977), including certain oomicetes, hyphomycetes, cytridis
actinomycetes, and bacteria.
A wide range of fungi is antagonistic to Phytophthora spp., and some of these
appear promising for biological control in greenhouse research. Trichoderma and
Gliocladium have proved effective in suppressing certain species such as P.
cinnamomi (Chamber & Scott, 1995). Preliminary tests carried out with T.
harzianum and T. viride have proved quite effective in the in-vitro suppression of P.
cambivora (Turchetti & Maresi, 2005). Hyphal lysis of parasitic mycelium is rapid
and, as observed with Trichoderma, involves contacting and coiling the parasite
around the hyphae. Trichoderma spp. are also able to produce either volatile or
soluble metabolites (Brasier, 1971; 1975).
The percentage of microbes antagonistic to P. cinnamomi is higher in

suppressive soils. These soils are inhospitable to some plant pathogens to the extent
that either the pathogen cannot become established or, if established, it cannot
initiate disease on hosts. Studies on the natural control of Phytophthora spp. were
reported by Broadbent, Baker and Waterworth (1971) and Broadbent and Baker
(1975). The latter showed that P. cinnamomi had been suppressed in certain soils.
Soil microorganism competition, similarly to ectomycorryzal action, may indeed
explain the recovering of P. cambivora even under asymptomatic trees, in several
parts of the C. sativa natural range (Vettraino et al., 2004). Ectomycorrhizae are
also reported to be effective in limiting infection by P. cinnamomi and other species,
for a wide range of host and symbionts.
In addition to the mechanical protection of apices by means of the mantle and the
dense hyphal network, mycorrhizal fungi produce antibiotic and mono-terpene
compounds that are inhibitory to P. cinnamomi (Erwin & Ribeiro, 1996).
Preliminary test carried out with ectomicorrhizal fungi against P. cambivora proved
effective in vitro in checking the growth of the parasite (Vrot & Grente, 1985;
Branzanti, Rocca & Zambonelli, 1994; Branzanti, Rocca & Pisi, 1999).
6. DISEASES MANAGEMENT
6.1. Blight, Silviculture and Biological Control

Fifty years after healing cankers were first reported (Biraghi, 1950), the dynamics of
chestnut blight is becoming stabilised in Italy and much of Europe, with a distinct
and constant predominance of hypovirulent strains. Studies carried out in the
intervening period have confirmed that a mixed inoculum of the pathogen that is
stable in space and over time is spreading naturally and on a large scale (Bonifacio
& Turchetti, 1973; Garbelotto, Frigimelica & Mutto-Accordi, 1992; Gurer et al.,
2001). This means that hypovirulent and non-hypovirulent strains co-occur in
chestnut orchards or stands and in the outbreaks of infection.
The pathogen has also the ability to produce new lines that can be detected by
means of vegetative compatibility grouping (Cortesi, Milgroom & Bisiach, 1996),
and that could render the hypovirus non-transmissible. However, the devastating
effects that would be produced by strains becoming incompatible have not been
observed so far in the field. The natural mixed inoculum containing various
hypovirulent strains apparently buffer any variations in the genome lines, by acting
through a feedback mechanism in the ecosystem. Therefore, hypovirulence appears
as a complex phenomenon which by now represents a stable part of the chestnut
ecosystems. In these, the effects of mixed pathogen inocula are conditioned by the
vegetative state of the host tree, and by interacting site factors depending on the
physiology of the tree, as well as on the interaction between the pathogen isolates.
Within this context, management measures need to examine the actual situation
in chestnut stands, and should take into account the natural dynamics of chestnut
blight. In any case, growers must take it for granted that blight will be a constant
presence in all stands, and that there will be a certain amount of wilting, which they
will have to put up with.
An evaluation of the damage due to blight is the first step in elaborating a
concrete management strategy aimed at decreasing the likelihood of infections with
a lethal outcome.
Various scenarios can be detected:
1) Reduced damage, concentrated in small branches or dominated sprouts in the

presence of old dead branches.
The re-growing of chestnut trees in orchards and stands is significant of the
favourable evolution of the disease. The different levels of re-growing in plants are
related not only to the impact of the disease, but also to environmental factors and to
treatments carried out in the past and present.
Management steps in the orchards will include the prompt elimination of any
branches that wilt as a result of recent chestnut blight attacks (these are easily
recognisable by the fact that the leaves that turn yellow still remain attached to the
branches). In contrast, it is easy to detect old branches that have died in the past as a
result of severe attacks: their presence is not symptomatic of the actual impact of the
disease.
The limited incidence of recent mortality emphasises the predominance of
hypovirulence. This can be assured by leaving healing and healed cankers intact
during pruning and thinning operations. Encouraging results have been achieved by
adopting these simple measures: for example, in some chestnut stands the disease
seems to have apparently disappeared, although the pathogen is still very much
present. Thus, in this context, the best strategy can be summarised in the
admonition: “Let hypovirulence work”.
For the management of coppices, there are several silvicultural options, as
reported by Amorini et al. (2001). In all of these, where hypovirulence is already
established and predominant, normal silvicultural practices aim at leaving the shoots
alive and infected with healing cankers, guaranteeing a continuous production of
mixed inoculum that contains hypovirulence over time. In this case, coppices can act
as a source of hypovirulence also for neighbouring orchards. In order to follow these
guidelines, forest workers and chestnut growers must be trained to recognise the
types of cankers that they may encounter.
2) Extensive recent damage involving stems and larger branches.
This situation may be due either to a predominance of virulent attacks of the
disease or to the action of constraint factors related to the site and capable of
weakening chestnut trees. Moreover, this may refer to the initial stage of the
introduction of the parasite and spread in healthy areas. Site conditions should be
examined where the damage is more severe. In addition, silvicultural choices could
aim at creating mixed forests that can be managed more in harmony with the
environment. If the site condition can be excluded as a disturbance factor, immediate
silvicultural treatments will be needed in order to eliminate all the branches that
have died because of virulent attacks.
The presence of healing cankers must be investigated in order to identify
possible natural foci of hypovirulent inoculum, and these cankers must then be
released.
In chestnut orchards and stands affected by severe damage, artificial inoculations
are advisable as a solution. It is assumed that treatments of natural infection with
hypovirulent compatible strains will resolve problems of blight. This curative
treatment of a developing infection, as suggested for the first time by Grente and
Sauret (1978) and still adopted (Diamandis & Perlerou, 2006), has shown a rather
poor performance when applied on a large scale, and appears to be overly expensive.
Its costs are increased by overly-long laboratory tests, by the need for specialised
personnel, and by the practical difficulties of working in tall trees. Moreover, as
suggested already by Shain and Miller (1991), curative inoculation cannot
completely change the inoculum produced from virulent infections, even if it is able
to stop their growth. Therefore, even the good results reported by some authors for
C. sativa were achieved in areas where hypovirulence was still spreading naturally.
On American chestnut trees, in the face of some good localised results, with curative
treatment performed by using hypovirulent strains containing Italian hypovirus
(Hogan & Griffin, 2008), relatively unsatisfactory results were achieved by means
of artificial inoculation with transgenic strains, which showed a reduced ecological
fitness (Root et al., 2005).
Combined artificial inoculations can act and/or increase sources of
hypovirulence. In this treatment, four hypovirulent strains - selected for their ability
to produce pycnidia and for their wide conversion spectrum - are inoculated in four
square holes (one strain per hole) made in the bark of selected shoots left on stumps
in a chestnut grove (Turchetti & Maresi, 1991; Antonaroli & Maresi, 1995). This
forms an infected area in the grove which produces fruiting bodies for new
hypovirulent infections for some two or three years, until the cankers are scarred
over by the host reaction.
Conidia produced by such artificial infections will produce further attacks
elsewhere and so on, in an exponential progression. This ensures that there will be a
copious supply of hypovirulent inoculum in the chestnut groves treated in this
manner. Hypovirulent isolates must be obtained from the local C. parasitica
population, through isolation or by means of the conversion of virulent isolates. The
choice of this strategy can also be proposed for areas in which the fungus is starting
to spread. In this case, it may be possible to change the starting inoculum of the
parasite, thus enhancing the hypovirulent infections. Combined inoculation has
proved to be effective and very inexpensive (Antonaroli & Maresi, 1995). It can also
be used by specially-trained but not (necessarily) specialised personnel.
Another key point in disease management is to protect all pruning wounds from
infection. Moreover, any waxes applied must not cauterise living tissue, as happens
in most cases. Application of biomastics, favouring callus growth on bark that has
been cut by pruning, is currently under study and at an advanced stage of testing.
Cankers could also be produced, with lethal effects on grafts, with the use of
hypovirulent isolates. The graft junction where the scion is grafted onto the stock
should hence be protected with wax containing biological additives (selected

bacteria able to stimulate tissues reaction) that favour wound healing. These waxes
are already on the market (CERAFIX PLUS) (Santagada, Maresi & Turchetti,
1996). All pruning wounds in all grafts should be protected in this way, to make it
impossible for the parasite to invade the host tissue.
Figure 4. Chestnut trees with a very reduced damage due to blight, because of the
predominance of hypovirulence.
6.2. Ink Disease Control

In chestnut orchards, the main objective is to lower prevalence of ink disease as
much as possible. Chemical control is difficult, because it cannot be applied in the
forest as it is in chestnut stands. Furthermore, the ‘naturalness’ of chestnut orchards
and trees should not be lost sight of, since they are a basic part of their commercial
image.
Biological control offers interesting possibilities if combined with silvicultural
and horticultural practices that aim at improving trees health (Bounous & Gomes
Abreu, 1998; Bounous, 2002). On sites subject to waterlogging, it will be necessary
to improve drainage and to aerate the soil, in order to stimulate the growth of
microflora. In the area of Mugello (Italy), integrated organic fertilisers have been
used for a number of years with very good results. Trees that were seriously affected
by ink disease not only recovered, but started once again to produce fair-sized
chestnuts, showing that control techniques linked to soil improvement really do
work (Turchetti et al., 2000, 2003).
Preliminary investigations in coppices and mixed stands, as well as in abandoned
chestnut orchards, have shown that competition with other, more invasive and frugal
tree species has given rise to an incidence of ink disease in chestnuts. In coppices
and stands, correct silvicultural practices that will guarantee the survival of the trees
and control their development are desirable: ink disease may well be an early sign of
change in the vegetation composition in a forest and of evolution towards a new type
of mixed forest, from which chestnut trees are slowly being squeezed out.
Preventive or curative treatments can be carried out in nurseries, which in
principle should produce disease-free plants. Propagation material from nurseries
should be carefully controlled for another important reason, namely to limit the
spread of P. cinnamomi which, due to its high polyphagy is an even more dangerous
parasite than P. cambivora. It is, unfortunately, already widespread in France, Spain
and Portugal.
From the management point of view, the continuous monitoring of the health
state of chestnut stands and orchards by forest technicians and growers, respectively,
remains a prime requisite, so that the way in which diseases, stands and orchards are
evolving can be safely predicted over time (Turchetti & Maresi, 2000).
7. PERSPECTIVES AND CONCLUSIONS

The improvement in the management of chestnut diseases needs a better
understanding of the ecological dynamic of the chestnut ecosystems. A holistic
approach that includes all the factors involved in chestnut tree ecology is
indispensable in planning the management of these ecosystems and in undertaking
the best measures of conservation and improvement. In particular, a comprehension
of the mechanism behind the spread and establishment of hypovirulence is possible
only by studying the site characteristics, the fungal inoculum, and the resistance of
the species. In this context, the approach based on hypovirus determination and on
the population genetics assayed by Vegetative Compatibility cannot be exhaustive
either for scientific purposes or as a practical approach. The maintenance of
hypovirulence seems to be related to a C. parasitica mixed inoculum (white,
intermediate, and virulent isolates spreading together), the action of which is
mediated by host resistance and site condition. Only by working on all the three
factors it will be possible to maintain, improve or reproduce the effectiveness of
hypovirulence.
Even now, several aspects of ink disease and C. parasitica biology are still not
completely understood and need more research work. However, current knowledge
can provide indications on general management that will make possible the “pacific
connivance” of chestnut cultivation with these diseases. For blight, the aim of all
silvicultural and agronomic practices should be to maintain abundance of healing
and healed cankers, removing other disturbance factors, such as competition, as
much as possible. Naturally, a low level of damage will always be present, but, as
observed in Italian woods, it will not be able to stop or menace cultivation.
Ink disease is more complicated to manage, due to a serious lack of knowledge
especially as concerns the pathogen and its competitors soil biology. In any case, the
aims of maintaining good soil functionality and managing water, organic matter and
manuring can unquestionably be achieved. In this context, the constant monitoring
of trees, in an attempt to detect the early symptoms of the disease, is important.
The main aim of the investigations carried out and of the expedients
recommended was to ensure the survival of chestnut orchards without diminishing
their productivity. The general indications given above represent a basic approach,
not only for researchers, but also for workers in the field, in as much as they can be
adapted to any area in which chestnut trees are cultivated. Within this wider
ecological context, biological control methods are an important means for managing
stands and ensuring a better exploitation of this economic resource in the mountain
regions.
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6
GIUSEPPE SURICO, LAURA MUGNAI AND GUIDO MARCHI
THE ESCA DISEASE COMPLEX
Dipartimento di Biotecnologie Agrarie, Sezione Patologia Vegetale,

Piazzale delle Cascine 28, 50144, Firenze, Italy
Abstract. Esca is a grapevine wood disease that seriously affects vine yield and longevity. Our
knowledge of this disease and its causes has changed profoundly in recent years, as it has become clear
that esca in fact comprises a number of distinct diseases and that the main fungal agents (primarily
vascular pathogens) invade the vines not only through wounds applied in the field but also as a result of
nursery practices. When vines become infected in the nursery, the diseases that develop may vary, from
Petri decline to full-blown esca, with or without white decay. With the banning of sodium arsenite no
chemical control is now available and sanitary practices in the nursery are suggested as being the best
approach to eliminate or at least reduce pre-planting infections from the tracheomycotic fungi. In the
absence of chemical prevention, some preventive and curative action can also be taken in the field to
reduce infections or to hamper symptom appearance in esca-infected vines, as will be described.
1. INTRODUCTION
Esca (also known as black measles in the USA) has long been considered a single
disease, that normally affects adult, or indeed old, vines. Its cause was considered to
be Phellinus igniarius, or sometimes also Stereum hirsutum, two known wood rot
fungi. Phellinus igniarius was seen, more often than S. hirsutum, as causing all the
typical symptoms of esca: white wood rot, followed by chlorosis and necrosis of the
leaves, as well as the apoplexy of those vines that suffered a sudden rapid wilting.
The central characteristic of esca thus was thought to be the white rot of the wood.
The term esca, which had thus been current for centuries in various Mediterranean
countries to denote both wood rot and the apoplexy of vines (as well as to denote
Fomes fomentarius, the tinder fungus used in the past to light fires, and P. igniarius,
the false esca fungus), started to circulate with its present meaning among plant
pathologists in France in the 1920s (Marsais, 1923; Viala, 1926).
Modern studies on esca initiated at the end of the 1980s, and have profoundly
changed our knowledge of the disease and its causes. Many studies carried out
especially by researchers in France, Italy and other countries (USA, Australia, South
Africa, New Zealand, Spain, Portugal, Germany and others) have established that
there are three main fungi, Phaeomoniella chlamydospora (Pch), Phaeoacremonium
119
120 G. SURICO ET AL.
aleophilum (Pal) and Fomitiporia mediterranea (Fmed), which are involved in five
syndromes, all of which are related, and which form the esca disease complex.
These syndromes are: brown wood streaking of rooted cuttings; Petri disease;
young esca; white rot, and esca proper (young esca plus white rot). More recently it
has been proposed to rename young esca phaeotracheomicosis (with the prefix
phaeo-, common to Phaeomoniella and Phaeoacremonium) or, more simply, to give
young esca the name esca (though, for the reasons indicated above, the term esca
would in fact be more appropriate to historically designate white rot) and to continue
using esca proper only to denote a combination of tracheomycosis and white rot in a
particular vine plant, the tracheomycosis caused by Pch, with or without the
concurrence of Pal, and the rot by Fmed. Esca proper is nevertheless the most
common affection encountered in mature or ageing vineyards. In that case, the dark
wood streaking of rooted cuttings (the occurrence of gum and dark streaks along
single or bundles of wood vessels) and Petri disease (a form of young vine decline
that can be lethal) are merely different forms of tracheomycosis.
Then it can be concluded that, in the light of current knowledge, the esca
complex comprises the following syndromes: brown wood streaking of rooted
cuttings, Petri disease, and esca. This complex of syndromes is caused by various
tracheomycotic fungi (especially Pch). White rot on the other hand is cuased by
various basidiomycetes (especially Fmed). The particular syndrome produced by
these fungi depends on the age at which the vine becomes infected; on what fungi
are found together in the same vine; on the symptoms exhibited by the infected vine,
and lastly on the concurrence of other external stresses. To these syndromes must
further be added apoplexy and (to consider the most common disorder in adult
vines) esca proper. This chapter will deal with the various syndromes exhibited by
infected vines, and will examine how these syndromes can be controlled.
2. THE PATHOGENS OF THE ESCA DISEASE COMPLEX AND OF WHITE ROT

2.1. Tracheomycotic Fungi
Two species of fungi are associated with the tracheomycosis/esca and its related
diseases (dark streaking of rooted cuttings and Petri disease): Pch and Pal. These
two fungi have been found to colonise the xylem tissues of vine plants (Eskalen,
Feliciano & Gubler, 2007; Mugnai et al., 1997; Larignon & Dubos, 1997; Sparapano
et al., 2000b), and they probably also cause (though the experimental demonstration
is as yet very scarce) the familiar leaf symptoms of esca, probably by a series of
concomitant activities such as the production of phytotoxins (Evidente et al., 2000;
Sparapano et al., 2000a; Tabacchi et al., 2000; Abou-Mansor, Couché, & Tabacchi,
2004; Bruno & Sparapano, 2006a), of pectinolytic (Marchi et al., 2001) and various
extracellular enzymes (Mugnai, Surico & Sfalanga 1997; Santos et al., 2006; Bruno
& Sparapano, 2006b), the occlusion of the vessels (Del Rio et al., 2001; Troccoli
et al., 2001; Edwards, Pascoe & Salib, 2007).
Phaeomoniella chlamydospora is common in all vine-growing areas, while Pal
is less widespread (with a very few exceptions in some limited areas) and can be
associated with other species of Phaeoacremonium. Moreover, while Pch has so far
ESCA DISEASE 121
at least been found only on grapevine, Pal also causes wood darkening on other
plants (Mostert et al., 2005; 2006a, b).
Phaeoacremonium is a recently-described genus, intermediate between
Phialophora and Acremonium (Crous et al., 1996), to which initially seven fungal
species were assigned: Pm. aleophilum, Pm. angustius, Pm. chlamydosporum (Pa.
chlamydospora), Pm. inflatipes, Pm. mortoniae, Pm. rubrigenum, Pm. viticola and
the type species Pm. parasiticum. With further surveys and samplings, more species
have been identified, among which 8 cause mycosis on human beings, 12 occur on
grapevine (Table 1), and 2 on other woody host plants. In addition, the original Pm.
chlamydosporum has been transferred with its specific epithet to a new genus,
Phaeomoniella (Crous & Gams, 2000), a genus which so far comprises only two
other species, Pa. zymoides and Pa. pinifoliorum, both saprophytes on plants (Lee
et al., 2006). Phaeoacremonium aleophilum, Pm. angustius and Pm. inflatipes have
also been found on other plants so far (Mostert et al., 2006 a, b).
The perfect form of Pch is not known, but that of Pal, Togninia minima, has
been produced in culture and also identified in the field (Mostert et al., 2003; Pascoe
et al., 2004; Rooney-Latham, Eskalen & Gubler, 2005). Pch and Pal, which
preferentially colonise the xylem tissues, have caused, in inoculation tests,
darkening of the xylem vessels and sometimes (Sparapano, Bruno, Graniti, 2001;
Table 1. Geographic distribution of reports on grapevine of species of Phaeoacremonium and

associated disease/symptoms, when reported. (Modified from Surico, Mugnai & Marchi, 2007).
Phaeoacremonium Geographic area of Associated diseases Wood symptoms

species isolated from reports on grapevine
grapevine
Pm. aleophilum North America, Decline, Esca Black streaking

Australia, Europe, South
Africa Brown-red wood
Brown necrosis
Pm. aleophilum South America Hoja de malvon
Pm. aleophilum Iran Esca
Pm. angustius Europe, North America Brown necrosis
Pm. australiense Australia
Pm. inflatipes South America None
Pm. iranianum Middle East Black streaking
Pm. krajdenii South Africa
Pm. mortoniae USA Brown necrosis
Pm. parasiticum South America, North Decline (?) Brown necrosis
America, Australia, Yellowish necrosis
Middle East, Sud Africa
Pm. scolyti Europe, South Africa None
Pm. subulatum South Africa Brown necrosis
Pm. viticola Middle East, Europe, Brown necrosis
South Africa, North (dark ring)
America
Pm. venezuelense South Africa
Eskalen, Feliciano & Gubler, 2007) the leaf symptoms of esca and decline
symptoms in young plants under water stress. Artificial inoculation with Pch and
Pal has also reproduced the symptoms of black measles on grape berries
(Sparapano, Bruno & Campanella, 2001; Feliciano & Gubler, 2001; Thind &
Gubler, 2007).
2.2. Basidiomycetes Causing White Rot

The name of the rot agent Fomitiporia mediterranea (Fischer, 2002) already gives
an idea of the range of this species, namely, the Mediterranean basin (up to the
western Asian countries). In other parts of the world, rot in grapevine, when it
occurs, is caused by other fungi (Table 2).
It has been found that F. mediterranea occurs not only on grapevine but also on
other plant species (Olea europaea, Acer negundo, Lagerstroemia indica, Actinidia
chinensis, Corylus avellana, Laurus nobilis, Ligustrum vulgare, Quercus ilex,
Cornus mas, Robinia pseudoacacia, Citrus spp.), and probably others as well
(Pilotti, Gervasi & Brunetti, 2005; Elena et al., 2006; Fischer, 2006), which can
therefore act as inoculum sources in the hypothesis, not yet verified, that isolates
from these plants are also pathogenic on grapevine.
Figure 1. Resupinate fruiting body of Fomitiporia mediterranea on a grapevine trunk: the

fungus forms a crust-like carpophore of a cinnamom colour, becoming greyish when it gets old.
Fomitiporia mediterranea forms woody and resupinate fruiting bodies (Fig. 1),
up to 15 mm thick, cinnamon brown in colour, with 6-8 pores per mm2. The fruiting
bodies are usually located on the upper part of the trunk. The basidiospores come in
two sizes: 6-7 × 5-6 μm and 5-5.5 × 4-5 μm, although the smaller basidiospores are
probably only immature. The mycelium is cottony and woolly, with yellowish or
brownish aerial hyphae. Some strains (type S, the ‘staining’ type) form scarce aerial
hyphae and give their growth medium (usually malt agar with yeast extract) a red-
dark brown colour; these strains grow more slowly in culture. The optimum growing
temperature is about 30°C and reproduction is homothallic (Fischer, 2002).
ESCA DISEASE 123
3. SYMPTOMS
3.1. Brown Wood Streaking of Rooted Cuttings
This disease, caused by Pch, presents no external symptoms, but if a cross or
lengthwise section is made, various types of wood deterioration are detected. In
lengthwise section the most conspicuous are dark streaks, single or gathered into a
blackish-brown bundle that sometimes starts at the graft junction and often extends
upwards and downwards to reach the lower end of the plant. More often, however,
the streaks leave from the bottom and move upwards. In cross-section the rooted
cutting shows black spots arranged in the form of an almost continuous ring around
the central pith, or scattered over the section surface. A gum-like exudate that is
almost black, often oozes from the vessels corresponding to these black spots
(Mugnai, Graniti & Surico, 1999).
Table 2. Basidiomycetes isolated from grapevine showing white decay in the trunk. The foliar
symptoms and/or the names of the diseases that different authors described as associated with
the wood decay are also reported. (Modified from Surico, Mugnai & Marchi, 2007).
Fungal species Geographic Associated Reference/Author

area where it foliar
was reported symptoms or
disease
Fomitiporia mediterranea Europe Esca Fischer, 2002
Stereum hirsutum Europe Esca Larignon and
Dubos, 1997
Fomitiporella vitis South Chlorotic leaf Auger, Aguilera, &
America roll Esterio, 2005
Inocutis jamaicensis South Hoja de Martínez, 2005
America malvon
Fomitiporia polymorpha North Esca (Black Fischer, 2007.
America measles)
Not identified taxa North - Fischer, 2006

America
Fomitiporia australiensis Australia - Fischer et al., 2005
Two not identified taxa Australia - Fischer et al., 2005
Three not identified taxa South Africa Esca Fischer, 2006
3.2. Petri Disease (or ‘Black Goo’)

Petri disease, also caused by Pch, was first reported in 1912 in Italy by the plant
pathologist Lionello Petri, and in more recent times (the second half of the 20th
century) in the USA. It was first noted in young graft vines in the field, and then also
in older mother vine stocks, in Australia, New Zealand and France. In the USA the
disease was initially called ‘black goo’ from its main symptom, a dark, gummy,
almost tarry substance that oozed from the wood vessels when they were cut
through. Petri disease attacks very young vines (from one year), and normally causes
stunting of the whole or part of the vine. Other symptoms are: complete cessation of
growth, moderate chlorosis of the leaves, loss of yield, and a gradual decline in
vigour. Sometimes the disease outcome is the death of the vine. In other cases
the vine continues to vegetate, and the disease most likely turns into esca, and/or
esca proper (this last with the concurrent action of Fmed) after some years.
The internal symptoms of Petri disease are: a darkened central pith; a black ring
around the pith, or else black dots scattered or arranged in a crescent in the wood of
the trunk (including that of the rootstock) and of the vine-canes; the exudation of
drops of a dark gummy liquid from cross-sectioned vessels, on which the drops
leave a tarry crust when they dry. Petri disease can arise in the field or be an
evolution of the dark wood streaking of rooted cuttings (Morton, 1995; Scheck et al.,
1998; Ferreira et al., 1999; Mugnai, Graniti & Surico, 1999; Pascoe & Cottral,
2000).
3.3. Esca (Young Esca)

This disease affects young, 2-3 year-old, vines, which externally exhibit the typical
(leaf) symptoms of esca, and internally the dark streaks already mentioned,
colonised by Pch, and sometimes also by Pal. It is possible that in the vineyard
phaeotracheomycosis marks a further stage in the development of the dark streaking
of rooted cuttings, or Petri disease, in those cases when it does not kill the vine. Esca
can also occur in the field however, as when infections arise on healthy nursery
material.
Slightly discoloured, roundish spots appear on the leaves of vines affected with
esca, between the leaf veins or along the edges. Gradually, the spots expand and
become confluent, and eventually they turn necrotic, at least in part (Fig. 2). When
the disease is fully developed, the leaf in the most typical cases assumes a ‘tiger-
stripe’ aspect, with wide chlorotic and necrotic stripes and only a narrow green
stripe along the midrib. On white-berried cultivars the discoloured areas are
yellowish, in black-berried vines they can be yellowish and/or reddish or dark red.
On the berries, especially those of table grapes, small spots sometimes appear.
These spots have a dark brown or a more or less intense purple colour. The spots are
usually more thickly scattered at the far end of the berry, but they can also be
distributed irregularly over the whole surface, or arranged in longitudinal stripes. In
the last case, surface cracks can appear that enable secondary rot agents to invade
the berry. In California, these spots on the berries (especially on the cv. Thomson
seedless) are much more common than the leaf symptoms of esca, and therefore esca
was originally called ‘black measles’ in this part of the world.
In cross section, besides the dark spots that are characteristic of the dark
streaking of rooted cuttings and of Petri disease, it is also possible to find areas with
darkened wood or brown or light brown necrosis (Fig. 3). These areas are usually
not very large, central (characteristic is the reddish-brown necrosis around the pith, a
sign of Pch) or sectorial, in the trunk and/or the branches, generally connected with
wounds (mostly pruning wounds). If the trunk or branches are stripped of their bark,
lengthwise dark or yellowish streaks can be seen in the cambium.
ESCA DISEASE 125
Figure 2. The leaves of esca affected vines show very typical tiger-like interveinal
necrosis surrounded by a yellow margin.
Figure 3. Cross section of a young grapevine showing dark spots and

reddish-brown necrotic wood.
3.4. White Rot

White rot occurs when the vine trunks, and often the main branches, but only rarely
the rootstocks, are invaded by Fmed. The rot caused by this fungus is a typical white
decay, where the wood is transformed into a spongy, friable, whitish-yellow mass
(Fig. 4). In cross-section the rotted area is often delimited by a dark line of varying
width that separates the rotted from the healthy tissue. The rot often starts from a
pruning wound and spreads out within the wood upwards and downwards until wide
sectors of the central cylinder are affected. In some cases the rot also reaches the
wood surface, when it causes cracks along the trunk (‘mal dello spacco’).
White rot normally forms more easily in wood affected by other types of
deterioration that have already been described: longitudinal dark streaks (showing
up as dark spots in cross-section), isolated or grouped together, occupying one of the
annual wood rings or near the pith; and reddish or dark areas, located in the central
cylinder or at the edge of necrotic or rotted tissues.
Rotted wood becomes completely non-functional; however, if only the last two
or three wood rings retain their capacity to transport water and solutes, the vine will
continue to vegetate normally (Pratt, 1974; Mullins, Bouquet & Williams, 1992).
This explains why vines with very extensive necrosis of the trunk will yet continue
to vegetate and produce yield, at least until they also become affected with
tracheomycosis or apoplexy. The external symptoms of white rot as such cannot be
described at the moment because no mature vines that have only rot of the trunk
and/or other plant parts, and no other wood symptoms, have yet been detected.
Young vines with small rotted areas on the trunk or at the graft union, but without
leaf symptoms, have however been found. On the other hand, artificial inoculations
with Fmed have reproduced the white rot without causing any esca leaf symptoms,
except perhaps in one case (Sparapano, Bruno & Campanella, 2001). Lastly, in some
countries, such as Australia, the leaf symptoms of esca have never been reported in
vines with trunk white rot (Edwards, Marchi & Pascoe, 2001).
Figure 4. A grapevine trunk section showing a large decayed area caused by

Fomitiporia mediterranea surrounded by dark necrotic areas.
3.5. Esca Proper

As mentioned, the esca proper syndrome is found when a vine shows the
superimposed effects of all the three main fungi associated with esca: Pch, Pal and
Fmed. The wood symptoms of esca proper are therefore simply those of
tracheomycosis, plus those of white rot.
ESCA DISEASE 127
In this as in the other disorders that have been described, other types of necrosis
may also occur, caused by other fungi or wood-inhabiting pathogens, such as species
of Botryosphaeria, Eutypa lata, Phomopsis viticola, etc. The occurrence of these
other pathogens and their incidence is dependent upon the existence of the particular
sources of inoculum in the area.
3.6. Apoplexy
Vines affected by apoplexy (traditionally considered the acute form of esca) may
show sudden wilting of all or part of the crown, as early as June. Sometimes such
vines resume growth in the same season or the next; more often, however, the plant
dies from this disease. The causes of apoplexy are not known. It has been observed
that heavy rainfall followed by hot winds in mid-summer favours the onset of
apoplexy. And in fact, after a heavy shower the vine opens its stomata to eliminate
the excess humidity. If a hot wind begins to blow when this happens, the vine should
close its stomata to adjust to the new situation, but if it still senses the previous
rainwater in the soil, it may not transmit the necessary signal (abscissic acid) early
enough to stop the loss of humidity by transpiration, leading to the rapid wilting of
the plant that is a characteristic of apoplexy. All this seems likely enough and may
well happen in nature; however, it has been ascertained that apoplectic strokes also
frequently occur at times when there is, or has been, no rainfall (Surico et al.,
2000b), and that apoplexy mostly strikes older vines that already exhibit very
extensive rot. And since moreover tracheomycosis can also affect vines of only two
years and without any rot, it seems reasonable to assume that apoplexy is above all a
condition associated with white rot, and that it is mainly caused by a dysfunction of
the conducting system of the plant.
However, since apoplexy almost always occurs in plants that are also affected
by tracheomycosis, it cannot be excluded that the disease is also favoured by the
activity of the two tracheomycotic fungi, Pch and Pal, perhaps by an accumulation
of phytotoxins (extracellular polysaccharides, scytalone, isosclerone, etc.) in the
leaves.
4. SOURCE OF INOCULUM AND SPREAD

Studies in California, France, Australia and South Africa have established that
propagules of Pch and various other species of Phaeoacremonium can be found on
the vine trunks, canes and berries, and even on old tendrils that have remained stuck
to the wires.
In California it has been found that Pch spores are mainly dispersed from
October-November til April, and sometimes also in other months, when rainfall
events occur, or after a shower with temperatures varying from 5°C in February to
20-25°C. In France, on the other hand, Pch spores were trapped during the entire
year, even in winter, and the optimal airborne spore dispersal and pruning wounds
infection were found to occur after a period in which temperatures averaged 7-15°C
with a maximum of 12-18°C, and when rainfall already had occurred. Pal, which
has been studied only in France, does not seem to infect pruning wounds since its
spores are never trapped in winter but only from early March to the first third of
April, or, more often, from mid-May to mid-June, depending on the year climate.
It has been found that the receptivity of pruning wounds to infection is greater,
and lasts longer, when pruning is carried out in winter (December and January).
Nothing is known about the source and spread of Fmed inoculum anywhere in the
world. The fruiting bodies of this fungus (from which the basidiospores arise) form
almost exclusively on very old vines. Therefore, it seems reasonable to assume that
Fmed inoculum reaches the vineyard from an external source: old vineyards nearby,
or carpophores that have grown on other hosts (olive, kiwi, oak, etc.). Within the
vineyard, it seems that Fmed inoculum is not easily spread from vine to vine along
the rows, though this is possible, and hence that it is not spread by the pruning tools
(Cortesi, Fischer & Milgroom, 2000; Surico et al., 2000a).
4.1. Infection Routes and Disease Distribution in the Vineyard

In order to explain the aetiology of the decline of young grapevines, researchers
directed their attention to vine propagation material and the tracheomycotic fungi of
esca. The first studies examined vine rooted cuttings (Bertelli, Mugnai & Surico,
1998), afterwards research was broadened to investigate how rooted cuttings were
prepared in the nursery, and lastly, the canes from mother vines were studied.
Initially, it was found in Italy that rooted cuttings ready for outplanting sometimes
had dark streaks that could extend along almost the whole length of the cutting, or
that were concentrated at the foot of the cutting, or near the graft junction, which
were colonised by Pch and Pal. These findings were confirmed by researchers from
other laboratories (Table 3).
Subsequently, it was found that a number of nursery practices were unsafe and
could allow these two fungi to become established (Zanzotto et al., 2001; 2006;
Ridgway, Sleight & Stewart, 2002; Retief et al., 2005). Later, it also became clear
that Pch and Pal could already occur in the canes of mother vines (Fourie &
Halleen, 2002; Edwards et al., 2004a; Larignon, Berud & Girardon, 2006). This was
not surprising, since the two fungi colonise the vessels and could thus be transported
upwards by the sap flow.
Healthy propagation material and the implementation of appropriate sanitary
practices, on the other hand, will lead to perfectly healthy rooted cuttings being
produced. When that happens, the first infections that could arise occur through
wounds, especially those caused by training system cuts and winter pruning,
removal of side-shoots, green pruning, and mechanical harvesting. Depending on the
age of the vine and the fungi involved, different types of disease may arise: Petri
disease, and then tracheomycosis, if the fungi colonising the vine wood are Pch
and/or Pal; white rot, if the first invading fungus is exclusively Fmed, and esca
proper, if all three fungi colonise the same vine plant. Among all these diseases,
only esca and esca proper certainly show the typical leaf symptoms of esca.
In conclusion, the progress of the symptoms connected with esca depends on
two orders of factors:
ESCA DISEASE 129
1. grapevine wounding. Every year the vine suffers wounds which, if not
protected, allow entry to Pch, Pal and Fmed, and to other fungi and
bacteria as well, most of which are saprophytes, constituting the endophytic
flora of the vine, but which are sometimes also pathogenic to vines;
2. the chronological order in which Pch, Pal, and Fmed invade the plant, in all
possible combinations.
The manner in which vines become infected is shown by their spatial

distribution. The first vines to become infected are located anywhere at random, and
a random distribution is maintained until the incidence reaches a certain level (at
least 10%) (Fig. 5). Only then, do infected vines begin to clump together without,
however, any preferential direction of spread becoming obvious (not even, as was
long thought, along the rows, following the route taken by the instruments used to
prune the vines).
Figure 5. Spatial pattern of esca-diseased plants in a vineyard of cv. Cabernet Sauvignon

in the province of Siena (Italy). Surveys to determine esca incidence began in 1995 and
proceeded yearly till 2006. Black squares show symptomatic plants in current year; gray
squares show symptomatic plants in current year or in one or more previous years; open
squares show asymptomatic plants; “X” show plants that died before start of survey
(cause unknown); “V” show plants that died during the survey period, with or without
esca symptoms in previous years.
Table 3. Incidence (%) of P. chlamydospora in grapevine propagating material obtained by

various authors (modified from Surico, Mugnai & Marchi, 2007)
Plant material Isolation (%) Country Reference

Rooted cutting 0-80 Italy Bertelli, Mugnai, &
Surico, 1998
Rootstock 7.5-38.4 Portugal Rego et al., 2000
Rooted cutting 20-60 Italy Zanzotto et al., 2001
Rooted cutting 12 Italy Sidoti et al., 2001
Rooted cutting and rootstock 0-37 California Stamp, 2001
Self-rooted cutting 30 Australia Laukart et al., 2001
Grafted and self-rooted cutting 28-31.5 Spain Aroca et al., 2006
Various propagation stages 0.1-5.4 South Africa Halleen, Crous &
Petrini, 2003
5. CONTROL
5.1. Control in the Nursery
Though it can no longer be doubted that nursery material has a role in causing
disease, it is not yet clear what measures should be taken to eliminate or at least
reduce Pch/Pal infections, when grapevines are being propagated.
A number of studies, especially in South Africa (Crous, Swart & Coertze, 2001;
Fourie & Halleen, 2004), Australia (Edwards et al., 2004b; Waite & May, 2005;
Waite & Morton, 2007) and California (Rooney & Gubler, 2001), have examined
the feasibility of using hot water treatment to sanitise propagation material. This
technique has long been used as a simple precautionary measure against various
parasites on vine, so much so that it is recommended by various bodies against both
specific pests, and pests in general: by the European and Plant Protection
Organization (EPPO) against phytoplasms; by the California Department of Food
and Agriculture (CDFA) against nematodes, and by the South African Plant
Certification Scheme for Winegrapes (SAPCSW) as a general treatment against
pests. However, this technique is not always exempt of collateral effects concerning
the viability of the plant material treated (Moretti, Gardiman & Lovat, 2005) and for
this reason a whole series of critical points must be strictly observed, if the
technique is to have any success: the relation between water temperature and
treatment duration, the relation between the volume of the plants to be treated and
the amount of hot water to be used, the physiological state of the plant material,
which must be completely dormant, etc. (Fourie & Halleen, 2004; Waite & May,
2005).
ESCA DISEASE 131
Hot water treatment has been used against the fungi causing esca and related
diseases with varying success. Some studies have been negative: Rooney and Gubler
(2001) found that after artificially inoculating scions of Cabernet Sauvignon, Pinot
noir and Thompson seedless with Pch and treating them with water at 51°C for 30
min, 80% of the Pch could be reisolated, which was not statistically different from
the uninoculated controls. With other studies results have been clearly positive.
Fourie and Halleen (2004) carried out various trials in different parts of South Africa
and at different times of the year. They found that hot water treament at 50°C for 30
min on the rootstock (Richter 110 and 101-14 Mgt) immediately prior to grafting
always significantly reduced the incidence of Pch and Pal at the base of the
rootstocks of uprooted grafted grapevines, compared to the untreated vines. In these
same trials, both benomyl and Trichoderma were also tested and gave interesting
results, although the effectiveness of the fungicide varied between years, whereas
that of Trichoderma varied among the nursery field locations.
Formulations based on Trichoderma to control Pch and Pal in the nursery have
also yielded promising results in other trials (Fourie et al., 2001; Di Marco, Osti &
Cesari, 2004). From these studies it appears that Trichoderma applied at various
stages of rooted cutting production does not induce a direct effect on the esca fungi,
but rather indirectly, it affects esca by enhancing the vigour of the vines.
Trichoderma applied at various vine growth stages made the vines more vigorous
and luxuriant, with a more voluminous root apparatus, even though the frequencies
with which Pch and Pal were reisolated from the rootstock and the roots did not
differ significantly from the controls, though they were slightly lower on average
(Fourie et al., 2001). This suggested that vines treated with Trichoderma are more
resistant to diseases related to stress, such as esca (Fourie et al., 2001; Di Marco,
Osti & Cesari, 2004; Fourie & Halleen, 2004). Di Marco et al. (2004) also found
that rooted cuttings inoculated with Pch, and whose root calli had then been treated
with Trichoderma, showed smaller necrotic areas.
They suggested that the Trichoderma induced resistance mechanisms in the
vines. Induction of resistance mechanisms also occurs in other crops (Fourie et al.,
2001; Di Marco Osti & Cesari, 2004).
5.2. Control in the Field

After the ban on sodium arsenite, the only control measures taken are of the
preventive kind. Some of these measures are designed to reduce the amount of
inoculum in the field:
- eliminating all vines with irremediable or very severe symptoms of esca;
- burning canes and dead stumps and all pruning residues, in order to reduce
the inoculum load in the vineyard;
- as part of a programme to control other vine diseases, applying fungicides

that are also effective against the esca fungi (such as folpet, kresoxim-
methyl, pyrimethanil, fludioxonil+cyprodinil);
- using healthy propagation material.
To reduce the risk of infection, the following steps are recommended:
- to identify and record all infected vines and prune them separately (even
though it has been established that the disease does not spread along the
rows) if they are not eliminated immediately;
- to use vine training that causes smaller wounds, which cicatrize more
quickly and hence offer less opportunity for the esca fungi to invade the
vines (for example vines trained to the vertical cordon are less affected by
esca, whereas those trained to double guyot are more affected);
- protect pruning wounds with appropriate sealant;
- avoiding recourse to mechanical harvesting.
As regards the time at which pruning should be carried out, two factors should
be considered: the time of the year when the esca fungal spores disseminate, and the
speed with which pruning wounds will cicatrize. Consequently, it is recommended
that in areas with more severe winters, pruning should be delayed as much as
possible to ensure that when the vines are pruned, the wounds produced will be
healing more rapidly; whereas where winters are milder, pruning should on the
contrary be carried out as early as possible, so that the wounds will heal before they
can become infected by the newly disseminated spores.
A fungicide replacing sodium arsenite, which was effective against the disease
even after it had started, has not yet been found. Sodium arsenite, as is known, did
not cure diseased vines so much as delay the onset of the leaf symptoms, and hence,
delay the damage caused to the vine plant (it should be remembered that vines that
are certainly diseased may not show any leaf symptoms suddenly for one or more
growing seasons in succession, during which time they appear completely normal
and undamaged). In France, only one product is approved against esca, called
Escudo®, a mixture of carbendazim and flusilazole, which protects wounds from
infection. Tests are now being undertaken in various countries to come up with other
fungicides (in particular the triazoles sprayed on the leaves or injected into the
trunk) but results have not always been encouraging so far.
As a result, the only practical possibility against esca at present is to prune the
vine in autumn or winter, leaving an ample margin (5-10 cm) between any necrotic
areas and the pruning cut below them (the surface of the pruning cut must not
exhibit any wood deterioration) and to grow a cane from the vine base that will in
time form the trunk of a new vine (Calzarano et al., 2004). An ancient custom, still
practised in some countries of the Mediterranean, is to open the trunk in the middle
and insert a stone as a wedge so as to leave the rotted tissue exposed to the air. It
appears that this practice delays the recurrence of symptoms for a number of years –
which is the same effect that sodium arsenite also had.
ESCA DISEASE 133
6. CONCLUSIONS
Although esca has been known for a very long time, probably ever since the vine
first began to be cultivated, and though much progress in understanding the disease
has been made even since the 1980s, the study of esca is still in its infancy. Even
today an understanding of esca and its control is greatly hampered by the difficulty
of reproducing the external symptoms of esca by artificial inoculation. These
difficulties derive not so much from the actual inoculation techniques employed in
the laboratory, as from the existence of unknown concomitant factors which must
concur if the disease is to manifest itself.
It necessarily follows that the mechanisms leading to the leaf symptoms of esca
are still in large part unknown, or at least have not yet been completely
demonstrated. The pathogenicity of the main fungi of esca is an unquestioned fact,
(Pch and Pal) being associated with tracheomycosis and (Fmed) with white rot, and
the manner in which the disease arises and develops is also no longer a subject of
debate. In this connection, ensuring the health of nursery propagating material is a
matter of vital importance. In the current state of our knowledge of the disease and
in the plant pathological context, it is generally believed that in many cases the
disease starts in the nursery and then proceeds in the vineyard with varying fortunes.
From this consideration, it follows that it is in the nursery that measures must be
taken to hinder or prevent those first pathological events that will, if they are not
prevented, continue later in the field, gradually becoming worse over the years,
though the course of the disease usually lasts for a long time. The possibility to
control esca in the field is still limited. At present there appears to be no active
ingredient among products commercially available, capable to cure a diseased vine.
Consequently, it is necessary to rely on other measures, mostly relating to cultural
practices, which on the whole can reduce the spread of the disease and hence its
incidence in a given area. In conclusion, the management of the esca complex of
diseases seems possible in the current state of our knowledge only by carefully
combining a variety of control measures, both in the nursery and the field.
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7
LEONARDO SCHENA1, FRANCO NIGRO 2
AND ANTONIO IPPOLITO 2
INTEGRATED MANAGEMENT OF ROSELLINIA

nECATRIX ROOT ROT ON FRUIT TREE CROPS
1
Dipartimento di Gestione dei Sistemi Agrari e Forestali, Faculty of Agriculture,
Mediterranean University of Reggio Calabria,
89122, Reggio Calabria, Italy
and
2
Dipartimento di Protezione delle Piante e Microbiologia Applicata,
University of Bari, 70126, Bari, Italy
Abstract. Rosellinia necatrix is a soil borne pathogen causing a disease commonly named “white root
rot”. The pathogen, widely distributed throughout temperate and tropical climates, recently showed an
increasing trend of attacks on a number of different host species. Economic losses are particularly serious
in the nurseries and on orchard trees, although field crops and weeds can also be severely damaged. The
pathogen is mainly disseminated by propagating materials and can survive in soil for many years. Control
strategies, which include cultural practices, soil disinfestations, chemical treatments, soil solarization and
biological control are expensive and not always resolutive. Therefore, white root rot control largely
depends on attempts to exclude the pathogen through the use of R. necatrix-free propagating material and
planting in non-infested soils. In this context a fundamental role is played by specific rules, promoting the
commercialisation of healthy propagating materials and the availability of new molecular detection
methods to exclude presence of the pathogen in soil and host tissues.
1. INTRODUCTION
Rosellinia necatrix Berl. ex Prill. (anamorph Dematophora necatrix R. Hartig) is an
emerging pathogen threatening a large number of species through tropical and
temperate climates. The range of hosts comprises more than 170 plant species,
grouped in 63 genera and 30 families of plants and algae. Frequent updates are,
however, required since several new hosts are continuously identified (see
http://nt.ars-grin.gov/fungaldatabases/index.cfm). For decades the “white root rot”
caused by R. necatrix has been erroneously considered of secondary importance,
when compared to other root rots like the “fibrous root rot” caused by Armillaria
mellea. Both microorganisms are soil borne pathogens which develop most of the
disease cycle underground, attacking the roots and the crowns of plants and
penetrating woody roots by invasion of aggregate organs (Delatour & Guillaumin,
137
138 L. SCHENA ET AL.
1985). Considering that symptoms caused on the canopy by these two pathogens are
indistinguishable and result very similar belowground, it is likely that they have
been frequently confused, with a consequent underestimation of Rosellinia root rot
incidence. Recent field surveys across Europe have revealed a wide diffusion of R.
necatrix, which is frequently more common than A. mellea, especially in intensive
irrigated plantations and in nurseries (Teixeira de Sousa, Guillaumin, Sharples &
Whalley, 1995; López-Herrera, Pérez-Jiménez, Zea-Bonilla, Basallote-Ureba &
Melero-Vara, 1998).
Recently, two extensive reviews have been published on the biology and
possible control strategies available against the white root rot (Ten Hoopen &
Krauss, 2006; Pérez-Jiménez, 2006). The present chapter will focus on the
management of the disease in intensive agricultural systems, typical of modern fruit
tree orchards.
2. TAXONOMY
The genus Rosellinia was first erected by De Notaris in 1844 and, according to the
9th edition of the Dictionary of the Fungi, is now classified as follows: Kingdom
Fungi, Phylum Ascomycota, Class Sordariomycetes, Subclass Xylariomycetidae,
Order Xylariales, Family Xylariaceae, Genus Rosellinia De Not. The genus
comprises more that 90 different taxa (Petrini & Petrini, 2005) which include well-
known root pathogens like R. necatrix Prill., R. desmazieresii (Berk. et Br.) Sacc.
(syn. R. quercina Hart.), mostly known from temperate zones, R. bunodes (Berk. et
Br.) Sacc., R. pepo Pat. and R. arcuata Petch, known only from the tropics (Ten
Hoopen & Krauss, 2006).
According to Petrini (1993) the earliest description of the anamorph D. necatrix,
known also as Rhizomorpha necatrix, was by Saccardo which erroneously assigned
the teleomorph to Rosellinia desmazieresii (Berk. & Broome) Sacc. Based on the
analysis of conidial morphology, Hartig (1883) speculated that the teleomorph of D.
necatrix might belong in Rosellinia or a closely related genus. Berlese (1892)
reinforced Hartig’s speculations but did not formally describe the teleomorph.
Prillieux (1902) originally published the name as R. necatrix without any other
author listed, but in 1904 he used the author attribution (R. Hart.) Berlese (Farr,
Rossman, Palm & McCray, 2006).
3. HOST RANGE AND GEOGRAPHIC DISTRIBUTION

Rosellinia necatrix causes severe root rots on several economically important crops.
Among the most susceptible hosts there are temperate fruit crops such as apple, pear,
plum, almond, peach, cherry, olive and grapevine (Sztejenberg & Madar, 1980;
Petrini, 1993; Holevas, Chitzanidis, Pappas, Tzamos, Elena, Psallidas et al., 2000).
Host plants include also subtropical species like avocado and mango, and tropical
species like coffee and tea (Saccas, 1956; Khan, 1959; Sivanesan & Holliday, 1972).
However, R. necatrix host range is much wider, including forest trees and
shrubs, field crops, weeds and algae belonging to 170 species, including 63 genera
and 30 families (Khan, 1959). A list of 109 fungus-host combinations with specific
ROOT ROT MANAGEMENT 139
references is reported by the U.S. National Fungus Collections (BPI) (Farr et al.,
2006). Sztejnberg and Madar (1980) found that the fungus attacked and killed
artificially inoculated deciduous trees (apple, pear, plum, almond), olive trees, citrus
rootstocks, grape rootstocks, avocado, mango, macadamia, field crops (cotton,
alfalfa, bean) and weeds (Prosopis farcta and Amaranthus gracilis). The same
authors suggested that weeds like P. farcta, A. gracilis and Conyza bonariensis
could promote the disease spreading. Lupinus luteus was proposed as a very
sensitive reference species to study the pathogen virulence (Uetake, Nakamura,
Arakawa, Okabe & Matsumoto, 2001).
Data about the centre of origin of this pathogen are very limited. Mantell and
Wheeler (1973) supposed that R. necatrix was introduced in the Scilly Isles
(Southern UK) at the beginning of last century on exotic ornamentals brought from
tropical or subtropical countries. In any case R. necatrix is now widely distributed in
five continents in temperate, subtropical and tropical zones (Saccas, 1956; Sivanesan
& Holliday, 1972; Anonymous, 1987; Petrini, 1993; Farr et al., 2006). The pathogen
is a limiting factor in apple and vineyard orchards in France, Portugal and many
other European countries (Guillaumin, 1986; Teixeira de Sousa et al., 1995).
Teixeira de Sousa et al. (1995) reported that in Alcobaça region (Portugal) 42% of
the orchards were infected by R. necatrix and 14% of the apples trees exhibited
advanced disease symptoms. In Italy the fungus represents one of the most
dangerous pathogens of root-rot in poplar (Anselmi & Giorcelli, 1990a) and is
widely diffused on fruit trees like sweet cherry, almond, peach, olive and grapevine
(Cellerino, Anselmi & Giorcelli, 1988; Amenduni et al., 2001; Schena & Ippolito,
2003).
In Southern Spain and in Israel the white root rot is one of the most important
diseases of apple and avocado crops, causing extensive wilting and tree death. Since
the first report in avocado orchards in Southern Spain in 1989, the incidence of the
pathogen has progressively increased and is now considered the most important
cause of endemic avocado root rot (López-Herrera et al., 1998). The fungus is also
responsible for important economic losses on tiger nut (Cyperus esculentus L.)
(García-Jiménez, Busto, Vicent & Armengol, 2004). The pathogen has been cited as
damaging coffee orchards in Africa (Saccas, 1956) and is widely diffused in
Southern America (Denardi & Bretón, 1995). In North America, the disease seems
to be less extensive, only causing significant losses in apple orchards in California,
although other plants, especially fruit trees, have been found to be infected by the
fungus (Thomas, Wilhelm & MaClean, 1953; Lee, Ko & Aldwinckle, 2000; Farr
et al., 2006).
In Asiatic countries, R. necatrix has been isolated in Japan on tea plants since
the middle of the last century (Abe & Kono, 1953) and is considered one of the most
serious pathogens of fruit trees such as grapevine (Kanadani, Date & Nasu, 1998),
Japanese pear and apple (Arakawa, Nakamura, Uetake & Matsumoto, 2002). In Iran
white root rot occurs frequently and has economic importance in nurseries and
orchards (Behdad, 1976). Similarly, in some regions in India it is considered the
most destructive disease of apple trees (Gupta, 1977). Recently R. necatrix has been
considered as a potential threat for Thailand (Thienhirun & Whalley, 2001).
4. SYMPTOMS
Rosellinia necatrix is a soil borne pathogen responsible for root and collar rots. As a
consequence of roots damage, affected trees show general declining symptoms
which are not distinguishable from those of other root rot pathogens such as A.
mellea and Phytophthora spp. Depending on environmental conditions and tree
species the disease can lead to a rapid decline of plants which quickly die (apoplexy)
or to a progressive weakening of trees, which can remain alive for several years.
Apoplexy is very common for seedlings in the nurseries and for new plantations in
irrigated orchards. Jung trees can die in few days after the appearance of the first
symptoms; leaves wilt, dry and remain attached to the tree for months. These
symptoms usually occur after a period of water or physiologic stress. In Southern
Italy, sweet cherry trees grafted on Prunus mahaleb L., commonly die after
vegetative flushing in spring or at the beginning of summer, as soon as temperatures
increase.
In the case of progressive weakening, the trees develop a generally unthrifty
appearance. Fruits are small and shrivelled, whereas leaves show incurved margin,
change of colour (yellowing and/or reddening), reduced size and premature fall. In
diseased trees there is absence of new shoots and root growth. Infected trees will
eventually die but can remain alive for several years. Infected olive trees can die
quite quickly. However, young and, in particular, adult trees usually remain alive for
several years. These symptoms worsen every year and when moisture and
temperature are unfavourable for growth, the tree eventually dies (Guillaumin,
Mercier & Dubos, 1982).
Figure 1. Soil pot artificially inoculated with Rosellinia necatrix and planted with Prunus
mahaleb. After 3 weeks, the root system was widely colonised by the pathogen and the plant
died; however, decayed rootlets were still visible since not completely destroyed.
Disease symptoms on small roots can be observed only during the early phases
of infection before rootlets are destroyed by the pathogen (Fig. 1). Infected rootlets
appear rotted and covered by a subtle layer of white mycelium. In advanced
infection phases, diseased trees are easily uprooted since all small and medium roots
are destroyed and the root system is very limited (Fig. 2A, B).
Infected roots are commonly covered by a white cottony mycelium and mycelia
strands coloured either white or black which also extend under the bark and into the
surrounding soil (Fig. 2A, B).
Figure 2. Advanced symptoms of white root rot on a 4 year-old peach tree (A) and detail of
a large almond root infected by Rosellinia necatrix (B). Small and medium size roots are
destroyed whereas large roots are covered by white cottony mycelium and by white or
black mycelia strands.
At the trunk base and on large roots the fungus colonises bark and external
wood parts. When environmental conditions are favourable, the white mycelium
may be visible on the root crown at ground level. In dry conditions the mycelium of
the pathogen is less evident, however, pieces of infected roots or of young plants
transferred to damp chambers, rapidly develop the characteristic white mycelium
and, eventually, sheets of microsclerotia (Fig. 3A). Rotted bark appears sunken and
dark-brown; a distinct margin is usually visible between the infected and healthy
bark. The most typical symptoms are white mycelial fans, which develop between
the epidermis and the bark on the crown and on large roots of dying trees by the end
of the infection (Fig. 3B).
Figure 3. Specific symptoms of white root rot on the crown of a 2-year old almond plant kept
for one week in a damp chamber. The white cottony mycelium is visible externally (A)
whereas typical white mycelial fans can be seen between the epidermis and bark (B).
On infected tissues at the base of dead plants and on roots maintained in damp
chamber, the pathogen quite commonly forms brown mycelial masses and sclerotia,
from which synnemata bearing conidia develop (Fig. 4 A-E). Synnemata arise as
tufts of several elements with a common base (Nakamura, Uetake, Arakawa, Okabe
& Matsumoto, 2000).
Sexual reproductive structures of R. necatrix have rarely been found. They
consist of single or linked stromata arising from a dark brown and felty subiculum,
containing a single perithecium with asci and paraphyses (Nakamura et al., 2000;
Pérez-Jiménez, Zea-Bonilla, & López-Herrera, 2003).
Figure 4. Agamic reproductive structures of Rosellinia necatrix. Synnemata arising from the
crown of a Prunus mahaleb plantlet (A), close-up of a group of synnemata (B), single
synnemata apex (C), close-up of a single synnemata apex (D) and conidia (E).
5. DISEASE CYCLE AND EPIDEMIOLOGY
5.1. Survival
Rossellinia necatrix is a soil-borne pathogen which can survive as mycelium for a

considerable long time, on its numerous woody and herbaceous hosts. Apart from
commercially relevant crops, there are a number of weeds that can host the pathogen
(Sztejnberg & Madar, 1980) and promote its spread in standing fruit trees as well as
its survival, after the uprooting of infected orchards. The weed Cyperus rotundus L.
was found to facilitate the pathogen survival and dissemination in Taiwan
plantations (Duan, Tsai & Tu, 1990). Furthermore, R. necatrix can survive in the soil
as saprophyte on dead roots and other plant debris, although fresh vegetable debris
rich in cellulose are required for the fungus to remain alive. In laboratory conditions
the pathogen survived on pear branches for a period of 18 months and died when the
cellulose content decreases to 50% of the original (Araki, 1967). However, quite
contrasting results were obtained in a similar study using apple branches, since the
fungus remained viable for 8 years (Thomas, Wilhelm & MaClean, 1953). Duan et al.
(1990) found that R. necatrix can survive in loquat (Eriobotrya japonica Lindl.)
infected roots and on the surface and inside the soil for 4 months and 3 years,
respectively. It has been also supposed that R. necatrix can survive in the soil as
microsclerotia produced in high quantities both in nature and in vitro (Sztejnberg,
Madar & Chet, 1980). However, data about microsclerotia survival are very limited.
Duan et al. (1990) suggested that microsclerotia have a minor role in the survival of
the pathogen since they are devitalised in approximately 1 month in soil and in no
more than 2 weeks under dry air conditions. Rossellinia necatrix can also produce
chlamydospores starting from pyriform swellings (Fig. 5) in which protoplasm is
condensed and a wall is formed that divides the spherical zone from the remaining
hypha (Khan, 1959). However, their role as resting organs is very limited, since they
are found only under exceptional environmental conditions and rarely in natural
conditions (Teixeira de Sousa & Whalley, 1991).
Figure 5. Hyphae of Rosellinia necatrix with typical pear-shaped swelling at septa.
5.2. Dispersal
Long distance dispersal of R. necatrix mainly occurs with infected propagating
materials, which frequently are asymptomatic during early phases of the infection
process. Furthermore, infested soils and infected plant debris can be distributed by
cultural practices or by water. Anselmi and Giorcelli (1990b) demonstrated that R.
necatrix can be diffused by river and irrigation water, since the pathogen can remain
alive for quite long time on poplar cuttings in running and standing water. Once in
the field, latent infections can became evident and the pathogen can diffuse to
surrounding healthy trees. Pathogen diffusion in the soil may occur through direct
root contact between host plants and by diffuse mycelium or by mycelial strands
which grow through soil cavities from infected plants to healthy ones. Due to this
mechanism of diffusion, Rosellinia root rots are often characterised by their
occurrence in patches, that extend in a circular pattern.
Several factors can influence the diffusion of R. necatrix in soil (Anselmi &
Giorcelli, 1990a). The pathogen spreads particularly easily in loose soil
characterised by high sand content and average quantity of water, being soil at field
capacity the best for fungal growth. The growth was insignificant at the maximum
water capacity of soil and decreased rapidly with decreasing moisture content, being
zero at wilting point. The mycelium grows well between 22.5 and 25.5°C, being
24°C the optimal temperature and its growth is higher in the dark, since the light has
a strong inhibitory effect. A minor role is exerted by the soil pH, since the pathogen
can grow well at a pH from 5 to 8 and continues to develop even at pH 4 and 9.
Using cuttings of sweet cherry branches we found that the growth of R. nectraix
in soil is not random but it is directed toward its specific host (Fig. 6). However,
how far the mycelium can grow through soil to reach specific hosts is not clear.
Mantell and Wheeler (1973) suggested that Rosellinia can grow in the soil from a
source of inoculum, but it is not able to survive for a long time without a food
source. They found that the mycelium grew sparsely in the fresh soil at first,
extending to about 10 mm after 5 days. However, there was no further growth over
the next 7 days and after 27 days the mycelium had disappeared. In this context,
several weeds commonly present in orchards can contribute to the diffusion and
survival of the pathogen.
Figure 6. Mycelial strands of Rosellinia necatrix growing from an artificially infected

sweet cherry cutting (black arrow) towards surrounding non-infected cuttings (grey
arrows). Scale bar: 1 cm.
Sexual and asexual spores (Fig. 4) have been historically considered not
important for the conservation and dissemination of the pathogen. Rosellinia
necatrix rarely produces ascocarps in nature and special techniques are required over
long times in order to induce their production experimentally (Teixeira de Sousa &
Whalley, 1991). Furthermore, ascospores of R. necatrix are hard to germinate
(Hansen, Thomas & Thomas, 1937; Nakamura et al., 2000). Similarly the ability of
conidia to germinate was doubtful (Khan, 1959) or differed from sample to sample
(Nakamura et al., 2000). However, Pérez-Jiménez et al. (2003a) found ascocarps of
R. necatrix in commercial avocado orchards in Southern Spain and speculated that
they may have important implications in the epidemiology of the disease. They
observed that the high pathogenic population diversity found in avocado crops is not
easy to explain, unless the occurrence of recombination is considered. Teixeira de
Sousa and Whalley (1991) suggested that the lack of ascocarps in nature results from
a number of physical and nutritional interactions, among which the lack of water has
a predominant role. Accordingly, continues summer irrigations were necessary to
stimulate the production of coremia-bearing conidiophores (Teixeira de Sousa &
Whalley, 1991). Therefore, it is possible to speculate about a new role of sexual and
asexual spores in the dispersal and variation of this species, in modern irrigated
orchards.
5.3. Infection Process
In the present paragraph a synthetic description of the infection process is reported;

for a more detailed description readers can refer to the review by Pérez-Jiménez
(2006). When the fungal mycelia make contact with the root surface a condensation
of the hyphae occurs. On young tissues, penetration occurs with the formation of
mycelial aggregates, defined as “cone of penetration”, which penetrates deeply into
the cortical parenchyma, without destroying cellular walls. On adult tissues, with
secondary growth, the penetration occurs by the formation of a “sclerotium of
penetration”, which has an external location and is in connection with the host
surface. This mycelium penetrates root tissues, destroying the suberized layers and
afterwards invades the phelloderm (Tourvieille de Labrouhe, 1982).
In addition to the mechanical action of the fungal aggregate organs, enzymatic
and toxigenic activities also play an important role during the infection process.
Rosellinia necatrix possesses high activity of cellulolitic enzymes, but low activity
of pectic enzymes (Araki, 1967; Sztejnberg, Azaizia & Lisker, 1989; Melo & Ferraz,
1990). Furthermore, it has been shown that it produces different metabolites with
phytotoxic effects (Abe & Kono, 1957). Sawai et al. (1982) isolated cytochalasin E,
which has been directly correlated with pathogen virulence (Tourvieille de
Labrouhe, 1986). More recently, new metabolites produced by R. necatrix have been
isolated, such as rosellichalasin (Kimura et al., 1989), diketopiperazines, rosellinic
acid and rosnecatrone (Edwards et al., 2001) and it has been demonstrated that
citochalasin E has a direct effect on photosynthesis (Kshirsagar et al., 2001).
6. CONTROL
Control of the white root rot is complicated by its ubiquitarian presence in the soil
and by a number of specific characteristics including: resistance to drought,
tolerance to a wide range of soil pH, high number of hosts, penetration deep into the
soil and resistance to various common fungicides (Khan, 1959). In general, soil
treatments are very expensive, are characterised by high environmental impact and
their efficacy is often not resolutive. Therefore, control of white root rot, like several
other diseases caused by soilborne pathogens, should be mainly based on the

avoidance of the pathogen through the use of certified healthy propagative materials
and new plantings in non-infested soils.
However, an important distinction needs to be made between nurseries and field
orchards. In the first instance vigorous control strategies like fumigation and steam
soil disinfection are economically arguable and necessary to avoid the production of
infected plantlets, which can be responsible of distant and large scale diffusion of the
pathogen. The entry of the fungus into nurseries with infested material (plantlets,
soils, tools, organic manure, etc.) has to be avoided and continuously checked
through specific tests on plant roots and soils.
When the pathogen is accidentally introduced, it must be eradicated. Control
strategies in the fields should be based on the avoidance of the pathogen through the
utilization of certified healthy propagating materials; however, once introduced in
the field control strategies should be directed to the management of the disease
rather than to the pathogen eradication, which is very difficult and too expensive to
be economically justifiable. In this context cultural control methods play a major
role.
6.1. Healthy Propagative Materials
6.1.1. Current Legislation in Europe

Based on the consideration that the production of healthy, high-quality fruits
depends to a large extent on the standard of the material used for fruit plant
cultivation, specific directives have been adopted by the European Union. The
basic Directive (Council Directive 92/34/EEC of April 28, 1992) established a
harmonised Community regime which ensures that growers throughout the EU
receive propagating material and fruit plants which are healthy and of good
quality. This stipulates that fruit plant propagating material, which are deemed to
be of major economic importance, may be marketed if they are either CAC
(Conformitas Agraria Communitatis), pre-basic, basic or certified material
(http://ec.europa.eu/food/plant/propagation/fruit/index_en.htm). To be classified as
such, the material must comply with the criteria for quality, plant health, testing
methods and procedures, propagation systems and varietal aspects laid down in
technical schedules and must have been recognised as satisfying these conditions
following official inspection. These schedules are laid down in Commission
Directive 93/48/EEC of June 23, 1993. Propagating material and fruit plants from
countries outside of the EU may only be marketed within the Community if they
offer the same guarantees as material produced in the EU, complying with Council
Directive 92/34/EEC. A complete list of the directives on this matter is consultable
on the web (http://ec.europa.eu/food/plant/propagation/fruit/index_en.htm).
More recently, the European and Mediterranean Plant Protection Organization
(EPPO) developed a number of schemes to promote the production of healthy plants
for planting across Europe (http://www.eppo.org/). In a typical EPPO certification
scheme, the certified material is descended by a fixed number of steps only, from
individual plants each tested and found free from pests. The certified material is then
maintained and propagated under rigorous conditions, excluding re-contamination.
Specific standards are available for the most important fruit tree species and
focus on diseases caused by mycoplasmas, virus and virus-like organisms
(http://archives.eppo.org/EPPOStandards/certification.htm). A reconsideration of
EPPO standards to include major plant pathogens among bacteria, stramenopiles,
fungi and nematodes which are mainly diffused trough propagating material is
probably advisable.
6.1.2. Diagnostic Tools

To ensure the health status of propagating materials and to facilitate reliable and
effective controls, the European Directives have encouraged the development of
detection methods suitable for large-scale analyses. Conventional methods to detect
R. necatrix in infected host tissues are accurate but not appropriate for large scale
testing. They involve detailed observations of the symptoms, damp chambers,
isolation of the pathogen on culture media (potato dextrose agar or malt agar)
containing one or more antibiotics (commonly streptomycin sulphate) and
microscopic observation of pathogen mycelia. The pathogen identification is
possible through light microscopy observations of the typical pear-shaped swellings
(Fig. 5). These procedures require skilled expertise to identify the pathogen (Petrini,
1993) and are laborious and time consuming (1-3 weeks).
A conventional baiting method based on the use of avocado leaf discs or
mulberry twigs has also been proposed to detect the pathogen from soil (Sztejnberg,
Freeman, Chet & Katan, 1987; Arakawa et al., 2002). However, this method is time
consuming (Freeman, Sztejnberg & Chet, 1986) and, based on experiments
performed in Southern Spain and Southern Italy, it displays a low sensitivity.
Recently, new molecular detection methods based on real-time PCR have been
developed and applied to a number of different hosts and matrices. A first method
was developed by Schena, Nigro and Ippolito (2002) who detected R. necatrix from
artificially inoculated soils. Subsequently, Schena and Ippolito (2003) proposed an
improved method which enabled the detection of the pathogen from several different
hosts and soils which were either artificially or naturally infected. This method,
which provided both qualitative and quantitative data, was further improved by
optimising the extraction protocols and was utilised for the analysis of a number of
naturally infected samples (avocado soil and roots) from Southern Spain (Ruano
Rosa, Schena, Ippolito & López-Herrera, 2007).
The availability of molecular detection methods opens new opportunities for the
control of the white root rot, since they can be used to exclude the presence of the
pathogen in soil or host tissues, certifying the health status of propagative materials.
Unlike conventional methods, they do not require accurate knowledge of the
pathogen and being rapid, effective and reliable are suitable for large scale analyses
by extension services (Schena, Nigro, Ippolito & Gallitelli, 2004; Schena, Hughes &
Cooke, 2006).
6.2. Cultural Control Methods

In the nurseries, as well as in open field, the first important step for an effective
control of the white root rot is to evaluate the health status of propagating materials
and soil as well, before new plantations. In this context the new molecular tools (see
paragraph 6.1.2) enable accurate analyses, avoiding unnecessary and costly
treatments. Once the pathogen is in the field it is necessary to uproot infected plants
and remove as much as possible contaminated roots and other plant debris from all
sites where the fungus was previously present. Infected plant debris must be burnt or
exposed to light and air to accelerate pathogen devitalisation. When economically
practicable, i.e. in the nurseries, infected soils can be treated by steam or with
chemicals (see paragraphs 6.3 and 6.6). The most advisable strategy in the field is to
wait 2-4 years before planting new orchards. Considering the debilitating action of
air and light on the mycelium and the need of plant residues for the pathogen
survival, this period should be enough to prevent or reduce new infections (Anselmi
& Giorcelli, 1990). Meantime, infested soils should be cultivated with non-host
species (cereals are commonly suggested as non-host species) or left uncultivated,
paying attention to the control of weeds that could favour the survival of the
pathogen. However, the suggested period (2-4 years) is only indicative, since current
knowledge about survival of the pathogen in soil without specific hosts is quite
limited, and available data are often contrasting (see paragraph 5.1).
Once a soil is infested by R. necatrix, the use of resistant varieties or species for
new plantations is strongly advisable, but it is very difficult to achieve due to the
very broad spectrum of hosts (see paragraph 3). As an example, in Southern Italy
some of the most important fruit crops which include table and wine grapes, olive,
almond, sweet cherries and peach are all very sensitive to the pathogen.
Furthermore, even when moderate levels of resistances are available, their utilization
is complicated by specific local vocations for one or few species. A moderate level
of resistance is known for Passiflora edulis Sims., and a number of rootstocks of
mango, grape, and citrus, killed by R. necatrix in greenhouse conditions, are able to
survive in the fields with infected soils, conditions which were prohibitive for a
number of other species (Sztejnberg & Madar, 1980). In the same situations,
persimmon and pecan trees did not show any disease symptom for more than four
years, due to high phenolic compounds within the root tissues (Sztejnberg, Azaizia,
& Chet, 1983).
Teixeira de Sousa (1985) analysed the sensitivity of a number of possible hosts
and grouped ligneous species as sensible, moderately sensible, moderately resistant,
and resistant. Among moderately resistant species there are important rootstocks like
Prunus marianna and P. myrabolan, whereas Diospyros lotus and D. virginiana
were classified as resistant. Interesting selection programmes are currently in
progress to identify Malus tolerant germplasm. Preliminary studies have shown
resistance to R. necatrix in some wild apple species (Johnson, 2000) and 5 clones,
out of 177, exhibited consistent resistance to R. necatrix among Malus germplasm
from Korea, Japan and the United States (Lee et al., 2000). Similar studies were also
carried out to identify resistant Malus germplasm in Brazil (Denardi & Bretón,
1995). In the avocado producing area of Spain, a tolerant avocado rootstock
selection programme is also being developed (Pérez-Jiménez, Zea-Bonilla, Imbroda-

Solano, López-Herrera & Barceló-Muñoz, 2003).
After planting, irrigation and soil management practices (tilling, mechanical and
chemical weed removing, cover crop, etc.) should be focused to avoid stresses due to
lack or excess of water (waterlogging). Drip irrigation is commonly preferable to
other irrigation methods. If new attacks occur, infected trees should be removed
immediately and diseased plants isolated. A detailed description of cultural methods
to isolate infected trees is reported by Ten Hoopen and Krauss (2006). However,
most of them appear anachronistic, requiring too much labour and then being
economically unacceptable in the short-medium term. Similarly, soil composition
and organic matter content can significantly influence the incidence of the white root
rot (Araki, 1967; Ten Hoopen et al., 2002). However, their modification to control
the disease seems to be unreliable, from a practical point of view.
6.3. Fumigation
In the past four decades, the eradication of R. necatrix and other soilborne pathogens
from infested soils was based on fumigations with methyl bromide. The pathogen
was efficiently controlled, for a period of at least 9 months, by applying methyl
bromide either by deep injection (up to 90 cm) of cold gas or by surface application
of hot gas at a dosage of 1500 and 1000 kg/ha, respectively (Sztejnberg et al., 1983).
Methyl bromide has been the fumigant of choice for many pre-plant soil applications
because of its broad spectrum of activity and its high vapor pressure facilitating
distribution through the soil profile, cost-effectiveness and comparatively short
plant-back intervals (Martin, 2003). However, methyl bromide is now classified as a
class 1 stratospheric ozone-depleting substance and according to the Montreal
Protocol (1997) its use in agriculture has been phased out by 2005 in industrialised
countries and would be completely phase out by 2015 in developing countries.
The substitution of methyl bromide poses a series of difficulties, especially in
the nurseries, and increases the risk of wide diffusion of R. necatrix through
propagating materials (Gullino, Camponogara, Gasparrini, Rizzo, Clini & Garibaldi,
2003). Many alternatives to replace methyl bromide have been proposed. Among
these, methyl-isothiocyanate and its generators (metham sodium and dazomet) are
often considered as the most suitable short-term solutions (Gullino et al., 2003;
Ruzo, 2006). However, they do not always provide complete disease control
(Minuto, Gilardi, Pomè, Garibaldi, & Gullino, 2000). Dazomet (marketed as
Basamid Granular®) provided good control against R. necatrix for periods up to two
years, when 100 g/m2 of a.i. were incorporated evenly in the top 50 cm of the soil
layer, followed by sprinkling with water (100 l/m3) and covering the soil with PVC
film for one month (Nitta, Hatamoto & Kurihisa, 2002). Three months were
necessary after removing the PVC film to enable complete release of all chemical
residues. In another study, metham-sodium (Vapam 4S®) and formaldehyde were the
most effective compounds in controlling the growth of R. necatrix in soil already
colonised by the pathogen (Mantell & Wheeler, 1973). Formaldehyde also favoured
the development of Trichoderma, a genus with significant biocontrol potential.
Chloropicrin showed high in vitro and in vivo activity against R. necatrix (Matuo &
Sakurai, 1959; Kubomura, Ieki & Itoi, 1970). In fine-textured and medium textured
soils, tilling was not necessary for good control, while on fine-textured soil it was
necessary to break up soil before fumigation to increase the amount of soil pores
which are not blocked by water, for effective soil fumigation (Kubomura et al.,
1970). More recently, preplant applications of chloropicrin by shank injection
proved to be a viable alternative to methyl bromide being effective against different
soilborne pathogens (Gullino et al., 2003). Other fumigants which have been
considered as possible replacements for methyl bromide include 1,3-dicloropropene
and other compounds whose registration process is still in progress, like methyl
iodine and sodium azide (Gullino et al., 2003; Ruzo, 2006).

Apart from fumigants, several chemicals have shown high in vitro efficacy against
R. necatrix and among these carbendazim (Gupta, 1977), benomyl and thiabendazol
(Behdad, 1976) stand out for their inhibitory effect. Field trials to control R. necatrix
are more limited and provided more variable results. Benomyl and thiophanate-
methyl were highly effective in controlling the disease in apple trees when they were
used in winter rather than in spring, and also when they were used as a preventive
treatment on healthy trees close to diseased loquat trees (Teixeira de Sousa, 1985).
Two field applications of carbendazim as a soil drench on 15–20 years old apples
infected by R. necatrix resulted in the recovery of all treated trees.
However, benzimidazoles are no longer used because they were banned in many
countries. A drench technique was also the best for the delivery of tridemorph to
mulberry trees in Japan and replaced the expensive painting of exposed roots
(Mappes & Hiepko, 1984). A detailed list of chemicals including antibiotics tested
against R. necatrix is reported by Ten Hoopen and Krauss (2006). However, most of
them have not been utilised in field trials or provided inconsistent levels of
protection or, finally, are not practically relevant, since their use is not permitted by
national specific legislations.
Worth of mentioning is the fungicide fluazinam which is applied to the soil
around individual diseased trees by drenching and is currently utilised in Japanese
orchards (Kanadani et al., 1998).
6.5. Physical Control

Steam soil disinfection has been proposed as a valid control strategy to devitalise a
number of soil borne pathogens in light of new technologies which may significantly
reduce the costs of applications (Runia, 2000). Although, no specific trials are
available about their efficacy against R. necatrix, the high sensitivity of the pathogen
to high temperatures (Sztejenber et al., 1987; Sharma & Sharma, 2002; García-
Jiménez et al., 2004) and the broad spectrum of activity of steam are encouraging.
Main constraints are the limited applicability on small surfaces (e.g. seedbeds), the
high costs and the high initial investment and energy consumption.
The use of a hot-water for treatment of Cyperus esculentus L. tubers was
studied as a control strategy to prevent the spread of R. necatrix through infected
tubers (García-Jiménez et al., 2004). The pathogen was completely devitalised in

tuber treated at 55°C for 10, 20 or 30 min and tubers did not show any reduction in
sprouting. In the field trials, hot-water treatments at 53°C for 25 min or 55°C for 25
min provided good control of the disease and did not affect normal plant
development and yield.
Soil solarization has been largely exploited all over the world wherever the
climatic conditions are favourable and proved to be effective and reliable against a
number of soil pests including R. necatrix. In Israel, soil solarization was found to be
very effective in controlling white root rot disease in established apple orchards and
in reducing the population or activity of R. necatrix in soil to the dept of at least 60
cm (Sztejnberg et al., 1987).
Soil solarization fulfilled the requirements for successful control of a soilborne
pathogen in existing orchard since: i) the trees were not damaged, ii) the inoculum
was controlled to a considerable depth, iii) soil reinfestation was delayed and iv) the
inoculum was also reduced in the shaded area. These Authors suggested that the
successful control of R. necatrix by solarization might be attributed to the extreme
sensitivity of the pathogen to high temperatures and to a variety of physical,
chemical and biological mechanisms activated by solarization.
Freeman et al. (1990) suggested that, in addition to the direct thermal
inactivation of R. necatrix, there are other mechanisms which contribute to the
pathogen control, i. e. the accumulation of volatile substances under the plastic film
and the activation of soil microorganisms.
Pathogens weakened by sub-lethal temperatures are more sensitive to
antagonists which can proliferate, contributing to induce suppressiveness to treated
soils and lasting reinfestation. Analysing solarised soils, Sharma and Sharma
(2002b) found an increased population of thermotolerant antagonistic
microorganisms, although, total microbial population including fungi, bacteria and
actinomycetes decreased. Furthermore, chemical characteristics of soil such as
available nitrogen, potassium, organic carbon contents, electrical conductivity and
pH increased after solarization, whereas the phosphorous content decreased.
Solarization also eliminated the weed population which could contribute to survival
and dispersal of the pathogen.
In established avocado orchards in Southern Spain temperature increases
attributable to soil solarization ranged between 4 and 8°C in unshaded areas,
whereas for shaded areas they were approximately 4°C (López-Herrera, Pérez-
Jiménez, Basallote-Ureba, Zea-Bonilla & Melero-Vara, 1999). Four to 8 weeks of
solarization were required for the elimination of the pathogen in root samples buried
at depths of 45 to 60 cm, and the pathogen was not recovered for at least 9 months
after solarization. More recently, the same Authors confirmed a direct correlation
among efficacy, timing of application and environmental conditions (López-Herrera
et al., 1999). One, 4, 5 and 6 weeks were required to devitalise R. necatrix at 15, 30,
45 and 60 cm depths starting solarization in early June, whereas, only 8, 10, 15 and
22 days of solarization were needed to achieve the same results at the same depths in
a second experiment starting by mid-July.
6.6. Biological Control

A number of biocontrol strategies have been investigated to control R. necatrix. A
dsRNA mycoreovirus (RnMYRV-3/W370) member of the newly established genus
Mycoreovirus, within the family Reoviridae, has been identified as the
hypovirulence factor in Rosellinia necatrix (Kanematsu, Arakawa, Oikawa, Onoue,
Osaki, Nakamura et al., 2004; Sasaki, Kanematsu, Onoue, Oikawa, Nakamura &
Yoshida, 2007) and the possible use of this and other mycoviruses has been
suggested to attenuate fungal virulence and to control the disease (Matsumoto, 1998;
Matsumoto et al., 2002). Inoculum containing an effective dsRNA is placed in
contact with the mycelium of the pathogen, so that the dsRNA may transfect the
pathogen. Such dsRNAs, replicating in the cytoplasm and organelles of fungal
hyphae, are thought to spread through the network of fungal mycelia. As opposed,
Arakawa et al. (2002) revealed that all isolates originating from ascospores were
negative and speculated that the teleomorph functions as a mechanism to eliminate
dsRNA in R. necatrix, as in other fungi.
Among fungal genera investigated as biocontrol agents against R. necatrix, the
genus Trichoderma received high attention. Trichoderma harzianum and T.
hamatum were assayed at different dosages of inocula in soils of apple orchards,
which were artificially or naturally infested by R. necatrix (Freeman et al., 1986).
These Authors concluded that the antagonistic control mechanism could either rely
on nutrient competence or mycoparasitic behaviour of the fungus. Mendoza, Ten
Hoopen, Kass, Sánchez and Krauss (2003) showed that mixtures of Clonostachys
and Trichoderma can be effective against Rosellinia bunodes in the greenhouse.
However, the effectiveness depended on soil pH and the amount of soil organic
matter. In another study, antagonistic effect of T. harzianum against R. necatrix was
inconsistent in field conditions and efficacy was influenced by a number of factors
including soil type, natural inoculum levels of the pathogen and sensitivity to
different temperatures (Sztejnberg et al., 1987). Isolates of the fungus Sordaria spp.,
investigated as biocontrol agents of R. necatrix, were able to increase seed
germination of different plant species (Watanabe, 1991). Finally, green amendments
and/or vesicular-arbuscular (VAM) mycorrhizal fungi of the genus Glomus were
able to reduce the incidence of the disease (Bhardwaj, Nag & Sharma, 2000).
Several bacterial species isolated from soil and rhizosphere have also been
investigated as biocontrol agents against R. necatrix (Cazorla-López, Bloemberg &
Lugtenberg, 2001; González-Sánchez, Cazorla, Ramos, De Vicente & Pérez-
Jiménez, 2004). Species of Agrobacterium and Pseudomonas showed a strong
antagonistic effect against R. necatrix and were able to colonize and survive on tree
roots (Yasuda & Katoh, 1989). Recently, a collection of 905 bacterial isolates from
the rhizospheres of healthy avocado trees was screened for antagonistic activity
against R. necatrix (Cazorla, Duckett, Bergström, Noreen, Odijk, Lugtenberg et al.,
2006). A set of eight strains belonging to the species Pseudomonas chlororaphis,
Pseudomonas fluorescens and Pseudomonas putida showed high inhibitory activity
against R. necatrix and, among these, P. fluorescens PCL1606 exhibited the highest
biocontrol efficacy in an avocado-R. necatrix test system. Biocontrol activity was
directly correlated to a new antifungal antibiotic which was identified as 2-hexyl
5-propyl resorcinol (HPR). Other bacterial metabolites with strong in vitro

inhibition against R. necatrix were obtained from Janthinobacterium lividum
(Shirata, Tsukamoto, Yasui, Hata, Hayasaka, Kojima & Kato, 2000), Bacillus
amyloliquefaciens (Yoshida, Hiradate, Tsukamoto, Hatakeda & Shirata, 2001) and
the cyanobacterium Nostoc, strain ATCC 53789 (Biondi, Piccardi, Margheri,
Rodolfi, Smith & Tredici, 2004). Furthermore, compounds which have shown
antimicrobial activity against R. necatrix include calixarenes, i.e. cavity-shaped
cyclic molecules made up of phenol units linked via the ortho positions by
methylene bridges (Lamartine, Tsukada, Wilson & Shirata, 2002) and products from
herbs fermentation by lactic acid bacteria (Kuwaki, Ohhira, Takahata, Hirota,
Murata & Mikiro, 2004).
In spite of the quite large number of reports about effective biocontrol agents
and/or natural compounds against R. necatrix, no practical examples of biocontrol
strategies are currently utilized to control R. necatrix in commercial conditions.
Most of the biocontrol agents and/or compounds have only been tested in vitro or in
confined greenhouse experiments, and verification of their potentials under field
conditions still remains to be done (Sztejnberg et al., 1987).
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Section 2
Diseases of Annual Crops

8
G. A. FORBES1, W. E. FRY2, J. L. ANDRADE-PIEDRA3
AND D. SHTIENBERG 4
SIMULATION MODELS FOR POTATO LATE BLIGHT

MANAGEMENT AND ECOLOGY
1
International Potato Center, Apartado 1558, Lima 12, Peru
2
College of Agriculture and Life Sciences Cornell University,
Ithaca NY 14853 USA
3
Papa Andina Initiative - International Potato Center (CIP),
Apartado 17 21 1977, Quito, Ecuador
and
4
Department of Plant Pathology and Weed Science, ARO, The Volcani Center,
Bet Dagan 50250, Israel
Abstract. Late blight of potato has been one of the most widely studied diseases and particular attention has
been given to the mathematical description of disease development. Several process based simulation models
have been developed and this paper focuses primarily on several versions developed at Cornell University,
and later through collaboration between that University, the International Potato Center and the Volcani
Center. The most recent version, LB2004, has been validated in the highland tropics and several other
countries. Historically, late blight simulators have been used to evaluate disease management scenarios.
However, they have also been used for other purposes, including, sensitivity analysis of resistance
components, comparative epidemiology, development of forecasting models and education. The potential for
using disease simulation has and will continue to expand as improvements are made in supporting
technology, both in computing power and acquisition of weather data.
1. INTRODUCTION
Late blight of potato and tomato (causal agent: Phytophthora infestans) has been and
continues to be one of the most intensively studied plant diseases. As a result, the
disease has served as a testing ground for much scientific development in plant
pathology, and particularly in epidemiology. The important role of late blight in
scientific development is due to several factors: the global importance of the disease
(Forbes & Landeo, 2006; Mizubuti & Fry, 2006), the “well behaved” nature of the
pathogen (readily cultured) and the disease (relatively uniform infection), and because
J. E. Vanderplank, the father of modern plant epidemiology, chose late blight to
illustrate epidemiological concepts in his early works (Vanderplank, 1963).
Both analytical and simulation models have been used to describe late blight
epidemiology and both approaches were recently reviewed by Mizubuti and Fry
161
162 G. A. FORBES ET AL.
(2006). In this paper we propose to extend that review by focusing in more depth on
late blight simulation. While we are unaware of new developments in analytical
modeling of this disease, a relative surge in simulation research in recent years
warrants a fresh look at the progress that has been made and the directions being taken.
2. PLANT DISEASE SIMULATION

Simulation has been described as a “process of designing a model of a real system and
conducting experiments with this model for purpose either of understanding the
behavior of the system or for evaluating various strategies for the operation of the
system” (Shannon, 1975). Thus, simulation is both a tool for fundamental studies on
the epidemiological process of a disease and for testing new management strategies.
One important advantage of simulation is that extensive experimentation can be carried
out that would be impossible, impractical or prohibitively expensive using the real
system in the laboratory, greenhouse or field.
Another characteristic of most plant disease simulators is that they are made up of
sub-models that represent steps in the infection process: spore germination,
penetration, colonization and sporulation (Campbell & Madden, 1990). These are
linked to produce the final output, which is generally disease development over time.
Data from the literature or further experimentation are used to parameterize these
processes, with respect to weather and other important variables.
2.1. Simulation vs. Forecasting

Simulators are related to, but should not be confused with, the much simpler
forecasters. As mentioned above, the purpose of simulators is to decompose the disease
process to provide greater insight or to allow for simulated experimentation. The
purpose of forecasters is to predict the initiation of a disease, or when the severity will
increase to a dangerous level. For example simulators can be used to predict the
relative efficacy of management tactics, but forecasters cannot.
Whereas the forecaster is generally the tool of the extension service or farmer, the
simulator is more often the tool of the researcher interested in developing management
strategies or evaluating components of the disease process. Since simulators can
decompose the disease progress, they can be used to develop forecasters. A large
number of late blight forecasting models have been developed. The IPM program of
the University of California at Davis maintains a useful catalogue of sixteen of them
(http://www.ipm.ucdavis.edu/DISEASE/DATABASE/potatolateblight.html) and several
models have been incorporated into much more complex decisions support systems
(DSS), involving monitoring and other disease management tactics (Schepers, 2004).
The purpose of this paper is twofold: i) to provide a detailed history of the
development of simulation of potato late blight – we are unaware of efforts to simulate
tomato late blight, and ii) to focus on potentially innovative uses of disease simulation
that will lend greater utility to this technology in the future.
MODELS FOR POTATO LATE BLIGHT 163
2.2. The Cornell Experience

Of the numerous disease simulators reviewed by Campbell and Madden (Campbell &
Madden, 1990), the late blight simulator developed by Bruhn and Fry (1981) (hereafter
referred to as LATEBLIGHT), and two others for other diseases, were identified as
having provided “valuable new understandings of epidemics ..... as well as the
development of improved disease management practices”. The success of these
simulators was assumed to derive from the fact that they were developed to solve
specific research questions, rather than as a goal in themselves. LATEBLIGHT has
been used to test numerous management strategies.
LATEBLIGHT was used to evaluate the precision of various disease forecast
systems (such as BLITECAST) and fungicide scheduling programs. In general, only in
few cases did the forecast systems achieve better disease control than weekly sprays,
and on the average the amount of fungicides was not reduced with forecasting (Fohner
Fry & White, 1984; Shtienberg & Fry, 1990). These predictions were corroborated in
the field (Fry, Apple & Bruhn, 1983; Spadafora et al., 1983; Spadafora, Bruhn & Fry,
1984). LATEBLIGHT was also used to develop a forecaster known as SIMCAST that
incorporated host resistance (Fry, Apple & Bruhn, 1983), which was subsequently
adapted for use in Mexico, with higher levels of resistance avilable in some Mexican
cultivars (Grünwald et al., 2002).
The potential effect of incorporating a weather forecast into potato late blight
disease forecasts, timing contact fungicide application, was also investigated using
LATEBLIGHT. The maximum contribution of a weather forecast occurred with a
perfect knowledge of future weather, one and two days in advance. Disease intensity
under these circumstances was reduced by 10%. However, simulated weather forecasts,
with the accuracy equivalent to those of real forecasts, improved disease control by
only 5% (Raposo, Wilks & Fry, 1993).
LATEBLIGHT was also used to demonstrate that applications of contact fungicide
for late blight suppression were not effective when applied 3 weeks before the end of
the season. In addition, it was shown that sprays applied close to late blight onset are
the most effective (Shtienberg et al., 1989).
The effects of host resistance, fungicide and weather on individual and combined
epidemics of early blight (a further disease caused by the fungus Alternaria solani) and
late blight were investigated using an improved version of LATBLIGHT (Doster
Milgroom & Fry, 1990a). To distinguish this model from the original version it was
referred to by Andrade-Piedra et al (2005c) as “LB1990”. The model showed that
moderate resistance to early blight had a 2-3 fold greater effects than did moderate
resistance to late blight. On the other hand, fungicides were 2-3 times more effective in
suppressing late blight than in suppressing early blight (Shtienberg & Fry, 1990).
These predictions were used to develop management program integrating host response
to both diseases and fungicides. The management program was evaluated in the field
and found accurate (Shtienberg et al., 1994).
The spray schemes utilizing the systemic compound metalaxyl, that would allow a
reduction in the amount of fungicide applied but still maintaining adequate suppression
of late blight, were examined using LB1990. Weather-dependent spray schemes did not
maintain disease control equivalent to standard schedule, when the total amount of
fungicide applied was reduced by amounts greater than 10%. However, some programs
in which spray dosage and intervals were adjusted according to the time in the season
resulted in much better disease suppression than the standard treatment (Doster & Fry,
1991).
LB1990 was also used to evaluate strategies for management of potato late blight
and resistance in the population of P. infestans to the systemic compound metalaxyl.
Using the model, metalaxyl and chlorothalonil mixtures performed better than
alternating application of these fungicides in suppressing both metalaxyl-sensitive- and
resistant-pathogens. Moreover, fewer applications of the mixtures (replaced by contact
sprays) achieved substantially improved management of metalaxyl resistance in the
pathogen population (Doster, Milgroom & Fry, 1990b).
Andrade-Piedra et al. (2005b) using a metamodelling approach (Kleijnen &
Sargent, 2000; Noordegraaf, Nielen & Kleijnen, 2003) demonstrated that late blight,
represented by the model version LB2004 (described below) was more sensitive to
changes of temperature, humidity and initial inoculum (initial lesions and time of
appearence) than to changes in fitness components (latent period, lesion growth rate,
sporulation rate and infection efficiency). Under the conditions in which the
metamodel was valid, the authors hypothesized that the disease could be best
controlled by delaying the appearance of initial inoculum and decreasing its level, or
by reducing the rates of disease increase by making the environment less favorable to
the disease and, to a lesser degree, by increasing the resistance level of the plant. They
mention that this insight can help to design strategies to ‘escape’ late blight through
early planting, growing the crop partly outside of the rainy season, and planting in
higher locations. All of these strategies are traditionally used by Andean farmers
(Thurston, 1992).
2.3. Globalizing LB1990

Prior to discussing efforts to make LB1990 more globally useable, it is worth noting
some aspects of late blight disease development in different agro-ecosystems. Late
blight is seasonal in the temperate zone, with the first symptoms starting after
emergence or sometimes even late in the season. During the winter months, the disease
virtually disappears. This phenomenon of seasonality means that any tactic that delays
the onset of the disease can be effective. If the initial amount of inoculum is reduced,
the disease will take longer to reach damaging levels. Therefore, reducing the sources
of initial inoculum (infected tubers, cull piles, neighboring fields, etc.) can be very
important in the temperate zone. The initiation of disease by germinating oospores is
also a threat in the temperate zone (Hannukkala et al., 2007).
In contrast, late blight is an endemic disease in many parts of the developing
world, particularly in the highland Tropics. In most cases, fields are surrounded by
numerous inoculum sources (other fields, volunteer plants, other hosts) throughout the
vegetative period. This means that any field unprotected by fungicide and/or resistance
will rapidly develop a severe level of infection. With this scenario, sanitation
procedures will not work very well. The approaches which should be successful here
are those that slow down the rate of the epidemic, such as host resistance, fungicides,
and a limited number of cultural practices that make conditions less favorable for
disease, including inter-cropping, weeding, drainage, fertility, etc. Of these, the two
which are most widely used are chemical control and, to a lesser degree, host resistance
(Forbes & Landeo, 2006).
In spite of its extensive use in research, LB1990 had until recently only been
validated in New York State, USA. Furthermore, there appeared to be several
limitations in LB1990 that needed to be resolved before the model could be used
outside its restricted area of development and validation. These limitations were
described in detail by Andrade-Piedra et al. (2005c), and primarily consisted of host
and pathogen parameters that were not realistic. These parameters had not been
measured experimentally (Doster, Milgroom & Fry, 1990a; Fry et al., 1991), but rather
estimated by calibration with field results (sensu Rykiel, 1996). The primary
inconsistency for pathogen parameters occurred with the period from infection to
sporulation, known as latent period (LP). The LB1990 version used a LP-value of 6
days (Fry et al., 1991), while it had been shown that LP varies between 2.4 and 4.1
days when measured in a susceptible potato cultivar under optimum environmental
conditions (Andrade-Piedra et al., 2005c).
LB1990 also had another apparent limitation: it was developed using experimental
data from studies involving the old population (sensu Spielman, et al., 1991) of P.
infestans. Most specialists now believe that P. infestans originated in Central Mexico,
and was restricted to that area until being introduced into US and Europe in the middle
of the last century (Spielman et al., 1991; Fry et al., 1992). That introduction led to the
Irish Potato Famine and greatly reduced production in many parts of the USA and
Europe (Bourke, 1993). Some of the earliest studies using genetic markers indicated
tremendous genetic variability in Mexico (Tooley, Fry & Villarreal Gonzalez, 1985),
with genotypes in Hardy-Weinberg equilibrium at most loci. This indicated that, in
Central Mexico, P. infestans is a highly variable, interbreeding population. Evidence
that the population was sexual was published in the 1950’s (Gallegly & Galindo, 1958),
and recent studies demonstrated that it remains sexual (Fernández-Pavía et al., 2004).
One seminal marker study using isolates of P. infestans from around the world
demonstrated that prior to the 1970’s, late blight in most, if not all areas, outside
Mexico was made up of a single clonal lineage with the A1 mating type, which was
designated US-1 because it was first found in the USA (Goodwin, Cohen & Fry, 1994).
This led to the chilling conclusion that the aggressiveness of this disease, generally
considered the worst on potatoes worldwide, was caused by a very small sample of the
pathogen’s gene pool. However, this situation did not last.
In the early 1980’s researchers in Europe published the discovery of the A2 mating
type in Switzerland (Hohl & Iselin, 1984), which meant they had found something
other than the US-1 lineage. Subsequent examinations of populations from several
continents including Europe, USA/Canada and Japan, led Spielman and co-workers
(1991) to postulate another “worldwide migration” of P. infestans, the first one having
caused the great Irish famine in the mid 1800’s. The second migration apparently
occurred in years just prior to the discovery of the A2 in Switzerland and, like the first
migration, originated in Mexico, spread to Europe, and now is spreading to other parts
of the world. More recently, independent migrations from Mexico directly into the
USA were also identified (Goodwin et al., 1994; 1995).
The recent migrations of P. infestans have changed the population structure of
pathogen in many parts of the world (Forbes & Landeo, 2006). Not only has the
original US-1 population been displaced (Drenth, Tas & Govers, 1994; Sujkowski
et al., 1994; Fry & Goodwin, 1997; Elansky et al., 2001), but the new populations in
Northern Europe (from the Netherlands to Moscow) are now sexual, with oospores
being produced and contributing to more severe epidemics (Drenth, Tas & Govers,
1994; Hermansen et al., 2000; Turkensteen et al., 2000; Elansky et al., 2001).
Epidemics are more severe because the new population is more aggressive (Day &
Shattock, 1997) and because the oospores overwinter in soil – providing a source of
inoculum not present in locations with only asexual populations. Repeated studies in
Europe and the USA have demonstrated that the new populations are more difficult to
manage (Day & Shattock, 1997; Kato et al., 1997; Miller, Johnson & Hamm, 1998;
Mizubuti & Fry, 1998; Carlisle et al., 2002) and this indicated that pathogen
parameters in LB1990 would have to be modified.
Additional limitations of early versions of LB1990 were related to initial inoculum
and the estimation of leaf wetness duration. These factors appeared highly relevant to
the discussion above of late blight development in different agro-ecologies. Initial
inoculum is a parameter of LB1990 that starts the simulation of disease, and it is
crucial for operational validation of the model. Previous versions of the model were
validated with data from field experiments located near Ithaca, New York, where the
potato plants were artificially inoculated (Bruhn & Fry, 1981; Doster, Milgroom &
Fry, 1990b). Thus, initial inoculum, expressed either as lesions per plant or sporangia
per plot, was known. In contrast, a version of LB1990 was needed that can use data
from field experiments located in the highland Tropics (e. g. Andes) where the plants
are infected by ‘natural’ inoculum since the pathogen is usually ubiquitous (Oyarzún,
Taipe & Forbes, 2003; Adler et al., 2004). Thus, for use in the highland Tropics, initial
inoculum would have to be estimated.
It is worth noting that despite the limitations in LB1990, the model was
successfully used to develop general strategies of fungicide utilization, as discussed
previously. Parameters derived from curve fitting were apparently not a major obstacle
because only general categories of resistance for the host, and a single population for
the pathogen, were usually considered and because its use had been restricted to the
region for which it was originally calibrated (Ithaca, New York, USA).
In order to deal with the limitations in LB1990, the International Potato Center
(CIP), Cornell University (USA) and the Volcani Center (Israel) joined forces in a
project to make the model a versatile tool for global application, but with particular
emphasis on the highland Tropics. This effort was funded by all institutions involved
and by a grant from the United States Agency for International Development. The
project’s general activities follow:
- Parameters for the host-pathogen interaction were measured using a “new” Andean
lineage of P. infestans and three Peruvian cultivars (Andrade-Piedra et al., 2005c).
- Improved equations for the effect of temperature on lesion growth rate and
sporulation rate were incorporated in the model. These equations were derived
from data measured using a new population of the pathogen (Andrade-Piedra
et al., 2005c).
- The model structure was modified to incorporate a temperature-dependent latent
period (Andrade-Piedra et al., 2005c).
- New parameters were incorporated in the model to create a new version,
designated LB2004 (Andrade-Piedra et al., 2005c). The new model was evaluated
by comparing simulated and real epidemics from several parts of the world. Data
were gathered from more than 50 real epidemics (controlled experiments) in Peru
(Andrade-Piedra et al., 2005a), Ecuador, Israel, Mexico and the United States
(Andrade-Piedra et al., 2005b). In Peru, where specific host and pathogen

parameters were known, the model performed very well (Fig. 1) and could be used
to answer specific questions about late blight management (Andrade-Piedra et al.,
2005a) (Fig. 1). For other locations, specific host resistance data were not
available, but the dynamics of epidemics were accurately simulated, given
reasonably accurate weather data (Andrade-Piedra et al., 2005b).
Figure 1. Observed (circles) and simulated (continuous line) disease progress curves of
epidemics with no fungicide applications of Phytophthora infestans in potato cultivars
Tomasa (A), Yungay(B), and Amarilis (C) in Comas, 1999; Tomasa (D), Yungay (E), and
Amarilis (F) in Oxapampa, 1999; Tomasa (G), Yungay (H), and Amarilis (I) in Huancayo,
2000; and Tomasa (J), Yungay (K), and Amarilis (L) in Oxapampa, 2000. The simulated
progress curves were obtained with the LB2004 version of LATEBLIGHT. Vertical lines
represent the standard deviation of the observed mean blight severity. All locations are in
Peru. Reprinted from Andrade-Piedra et al. (2005a).
In order to facilitate the utilization of LB2004 for research and training, computer code
from the research version of the simulator (in SAS®) was translated into Delphi® to
produce a compiled program that can be used independently of other software (a major
disadvantage of the SAS® code for training). The new version of the simulator, known
as POLUX, is powerful enough to meet the needs of end-user researchers, but easily
copied and installed, because of its light size. POLUX has a graphical user interface
(GUI) and does not require prior familiarity with a statistical language, as does the
LB2004 SAS® version. Nonetheless, LB2004 remains the most powerful tool for
many research purposes, because of the intrinsic capability of SAS® to analyze output
from the simulation exercises and the capacity to run multiple simulations
simultaneously.
LB2004 did not work as well as expected for plots that had been treated with
fungicides, even in Peru. This was somewhat of a surprise because the fungicide
component of LB2004 is one of the simplest, involving just two functions: fungicide
efficacy (as a dose response curve) and wash-off (as a rainfall residue curve).
Because of the simplicity of this part of the model, researchers hypothesized that
fungicide functions were not accurately representing reality and experiments were
initiated in CIP-Lima to validate the data. These experiments, now under way, are
generating data to incorporate into the simulator. Initial experiments focus on the
contact fungicides mancozeb and chlorothalonil, because they are the two a. i. most
commonly used in potato production in the Andes (Crissman et al., 1998; Ortiz et al.,
2003), and because they were employed in several field trials that can be utilized for
model validation. Subsequently, data will be generated for copper-based fungicides
used by organic potato growers, as well as for the most important translaminar and
systemic compounds.
3. OTHER SIMULATION MODELS

LB1990 and LB2004 represent the most widely used suite of late blight simulation
models, but other simulators have also been developed for blight. One was developed
by Kluge and Gutsche (Kluge & Gutsche, 1990) which is based on symptomatic
aspects of the host plant. The total area of foliage is divided into an uninfected part,
latent part, sporulating part the dead part. This model is simple to use and validate, but
it gives little insight into the effects of weather and technology (fungicides and host
resistance) on important aspects of the disease, such as lesion expansion, dispersal,
spore germination.
Another simulator was developed by Van Oijen (Van Oijen, 1992a; 1992b) in the
Netherlands. The model was used to evaluate breeding strategies for resistance and
tolerance to late blight in potato. This was done by using the model to identify the
resistance components that were most important in resistant cultivars. Based on a
sensitivity analyses, lesion growth rate was found to be the most important, followed
by infection efficiency, infectious period and finally latent period.
Van Harren and Jansen (2003) took a completely different approach to simulating
potato late blight when they developed LINBAL. This model was developed by
introducing a late blight limiting extension into the potato simulation model LINTUL
(Spitters & Schapendonk, 1989). The effort admirably addressed the conspicuous
absence of good growth and yield models in previous disease simulation models.
Unfortunately, LINBAL did not incorporate weather based functions to drive the
epidemiological part of the model. The successful integration of accurate disease
development and host growth into one late blight model remains an unfulfilled
challenge for plant scientists.
4. INNOVATION AND FUTURE DIRECTIONS FOR LATE BLIGHT

SIMULATION
To date, late blight simulation has been used primarily to explore disease management
scenarios that are then evaluated in field trials (Andrade-Piedra et al., 2005c).
However, disease simulation is a powerful tool that can be used to enhance other areas
of biological interpretation related to plant disease. Below we discuss some of the
areas, other than disease management, where simulation has been used, or could be
used, in an effort to stimulate innovation with this technology in the future.
4.1. Comparative Epidemiology

Multi-locational trials may give conflicting results. For example, one cultivar may be
resistant to a disease in one location, but it may result susceptible in another, and this
may occur for a number of reasons (e.g., host resistance, weather, pathogen diversity,
soil and water characteristics). Disease simulation is one way of decomposing the
many factors that interact to affect disease severity. With disease simulation, we can
“control” one or more factors in the different locations, thus allowing for a more
precise comparison of other factors.
In one recent exercise, researchers at CIP compared late blight severity in the
Andes and sub Saharan Africa (SSA) using simulation. The simulation exercise was
stimulated by the observation that farmers in SSA sprayed less frequently then farmers
in the Andes, even though conditions in both countries appeared to favor disease
development. Himans et al. (2000) also noted an apparent sub-utilization of fungicides
in SSA and attributed it to socio-economic factors (reduced access to products and/or
insufficient farmer liquidity). However, an alternative hypothesis was that late blight
development in SSA, still characterized by the old population (sensu Spielman et al.,
1991) of P. infestans (Vega-Sanchez et al., 2000), is slower than in other parts of the
world. As discussed earlier, the old population is considered less aggressive than new
populations currently present in the Andes and elsewhere.
CIP researchers found that epidemics with no fungicide application in SSA were in
most cases overestimated by LB2004. To perform this analysis, the researchers had
parameterized the model with Peruvian cultivar Yungay and the Andean pathogen
population, and compared it to field data of the Kenyan cultivar Asante. A direct
comparison of levels of resistance of Asante and Yungay was not available. However,
by comparing each one to a third cultivar, Dutch Robijn, for which data did exist (M.
Olanya, personal communication; Forbes et al., 2005), the authors were able to
determine that Asante and Yungay have roughly the same level of resistance to P.
infestans. Therefore, it is likely that the differences between the observed and
simulated progress curves in epidemics with no fungicide use were due to differences
in aggressiveness of the pathogen population (Fig. 2 A, B).
In another case of comparative epidemiology, disease development in two potato
producing areas of Israel were evaluated with LB2004. Late blight is the most
destructive foliar pathogen of potatoes in Israel. All potato fields grown in Israel are
irrigated but the source of the irrigation water varies among the different production
areas. Fresh water is used in the coastal plain and the Khula valley, while municipal
recycled water in the northern Negev, and saline water are used in the Arava valley.
Observations made in potato fields in the different regions suggested that the potential
intensity of the disease differs in the different regions. Whereas severe epidemics
occasionally develop in the coastal plain and the Khula valley, epidemics in the
northern Negev are usually moderate and late blight seldom develops in potatoes
grown in the Arava valley. Several factors (and their interactions) may contribute to
these differences, including production practices, microclimatic conditions and
variation in the aggressiveness of the prevailing P. infestans isolates. However, as the
different sources of the irrigation water coincided with the variable intensity of late
blight epidemics in the different regions, it also seemed a plausible factor. The late
blight simulator was applied as a first test of this hypothesis.
Figure 2. Late blight epidemics on potato cultivar Asante in Kalyngere (Uganda, 2005) (A)
and on potato cultivar ‘Yungay’ in Oxapampa (Peru, 1999). Empty circles represent observed
mean blight severity and continuous lines represent simulated blight severity in epidemics
with no fungicide. Black circles represent observed mean blight severity with the protectant
fungicide mancozeb, and broken lines represent simulated blight severity with the protectant
fungicide chlorothalonil. Black arrows near the horizontal axis represent dates when
mancozeb (observed) or chlorothalonil (simulated) was applied. Vertical bars show the
standard deviation of the mean.
LB2004 was first used to simulate the disease in Northern Negev region, where the
fields are irrigated with municipal recycled water. Two trials were carried out for thus
purpose in autumn of 2001. Visual comparison of observed and simulated epidemics
revealed that the predictions issued by the simulator overestimated the epidemics that
actually developed in the fields. Comparing all observed and predicted epidemics
corroborated these conclusions: the slope of the regression equation (1.34) was
significantly higher than 1 (t- test; P<0.05) (Fig. 3).
Figure 3. Comparison of observed and predicted late blight severities in two field
experiments.Dots represent plots with different levels of disease. The 1:1 line is a
theoretical representation of perfect coincidence between observed and predicted
values. The thick line represents the regression equation of predicted values regressed
on observed. Over prediction by the late blight simulation model LB2004 in this case
indicated that some factor was limiting disease in this location.
In follow-up studies stimulated by this simulation exercise, researchers found that

boron, a microelement that exists in high concentrations in recycled water, induces
systemic acquired resistance against P. infestans and also against A. solani.
In another example of comparative epidemiology, LB1990 was used to
quantitatively explain changes in disease dynamics over time. After the introduction of
new pathogen strains into the USA in the 1990s, farmers had much greater disease
management problems (Fry & Goodwin, 1997). Researchers at Cornell, re-
parameterized LB1990 to represent the new pathogen population and compared output
with that of the original LB1990 (Figure 4). The analysis indicated that the new
aggressive population required about 25% more fungicide than that required to
suppress the “old” population, which was generally in agreement with observations in
the field (Kato et al., 1997).
Figure 4. Simulation model predictions of the effect of different fungicide application

frequencies to suppress epidemics of late blight caused by the US-1 and US-8 clonal lineages.
The application frequencies are described according to the number of days between
applications. Thus, application every 2 days involved the greatest amount of fungicide and
application every 14 days involved the least. The lineages differed only in terms of the lesion
expansion rate in the simulation model as described in the text. All other factors were the same
for each lineage and are described in the text. Each data point resulted from 50 independent
simulations—one for each of 50 years of weather data from upstate New York. Linear
regressions for the lineages were US-1, AUDPC = –2.51 + 0.67x, R2 = 0.962; and US-8,
AUDPC = –2.50 + 0.90x, R2 = 0.970. Using indicator variables to represent the lineages, the
difference between the slopes of the lines was significant (P = 0.026). From Kato et al., 1997.
4.2. Biological Control

As with many biological phenomena, the extrapolation of biocontrol results from
greenhouse or laboratory studies to the field is difficult (Stephan et al., 2005).
Nonetheless, McClay and Balciunas (2005) argued that pre-release efficacy assessment
of biocontrol agents would reduce risk of high cost and even potential negative
collateral effects of biocontrol, by helping to identify ineffective agents prior to release.
Simulation technology, like LB2004, appears to be highly appropriate for such an
assessment, although we are not aware of examples in the literature. The effect of
biocontrol on disease is analogous to that of a chemical disease control product and
could be quantified in greenhouse experiments by measuring a dose-response change in
epidemiological parameters (latent period, lesion expansion, sporulation, etc.) as well
as persistence of the agent. The change in epidemiological parameters caused by the
biocontrol agent would then be introduced into the simulator (LB2004) and, using
historical weather data, the efficacy of the agent would be predicted just as one would
predict the efficacy of a fungicide. After field validation of the process, disease
simulation could be a cost effective way of screening biocontrol agents.
4.3. Geographic Zonation and Impact Assessment

Because of the high spatial and temporal variability of late blight incidence and
severity, there was no way of predicting relative late blight severities on a global scale.
Potential severity, which ignores the effect of interventions such as fungicide spraying,
resistance and cultural controls, was estimated applying a GIS framework using late
bight forecasting models (Hijmans, Forbes & Walker, 2000). This technology leads to
geographic zonation based on potential late blight severity (Fig. 5) and can be useful
for comparative epidemiology, resource allocation and impact assessment. As noted
earlier, forecasters are different from simulation models, but one model the forecaster
used in this case was developed from LB1990.
Figure 5. Map depicting global zonation of potato production zones based on predicted number
of protectant fungicide sprays needed to control late blight. Predictions based on BLITECAST, a
late blight forecasting model, run within a GIS (from Hijmans Forbes & Walker, 2000).
The strength of the GIS forecasting approach lies in this link between simulation
model, forecasting rules and GIS zonation. The link to simulation can ultimately
provide much greater power for biological interpretation by enabling geographic
zonation based on parameters that are specific to a cultivar, pathogen population or
both. Zonation can be done with geo-referenced historical weather data sets, but also
with weather data generated from climate change models. This was done in CIP for late
blight (unpublished), has been done for rice leaf blast caused by Pyricularia oryzae
using disease simulation models linked to GIS (Luo et al., 1998) and, more recently,
when researchers predicted increased range of oak disease as a result of climate change
(Bergot et al., 2004).
4.4. Plant Breeding - Predicting Resistance Performance

As noted above, Van Oijen (1992a) used a late blight simulation model to evaluate the
relative importance of different epidemiological components, which can then be used
for plant breeding. This approach could be extended to evaluate the potential utility of
wild sources of resistance that may comprise different components (e.g., infection
efficiency vs. sporulation). Similarly, simulation could be used tp predict the utility of
genes that confer partial resistance, such as those from Solanum bulbucastanum that
apparently confer high levels of partial resistance (Van der Vossen et al., 2002; Song
et al., 2003).
4.5. Training
LB2004 represents one of the most complete process oriented models for
polycyclic foliar diseases and for that reason it could be useful in teaching
epidemiological concepts. The availability of data from a large number of experiments,
which have now been compiled by CIP, Cornell and other workers, will increase the
credibility of the model by allowing learners to compare simulated and real data for
diverse situations. The graphical user interface of POLUX, discussed above, allows for
workshop participant to quickly learn how to incorporate their own field data, thus
making workshops more pertinent to participants.
While simulation in itself is an excellent tool for learning epidemiological
concepts, it also can help generate non-computer based materials and exercises that are
highly illustrative of epidemiological processes (e. g., quantitative relationships
between weather variables and disease growth) that can be used without the simulator.
Farmers in developing countries cannot realistically use a computer-based simulation
model but the tool can be used to verify our knowledge of how disease progresses in a
particular situation – prior to transmitting that knowledge – and to test participatory
learning activities that have been designed to help farmers learn epidemiological
concepts.
We have identified some innovative uses of plant disease simulation but it would
appear that this technology could be applied to a greater span of plant science
problems. Researchers should focus on several outstanding issues to improve utility of
simulation, including the incorporation of effects of new fungicide chemistry and
accurate estimation of yield loss, due to disease. The effort to “globalize” LB2004
described above was only rendered feasible because of changes in technology that
facilitate this type of work. Both high speed computing and accurate logging of precise
weather data can now be done for very modest sums. This trend will continue, which
will only increase the potential of simulation as a useful tool.
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9
JAN HADDERS
AN EXAMPLE OF INTEGRATED FORECASTING

SYSTEM FOR PHYTOPHTHORA INFESTANS
ON POTATO
Dacom PLANT-Service BV, P.O. Box 2243
7801 CE Emmen, The Netherlands
Abstract. PLANT-Plus® was initially developed by Dacom as a Decision Support System (DSS) for
management of Phytophthora infestans and has been used on-farm since 1994. The system has been
extended with models for Alternaria solani and several other fungi or insect pests in different crops.
Another modules include the irrigation management system based on ET data and soil moisture sensors,
and models for potato tuber infection and fertilizers management. This integrated suit of DSS models
offers a turn-key solution for the plant grower. The PLANT-Plus platform enables communication of data
between farmer, consultant, processors and other accredited users in the food chain. The users can choose
the most appropriate interface, and can configure a variety of output types such as SMS text messaging,
fax and e-mail warnings. The system offers an integrated ten day weather forecast which provides a
predictive risk assessment for the coming days. The disease models require the availability of on-farm,
automatic, weather data and were developed in cooperation with experts from different areas and
countries. In contrast to most of the other available models, PLANT-Plus is based on the lifecycle of the
P. infestans, combining infection events with the unprotected part of the crop. The model will recommend
when to apply a new spray and what type of chemical to use, i.e. contact, translaminar or systemic. The
benefits of the models are clearly demonstrated in field trials and commercial evaluations all over the
world and provide safe spraying programmes with the lowest possible use of chemicals for the control of
late and early blight in potato crops.
1. INTRODUCTION
Dacom PLANT-Service BV commercially developed and operates the PLANT-Plus
integrated system for crop management. Development of this system initiated in the
early 90’s as a crop recording system. It was already then envisaged to provide a
platform to be able to exchange data and information between various partners in the
agribusiness. On top of this platform a disease model was developed to optimise the
control of Phytophthora infestans in potatoes. Given the state of technology at that
moment it was possible to build a biological model based on fuzzy-logic principles
and the use of hourly weather data, both retrospective and as a forecast.
The PLANT-Plus databank uses a climate data interface that can import and
distribute data of different types of on-farm weather stations (i.e. Adcon, Skye,
Metos, Campbell and Hardi Metpole). Meteo Consult of Wageningen provides
179
180 J. HADDERS
Dacom with a high-quality, local weather forecast for the coming 10 days for any
desired location in the world. Furthermore, scientific information about the diseases
cycles, product activity and variety characteristics are added to the databank. During
the growing season, the grower adds current crop information to the data set. (Fig 1)
Figure 1. Input parameters of the Plant-Plus system.
The current output (Fig. 2) consists of a number of DSSes for a large number of
fungal diseases in different crops. For potatoes, a tuber infection risk model has been
added. and a number of insect models have been operational over the last number of
years. The irrigation advisory modules in the system are becoming more and more
important to the PLANT-Plus users around the word and, in 2006, a fertilizing
module has been added to the system.
Figure 2. Output module of the PLANT-Plus system.

PHYTOPHTHORA DSS ON POTATO 181
The use of a databank platform also allows for the use of different user
interfaces like old-fashioned PC-based MS-DOS applications, sophisticated PC-
based MS-Windows applications and simple Internet Server Applications. The
system can also deliver specific additional outputs like SMS text messages, e-mails
and fax. The platform was subsequently extended with more regional applications:
telephone-based, like ALPHI (Bouwman & Raatjes, 2000) and ALERT (Hadders,
2002) or like the web-based Syngenta Blight Forecaster (Hinds, 2003). The
combination of crop records, weather data and treatment recommendations provides
an excellent tool for crop assurance schemes.
2. DISEASE FORECASTING MODELS & PRINCIPLES

The PLANT-Plus disease forecasting models have been developed in close
cooperation and harmony with experts on plant diseases. For potatoes the model for
late blight (Phytophthora infestans) was started in 1994 (Hadders, 1997) and the
model for early blight (Alternaria solani) was started in 1999 (Hadders, 2003).
All model inputs and outputs are evaluated and calculated on hourly basis or
three hourly for the forecast data. This implies the need for high quality continuous
weather monitoring stations and regular updated weather forecasts.
In contrast to most other systems the PLANT-Plus model is a biological model,
that evaluates the complete life cycle of the fungus (Raatjes et al, 2004).
Additionally, any infection events are related to the degree of protection of the crop
by chemicals and new growth of leaves.
The model can be divided into the following sub-models:
1. Unprotected part of the crop
a. Growth of new leaves
b. Degradation and wear off of chemicals
2. Infection events of the disease
a. Formation of spores on each infected leaf
b. Ejection and dispersal of spores into the air
c. Germination of spores and penetration into unprotected leaves
3. Combination of un-protected leaf area and infection events into treatment
recommendations
2.1. Sub Model 1a - Unprotected Crop by Growth of New Leaves

This factor is the most underestimated one in standard crop spraying regimes. Rapid
growth in early season can cause crops to be vulnerable to infection from 3 days
post fungicide treatment onward (Flier, unpublished). The assessment of growth of
new leaves is dependant on field scout reports, which means the farmer or his
consultant will have to go out into the field and score the development of new leaves
compared to the last measurement on a very regular base (Hadders, 1997).
182 J. HADDERS
2.2. Sub Model 1b - Unprotected Crop by Degradation and W ear-Off of Chemicals

PLANT-Plus includes information about chemicals as part of the base of the system.
This information includes active ingredients, recommended dose rates, a.i. efficacy
against the disease and factors for wear-off influenced by precipitation and solar
radiation. The background data is mainly derived from independent trials that were
recently carried out by Schepers and his colleagues at Plant Research in the
Netherlands (1996-2002) and from the agrochemical companies.
2.3. Sub Model 1- Unprotected Part of the Crop

The results of sub models 1a and 1b together represent the unprotected leaf area.
Basically it is of no concern if the crop is unprotected, as long as the there are no
possibilities for the fungus to infect the crop. Trials have demonstrated that the spray
interval can be stretched to 2-3 weeks without any problems under such
circumstances (De Visser & Meijer, 2000; Raatjes et al, 2001; Kessel, Wander &
Spits, 2003).
2.4. Sub model 2 - Infection Events of the Disease

This sub model replays the life cycle of the pathogen on an hourly basis. The
development of ‘new blight’ with sexual reproduction and more aggressive strains
of P. infestans (Flier et al, 2002) is integrated into the system. The epidemiological
soundness and accuracy of the Dacom models for Phytophthora and Alternaria have
been successfully evaluated in numerous field trials (Hadders, 2003; Denner &
MacLeod, 1998; Marquinez, 1999; De Visser & Meijer, 2000; Spits & Wander ,
2001). Whenever necessary the models will be updated to implement the most
recent scientific background. The relation between a general fungus life cycle and
the PLANT-Plus sub-models is presented in Fig. 3.
Figure 3. Fungus life cycle and PLANT-Plus sub-models

2.5. Sub Model 2a - Formation of Spores on Each Infected Leaf

Sub model 2a calculates the number of viable spores on an imaginary lesion using
temperature and relative humidity ranges for growing and dying off. In effect it can
be compared to the size of the grey ring of sporangiophores present around a
developing P. infestans lesion. The source of the lesion can either be from infected
seed, resulting in a primary infected shoot or from secondary leaf infections. For
Alternaria it can also come from infected debris.
2.6. Sub Model 2b - Ejection and Dispersal of Spores into the Air
After formation the spores can be dispersed into the air. This can be caused by either
climatic conditions like a (sudden) drop in the relative humidity, wind or rain. But
the lesion itself also purges out spores, a process called leakage (Turkensteen, 1995,
unpublished). The inputs for this model are the output from sub model 2a and the
presence of the disease in the vicinity of the field, provided by field scouts (Hinds,
2000) or a disease mapping system (Hendriks, 1999; Hadders, 2002). This sub
model has been evaluated with spore traps. The graph in figure 4 compares the
PLANT-Plus output with spore trap readings near a trial carried out by Schlenzig
from TU, München-Weihenstephan. Note that although the absolute spore counts
can not be compared with the PLANT-Plus output as it calculates an arbitrary figure,
the trends match closely.
The information about the presence of the disease is vital for an accurate
calculation. The model will, nonetheless, calculate spore flights based on a very low
(standard) presence, but not at accurate levels. Recent research has revealed the
effect that different sources of infection can have on Late blight epidemics, like
infected mother tubers, dumps, (excessive) distant sources and volunteers (Flier et al.,
2002; Raatjes & Kessel, 2003; Van Baarlen & Raatjes, 2001). For example in the
Netherlands in 1999 one field with a severe infection caused secondary infections in
other fields up to 30 km away. In 2000 and 2001 the effect of infected seed was
clearly demonstrated and in 2002 early volunteers had great effect on primary
inoculum levels. Sub model 2b results in a fictitious concentration of airborne spores
to be deposited on the foliage of the potato field.
2.7. Sub Model 2c - Spores Germination and Penetration into Unprotected Leaves
The next step in the life cycle is to calculate the germination and infection of the
spores on a leaf and it completes the infection event. This part is based on
temperature, leaf wetness and variety resistance. Leaf wetness enables the spores to
germinate. PLANT-Plus has a specific model that calculates the leaf wetness of the
crop, based on climatic conditions and the latest observation for crop density.
Temperature and variety resistance influence the speed of germination, penetration
and incubation. Sub model 2c results in the fictitious number of spores that can
infect an unprotected leaf.
The accuracy of this sub model is demonstrated in the correspondence with
outbreaks in the fields (Van Baarlen & Raatjes, 2002). Based on late blight surveys
184 J. HADDERS
age and size of lesions are associated with the timing of the infection events
according to the model.
In a broader range the infection events for P. infestans were compared to the
number of outbreaks reported to the disease mapping system in the Netherlands. The
graph in Fig. 5 shows the timing of the infection events compared to actual reported
late blight outbreaks. The arrows depict the delay in onset, caused by the incubation
of the disease.
Figure 4. Comparison between output from PLANT-Plus sub-model 2b and spore trap data.
Figure 5. Relation between timing of PLANT-Plus infection events and reported

outbreaks in the period June 8th - July 7th, 1997.
Raatjes & Kessel (2003) have also demonstrated that the accumulated PLANT-
Plus infection index over the seasons reflects the accumulated number of outbreaks.
The outputs of model 1 and 2 are combined into one simple graph (Fig. 6) that
reveals all the necessary information. The model run always starts with the date of
crop emergence or the most recent chemical treatment (left of the graph) and uses
retrospective weather station data until the current point of time (arrow) and
continues with five days of forecast data.
Figure 6. Example of PLANT-Plus disease model output.
2.8. Sub Model 3: Combination of Unprotected Leaf Area and Infection Events into
Advice
Sub model 3 interprets the unprotected leaf area and the infection event and provides
a recommendation if and what type (contact, translaminar or systemic) of chemical
to use (Fig. 5). The choice of the chemical is left up to the grower or his advisor.
Within the recommendation the system also specifies the relative need for a
treatment: not needed, to be considered or necessary. The following
recommendations for chemical types are feasible:
- no treatment needed
- treatment with contact fungicide to be considered / necessary
- treatment with translaminar fungicide to be considered / necessary
- treatment with systemic fungicide to be considered / necessary
This recommendation is influenced by the timing of the infection event related

to the current point of time. An infection in the previous 12 or next 24 hours will
have to be treated with a contact fungicide. Depending on timing, temperature and
186 J. HADDERS
variety resistance an ‘older’ infection event will have to be cured with either a
translaminar or a systemic fungicide. The last three days of the forecast are not
converted into an advice, but the user can view it in the graphical output.
Trials and projects have demonstrated that PLANT-Plus strategies rely heavily
on contact fungicides as recommendations are often triggered before or during the
infection event (Bouwman & Raatjes, 2000; Kleinhenz & Jörg, 2000; 2001).
Continuous, daily consulting of the system will however be necessary to achieve this
status.
The PLANT-Plus model is not ‘blocked’ on short intervals or long intervals.
This means that a new recommendation to treat can be given after 2-3 days, when
conditions are dangerous and the crop is growing rapidly (Fig. 7). Recommendations
not to spray can continue to be given for 3-4 weeks under dry conditions. As
McGrath (2000) reports: farmers would normally never dare to wait that long.
All recommendations include an outlook for the spraying conditions for the next
five days, based on expected rainfall and wind speed. This provides the user with a
tool for advanced planning.
Figure 7. Example recommendation: Sub model 3output of Dacom disease forecasting model.
3. QUALITY OF WEATHER FORECASTS

One of the key advantages in PLANT-Plus is the delivery of a local ten day weather
forecast anywhere in the world. This implies a strong dependency on the forecast,
that must therefore be of high quality. Dacom is continuously evaluating the
accuracy of the forecast data by comparison to the on-farm meteorological stations.
In 1997 a study was done in the Southern part of the Netherlands (Maastricht
area) to evaluate the results of PLANT-Plus (Geelen, 1997). Relative humidity is a
crucial factor in the epidemic of fungus diseases. In the comparison the forecast
underestimated the readings for the humidity by approx. 13%, which meant that the
realised humidity was always higher. Raatjes et al. (2001) have made the same
comparison for Egypt and find more or less the same results, but this has not
resulted in (more) curative sprays or missed infection events. This means that for
critical conditions the forecast will indicate the infection event in time, although the
details of the forecast may not always be fully correct. The conclusion is supported
by the large share of contact fungicides that PLANT-Plus users generally apply.
4. FUTURE DEVELOPMENTS AND CONSTRAINTS

The previous results and experiences might lead to the conclusion that every farmer
should be ready to use the PLANT-Plus system as there is a clear economic benefit.
Yet, even in the Netherlands there is only a small but steadily increasing proportion
of the ‘professional’ farmers using the models. A market survey showed that farmers
have different kinds of excuses why not to use the models. One of them is the
complexity of the subject and the difficulties farmers find in understanding the
recommendations. To change the farmer’s mind from an easy-going, prophylactic
strategy to an alert, infection event reactive approach has always been the toughest
challenge.
The contradictory recommendations or even opposition from other influencers,
like chemical resellers or governmental bodies is another threat for successful
implementation.
A successful introduction of PLANT-Plus furthermore requires the availability
of an effective range of fungicides for the control of the disease. In some countries
like Sweden and the Netherlands there are only a few fungicides registered and this
sometimes leaves few options to choose products, especially translaminars and
systemics. With regards to Alternaria it is of general concern that for most of the
registered chemicals the efficacy against this disease is not full, but partial at best.
Another point of concern is the effort that farmers have to deploy to provide
crop informations. In essence this is a positive factor, since farmers will have to go
out in the field to observe their crops, but it is often recognized as too much trouble.
Dacom is therefore seeking alternatives such as Remote Sensing images or
automated snapshot cameras. Bakker (1999) has found that processing RS images
into crop cover maps can provide accurate information. The PLANT-Plus feasibility
study in Egypt (Raatjes et al., 2001) however, revealed significant problems with the
availability of useful, cloud free images.
The PLANT-Plus system was extended with a large and successful range of
disease models for other crops like vegetables, fruits and vines and additional
models for insect management are also under development. Currently, new models
are developed to minimize the input of crop data by the grower and to estimate yield
and harvest product quality.
188 J. HADDERS
REFERENCES
Baarlen, P. van, & Raatjes, P. (2001). Karakterisering van primaire haarden van Phytophthora infestans
tijdens het teeltseizoen 2001. Project Report Masterplan Phytophthora. LTO-Nederland, Den Haag,
The Netherlands.
Bakker, A. (1999). PHYTOGIS. Integrated service of using earth observation in optimisation of services
in Phytophthora disease control. ITT RGC, Project report 11/99.
Bouwman, J. J., & Raatjes, P. (2000). ALPHI Actual Local PHytophthora Information Line based on
PLANT-Plus. In: Schepers, H. (Ed.). Proceedings of the Workshop on the European network for
development of an integrated control strategy of potato late blight, Oostende, Belgium, September -
October 1999. PAV Special Report, 6, 90-93 (http://www.ppo.wur.nl/uk/publications /publications
+ppo/agv/).
Denner, F. D., & McLeod, A. (1998). Evaluation of disease forecasting models for control of potato late
blight. Report ARC Roodeplaat, South Africa.
Flier, W. G., Kessel, G. J. T., Bosch, T. van den, & Turkensteen, L. J. (2002). Impact of new populations
of Phytophthora infestans on integrated late blight management. In: Westerdijk, C. E. and Schepers,
H. T. A. M. (Eds.). Proceedings of the Workshop on the European network for development of an
integrated control strategy of potato late blight, Edinburgh, Scotland, September 2001. PPO Special
Report, 8, 193-201 (http://www.ppo.wur.nl/uk/publications/publications+ppo/agv/).
Geelen, J. L. M. (1997). Het gebruik van het Dacom adviesmodel onder Zuid-Limburgse
omstandigheden. In: 1997 Trial report. LLTB, Tilburg–Roermond, NL.
Hadders, J. (1997). Experience with a late blight DSS (PLANT-Plus) in starch area of the Netherlands in
1995 and 1996. In: Bouma, E. and Schepers, H. T. A. M. (Eds.). Proceedings of the Workshop on
the European network for development of an integrated control strategy of potato late blight,
Lelystad, The Netherlands, September-October 1996. PAV Special Report, 1, 96-100.
Hadders, J. (2002). Don't call us, we call you. In: Westerdijk, C. E. and Schepers, H. T. A. M. (Eds.).
Proceedings of the Workshop on the European network for development of an integrated control
strategy of potato late blight, Edinburgh, Scotland, September 2001. PPO Special Report, 8, 93-102
(http://www.ppo.wur.nl/uk/publications/publications+ppo/agv/).
Hadders, J. (2003). Control of Early Blight (Alternaria) with Plant-Plus. In: Westerdijk, C. E., and
Schepers, H., T., A., M. (Eds.). Proceedings of the Seventh Workshop of an European Network for
development of an Integrated Control Strategy of potato late blight, Poznan, Poland, October 2002.
PPO Special Report, 9, 95-104 (http://www.ppo.wur.nl/uk/publications/ publications+ppo/agv/.
Hendriks, (1999). Waarnemen en registreren seizoen 1999. Project Report Masterplan Phytophthora.
Dutch Growers Association (LTO), NL.
Hinds, H. (2000). Using disease forecasting to reduce fungicide input for potato blight in the UK. In:
Schepers, H. (Ed.). Proceedings of the Workshop on the European network for development of an
integrated control strategy of potato late blight, Oostende, Belgium, September-October 1999. PAV
Special Report, 6, 82-89 (http://www.ppo.wur.nl/uk/publications/publications+ppo/agv/).
Hinds, H. (2001). Can blight forecasting work on large farms? In: Westerdijk, C. E. and Schepers, H. T.
A. M. (Eds.). Proceedings of the Workshop on the European network for development of an
integrated control strategy of potato late blight, Munich, Germany, September 2000. PAV Special
Report, 7, 99-106 (http://www.ppo.wur.nl/uk/publications/publications+ppo/agv/).
Hinds, H. (2002). A late blight forecasting project for FL1953 (Courlan), a blight sensitive processing
cultivar. In: Westerdijk, C. E. and Schepers, H. T. A. M. (Eds.). Proceedings of the Workshop on
the European network for development of an integrated control strategy of potato late blight,
Edinburgh, Scotland, September 2001. PPO Special Report, 8, 131-136. Available on-line at
internet website http://www.ppo.wur.nl/uk/publications/publications+ppo/agv/.
Hinds, H. (2003). Blight Forecaster - a web based DSS using UK local and forecast weather data. In:
Westerdijk, C. E., and Schepers, H., T., A., M. (Eds.). Proceedings of the Seventh Workshop of an
European Network for development of an Integrated Control Strategy of potato late blight, Poznan,
Poland, October 2002. PPO Special Report, 9, 211-216 (http://www.ppo.wur.nl/uk/publications/
publications+ppo/agv/.
Kessel, G. J. T., Wander, J. G. N. & Spits, H. G. (2003). Evaluatie van beslissingsondersteunende
systemen voor bestrijding van Phytophthora infestans in aardappel. Project Report Agrobiokon, NL.
(http://www.agrobiokon.eu/).
Kleinhenz, B., & Jörg, E. (2000). Results of validation trials of Phytophthora DSS in Europe in 1999. In:
Schepers, H. (Ed.). Proceedings of the Workshop on the European network for development of an
integrated control strategy of potato late blight, Oostende, Belgium, September - October 1999. PAV
Kleinhenz, B., & Jörg, E. (2001). Results of validation trials of Phytophthora DSS in Europe in 2000. In:
Westerdijk, C. E. and Schepers, H. T. A. M. (Eds.). Proceedings of the Workshop on the European
network for development of an integrated control strategy of potato late blight, Munich, Germany,
September 2000. PAV Special Report, 7, 23-30 (http://www.ppo.wur.nl/uk/publications/publications
+ppo/agv/).
Marquinez, R. (1999). Blight forecast and chemical control in Spain, experience in 1998. In: Schepers,
H., and Bouma, E. (Eds.). Proceedings of the Workshop on the European network for development
of an integrated control strategy of potato late blight, Uppsala, Sweden, September 1998. PAV
McGrath, P. (2000). Beating blight saving sprays. The Grower, April 6, 2000, UK.
Raatjes, P., Germs, F., Verbeek, M., & Loon, J. A. A. van. (2001). Phytophthora disease forecasting in
Egypt. Pesticide Environmental Stewardship Program (PESP). Project report 00066.
Raatjes, P., & Kessel, G. J. T. (2003). Phytophtora hotspots in de Veenkoloniën. Project Report
Agrobiokon, NL (http://www.agrobiokon.eu/).
Raatjes, P., Hadders, J., Martin, D., & Hinds, H. (2004). PLANT-Plus: turn-key solutions for disease
forecasting and irrigation management. Decision support systems in potato management: bringing
models into practice. Wageningen, Netherlands: Wageningen Academic Publishers. 169-185.
Spits, H. G., & Wander, J. G. N. (2001). Field evaluation of four decision support systems for potato late
blight in the Netherlands in 2000. In: Westerdijk, C. E. & Schepers, H. T. A. M. (Eds.), Proceedings
of the Workshop on the European network for development of an integrated control strategy of
potato late blight, Munich, Germany, September 2000. PAV Special Report, 7, 77-90
(http://www.ppo.wur.nl/uk/ publications/publications+ppo/agv/).
Visser, C. L. M. de, & Meijer, R. (2000). Field evaluation of four decision support systems for potato late
blight in the Netherlands in 2000. In: Schepers, H. (Ed.), Proceedings of the Workshop on the
European network for development of an integrated control strategy of potato late blight, Oostende,
Belgium, September - October 1999. PAV Special Report, 6, 135-152 (http://www.ppo.wur.nl/uk/
publications/publications+ppo/agv/).
10
RON R. WALCOTT
INTEGRATED PEST MANAGEMENT OF

BACTERIAL FRUIT BLOTCH OF CUCURBITS
Department of Plant Pathology, The University of Georgia,
Athens Georgia 30607 USA
Abstract. Bacterial fruit blotch (BFB) is the most economically important bacterial disease of cucurbits
worldwide. The pathogen is seed transmitted and affects all stages of many cucurbit crops eventually
causing destructive fruit rots. Like many phytobacterial diseases, the chemical options for BFB
management are limited and primarily include copper based compounds. Additionally, the unavailability
of commercial cucurbits cultivars with BFB resistance makes disease management difficult. Out of
necessity, BFB management involves an integrated approach that seeks to exclude primary inoculum
through the production of clean seeds by isolating seed production fields seed field inspection and
certification, seed health testing, seedling inspection, and prophylactic copper-based management. Thus
far, despite the efforts to exclude Acidovorax avenae subsp. citrulli, BFB outbreaks continue to occur
sporadically worldwide. To develop a more effective integrated disease management strategy, a better
understanding of BFB epidemiology and pathogenesis is needed in fruit and seed production
environments. Additionally, understanding the role of blossoms in seed infection has revealed potential
avenues for integrated disease management. This chapter will explore the current understanding of the
biology and epidemiology of BFB and the integrated disease management strategies currently employed.
1. INTRODUCTION
Bacterial fruit blotch (BFB), caused by the Gram negative bacterium Acidovorax
avenae subsp. citrulli (Schaad et al. 1979) Willems et al. 1990 (Schaad et al., 1978;
Willems et al., 1992), is the most economically important bacterial disease of
cucurbits worldwide. It is arguably the largest threat facing commercial vegetable
seed producers. Since the initial reports of BFB in commercial watermelon fields in
1988 (Latin & Rane, 1990; Somodi et al., 1991; Wall et al., 1990) the disease has
been a perennial threat to cucurbit seed, transplant and fruit producers. Like other
phytobacterial diseases, BFB outbreaks are heavily dependent on temperature and
moisture conditions, and can develop sporadically and rapidly. Additionally, there
are few options for chemical-based management once epidemics are initiated.
It is likely that changes in commercial cucurbit production trends are
responsible for increases in BFB incidence in the US and other countries.
Consolidation of vegetable seed companies and increased demand for hybrid diploid
and triploid (seedless) watermelons has lead to seed production outside the USA.
191
192 R. R. WALCOTT
Additionally, poor germination of triploid seeds necessitates transplanting, which

predisposes seedlings to infection. In the absence of BFB-resistant cucurbit
cultivars, integrated disease management is the most appropriate control strategy for
BFB. Through a combination of exclusion of primary inoculum, avoidance of high
risk locations and environmental conditions, preventative antibacterial chemicals
and eradication, cucurbit producers seek to limit the losses caused by BFB. This
chapter will discuss the history, biology and epidemiology of BFB and outline the
current IPM strategies that are being employed. Additionally, the challenges and
opportunities for improved IPM of BFB will be discussed.
2. BACKGROUND
2.1. Brief History

Bacterial fruit blotch of cucurbits was first reported on watermelon seedlings at the
USDA regional plant introduction (PI) station in Griffin GA, USA in 1965 (Webb &
Goth, 1965). Seedlings of two plant introductions, PI1714103 and 174104, displayed
water-soaking on their cotyledons, followed by necrosis and premature death. The
causal agent was determined to be seedborne and seed transmissible with the ability
to survive for up to 7.5 years in stored seed. In addition to watermelon, the pathogen
infected squash and melon by artificial inoculation. The symptoms of this disease
only included a seedling blight; however in 1969, a disease that caused fruit
symptoms was observed on watermelons at a research farm in Leesburg, FL, USA
(Crall & Schenck, 1969). Typical fruit blotch symptoms were also reported in
Queensland, Australia in 1978 (Vock, 1978), and the causal agent was identified as a
Pseudomonas spp. Studying strains of the seedling blight pathogen at the USDA PI
station, Schaad et al. identified the causal bacterium as Pseudomonas
pseudoalcaligenes subsp. citrulli (Schaad et al., 1978) and its ability to survive in,
and be transmitted by seed was confirmed (Sowell & Schaad, 1979).
In 1987 the first outbreak of BFB in commercial watermelon fields was
observed on Guam and Tinian (Mariana Islands). Fruit rot was extensive and the
pathogen was identified as P. pseudoalcaligenes subsp. citrulli (Wall et al., 1990).
This represented the first time that P. pseudoalcaligenes subsp. citrulli was
associated with a natural BFB and watermelon seedling blight outbreak (Wall &
Santos, 1988; Wall, 1989). In 1989, some 20 years after the disease was first
observed at the USDA plant introduction station in USA, BFB outbreaks occurred
in major watermelon production regions including Florida, South Carolina and
Indiana (Latin & Rane, 1990; Somodi et al., 1991). Because the pathogen is
seedborne, many watermelon growers in the USA filed costly lawsuits against seed
producers, forcing many of them to restrict the sale of seeds in certain high risk
states. Growers were also required to waive their rights to sue seed companies if
BFB developed in their fields. This created an antagonistic relationship between
seed companies and fruit producers.
Between 1990 and 1995 devastating BFB outbreaks occurred sporadically
across major watermelon producing regions in the midwestern, northeastern and
IPM OF SEEDBORNE BACTERIAL DISEASES 193
southern USA causing significant economic losses. By 1995 university and industry
plant pathologists developed a comprehensive set of BFB management guidelines
for seed, transplant and fruit producers. A central aspect of these guidelines was
stringent seed health testing and this strategy was in part responsible for the decrease
in BFB outbreaks between 1995 and 1998. However, by 1999, the incidence of BFB
outbreaks increased with increased occurrences in cucurbits other than watermelon
including melons, cucumber, honeydew and pumpkin (Langston et al., 1999; Martin
& Horlock, 2002; Martin & O'Brien, 1999; Walcott et al., 2000; Zhao et al., 2001).
Additionally, reports of BFB around the world increased noticeably. Currently, BFB
outbreaks continue to occur sporadically in transplant and fruit production systems
worldwide (with the exception of Europe) and all commercial cucurbits appear to be
susceptible to the disease.
2.2. BFB Etiology and Symptomatology

Bacterial fruit blotch is caused by Acidovorax avenae subsp. citrulli Willems et al.
(Willems et al., 1992), a Gram negative bacterium that is a member of the β-
Proteobacteria. Originally classified as P. pseudoalcaligenes subsp. citrulli (Somodi
et al., 1991), the bacterium was reclassified in the genus Acidovorax in 1990
(Willems et al., 1990) based on DNA:rRNA hybridization, DNA:DNA hybridization
and protein and phenotype analyses (Willems et al., 1992). The pathogen is non-
fluorescent on King’s medium B (King, Ward & Raney, 1956) oxidase positive;
arginine dihydrolase negative; motile by a single polar flagellum and capable of
growing at 40°C (Schaad et al., 1978).
Discrepancies between the American type culture collection (ATCC) A. avenae
subsp. citrulli type strain (ATCC 29625) (Schaad et al., 1978) and the strains
associated with initial BFB outbreaks (Somodi et al., 1991), lead to confusion about
the etiology of the BFB pathogen. In particular, the ATCC type strain which was
recovered from seedlings at the USDA PI station failed to cause a hypersensitive
reaction on tobacco (Schaad et al., 1978). One possible explanation for this
discrepancy was differences amongst A. avenae subsp. citrulli populations, which
was suggested by O’Brien and Martin who found that A. avenae subsp. citrulli
strains from North and South Queensland, Australia differed in their ability to utilize
L-leucine and 2-amino ethanol (O'Brien & Martin, 1999).
South Queensland strains, primarily from watermelon, used L-leucine but not
2-amino ethanol and were reduced in virulence on melons. On the other hand, melon
strains from North Queensland utilized 2-amino ethanol but not L-leucine and were
virulent on melons and watermelon. These observations were subsequently
confirmed by pulse field gel electrophoresis DNA fingerprinting, fatty acid methyl
ester analysis, single carbon substrate utilization profiling and pathogenicity assays
of a population of A. avenae subsp. citrulli strains in the USA (Walcott, Fessehaie &
Castro, 2004; Walcott et al., 2000).
Walcott et al. defined the subpopulations as groups I and II, of which group II
strains were aggressive on watermelons but weakly so on other hosts. Group II
strains on the other hand were recovered from a wider range of hosts and were
equally aggressive on melon, pumpkin and squash (Walcott, Fessehaie & Castro,
2004). These finding provide a possible explanation for why BFB initially was
194 R. R. WALCOTT
restricted to watermelons. However, it does not explain the origin of the group I
isolates.
Acidovorax avenae subsp. citrulli can attack all stages of cucurbit plants.
Symptoms start as water-soaked lesions on the undersides of cotyledons. These
lesions may be on the edges but are commonly aligned along the mid-veins of the
cotyledons (Fig. 1A). Over time the lesions dry and develop into reddish to dark
brown lesions along the leaf venation. These types of symptoms are typical to most
cucurbits and in severe cases, coalescing lesions on cotyledons, hypocotyls and
stems cause seedlings to collapse. On true leaves symptoms include water-soaking
and reddish-brown to dark-brown water-soaked lesions that run along the leaf veins
(Fig. 1B). However, as the lesions age, they become inconspicuous and can be easily
mistaken for those associated with fungal diseases.
BFB symptoms on cucurbit fruits range from obvious to inconspicuous. On
watermelon, BFB symptoms initially appear as small, dark olive, greasy, irregularly
shaped, blotch-like water soaked spots on the rind on the upper surface of the fruit.
These lesions may expand rapidly to cover the entire upper surface of the fruit. At
advanced stages, distinctive cracks develop in the fruit lesions and from these
cracks, amber-colored ooze may be released (Fig. 1C). In some cases, effervescent
ooze may also be produced by infected fruit (Fig. 1D). The lesions may also
penetrate into the flesh of the fruit, which eventually may lead to fruit rot. In
contrast, BFB symptoms on melons, especially those with netting (e.g. muskmelon),
start out as small water soaked spots (Fig. 1E). In the case of muskmelon, the netting
does not develop over lesions and lesions do not expand on the rind surface but
appear as small inconspicuous pits (Fig. 1F). These lesions extend into the fruit
resulting in rotten cavities.
Figure 1. Bacterial fruit blotch symptoms including: A) water-soaked lesions on cantaloupe

cotyledons; B) reddish brown lesions on cantaloupe leaves; C) water-soaking and cracks on
watermelon fruit; D) advanced fruit rot with effervescent ooze; E) small water-soaked lesions
on honey dew fruit; F) sunken surface lesions and internal rotten cavities on muskmelon.
2.3. Host Range and Geographic Distribution

Prior to the late 1990s, BFB outbreaks were reported primarily on watermelon in the
USA and Guam. Outbreaks were reported throughout the central, eastern and
southeastern USA in states including Florida, Texas, Georgia, South Carolina, North
Carolina, Illinois, Iowa, Missouri, Delaware, Oregon, Oklahoma (Black et al., 1994;
Hamm et al., 1997; Jacobs, Damicone & McCraw, 1992; Latin & Rane, 1990;
Somodi et al., 1991).
In general, the disease was limited to regions with hot and humid growing
seasons and it was not observed in cooler, drier regions including California. More
recently, however, the disease has been reported in many cucurbit producing regions
around the world on a range of hosts. BFB has been reported in the USA on
honeydew citron, pumpkin, cantaloupe and wild bur gherkin (Isakeit, et al., 1997;
Isakeit, Black & Jones, 1998; Langston et al., 1999; Walcott et al., 2000); in Canada
on watermelon (D. Cuppels, Agriculture and Agri-Food Canada, personal
communication); in Australia on watermelon, rockmelon, prickly paddy melon,
honey dew, gramma and cucumber (Martin & Horlock, 2002; Martin & O'Brien,
1999; O'Brien & Martin, 1999); in Mexico, Honduras, Brazil, Nicaragua and Costa
Rica on watermelon and melon (Assis et al., 1999; Macagnan et al., 2003; Mora-
Umana & Araya, 2002; Munoz & Monterroso, 2002); in Taiwan on melon and
watermelon (Macagnan et al., 2003); in Turkey on watermelon (Demir, 1996; Mirik
et al., 2006); in Israel on watermelon and melon (Burdman et al., 2005); in Japan on
watermelon (Shirakawa et al., 2000); in Thailand on watermelon (P.
Thummabenjapone, Khon Kaen University, personal communication) and in China
(Xinjiang, Neimenggu, Fujian, Jillin) on Hami melon and watermelon (Cai et al.,
2005; Fan & Ma, 2004; Jin et al., 2004; Ren et al., 2006; Zhao et al., 2001).
Acidovorax avenae subsp. citrulli has also been reported to be isolated from tomato
seeds in Israel and to cause disease on several solanaceous hosts by artificial
infection (Assouline et al., 1997; Nascimento, Mariano & Silva, 2004). It is likely
that the expanding geographic range of A. avenae subsp. citrulli is due to its
seedborne nature and the worldwide cucurbit seed trade. At present, the
geographical center of origin for A. avenae subsp. citrulli is not known.
2.4. Epidemiology
2.4.1. Seed Production

Acidovorax avenae subsp. citrulli is seedborne and seed transmitted in many
cucurbits. It is well established that infested seeds represent the most important
source of primary inoculum (Hopkins & Thompson, 2002; Rane & Latin, 1992;
Sowell & Schaad, 1979; Webb & Goth, 1965). However, little is known about BFB
epidemiology in seed production environments. In the arid and cool environments
that are routinely used for cucurbit seed production, inoculum could be introduced
on stock seeds or the bacterium could be endemic, originating from indigenous
weeds or local irrigation sources.
196 R. R. WALCOTT
It is likely that the low moisture conditions in seed production fields prevent the
development of typical fruit and foliar symptoms, making it difficult to visually
observe BFB development. Indeed, BFB symptoms are not usually observed in seed
production fields at the time of seedling transplant or when fruits reach harvest
maturity (P. Guzman, California Crop Improvement Association, personal
communication). Despite the lack of obvious fruit or foliar symptoms, infested seeds
are still produced, suggesting the need for a better understanding of BFB
epidemiology in seed production environments.
To start to understand the biology of seed infection, Walcott et al. investigated
the role of blossom invasion in seed infection (Walcott, Gitaitis & Castro, 2003).
Under greenhouse conditions, female blossoms inoculated with A. avenae subsp.
citrulli cells resulted in the production of infested seeds within symptomless fruits.
Acidovorax avenae subsp. citrulli rapidly colonized the stigmas of female
watermelon blossoms and penetrated through the styles via the transmitting tract
tissues to enter the ovaries, seven days after inoculation (Lessl, 2003; Lessl,
Fessehaie & Walcott, 2007). Seeds infected in this manner were capable of
transmitting BFB to watermelon seedlings.
Additionally, in field experiments conducted in Georgia, USA, honey bees that
were allowed to forage in A. avenae subsp. citrulli-infected watermelon plants
transmitted the pathogen to female watermelon blossoms leading to infection of
25% of the seedlots even though no fruit symptoms were observed (Fessehaie et al.,
2005). While honey bees are not employed for pollination in commercial seed
production, these data indicate a need for further investigation of the role of
blossoms as infection courts for infection, as it may provide an avenue for integrated
pest management.
2.4.2. Transplant Production

Typical conditions in watermelon transplant houses are highly conducive for BFB
development. These conditions include high watermelon seedling populations, high
temperatures, high relative humidity, and overhead irrigation that leads to splash
dispersal of bacteria. Infested seeds are the primary inoculum source in transplant
houses and even low levels of infested seeds can initiate severe BFB epidemics.
Seedborne inoculum leads to infected seedlings and overhead irrigation diseminates
A. avenae subsp. citrulli cells to adjacent plants. Additionally, aerosols are generated
by the impact of droplets on infected tissues which also spreads cells throughout the
greenhouse.
Bacteria landing on uninfected seedlings penetrate cotyledons and leaves via
stomata and multiply rapidly in intercellular spaces. Splash-dispersed cells lead to
numerous secondary infection cycles. Under typical greenhouse conditions
asymptomatic plants may carry epiphytic populations of A. avenae subsp. citrulli
that can initiate disease outbreaks under field conditions.
Secondary spread of A. avenae subsp. citrulli can also occur between
proximal greenhouses by movement of contaminated irrigation equipment or by
handling infected plants. Seedlings originating in contaminated greenhouses have
a high risk of developing BFB epidemics in the field.
2.4.3. Fruit Production Fields

In commercial fruit production fields, the most important sources of primary
inoculum are infested seeds (in the case of direct seeded crops) or infected seedlings.
Inoculum may also originate from the previous season’s plant debris, volunteer
watermelon seedlings or from cucurbitaceous weeds (Isakeit, Black & Jones, 1998;
Latin & Hopkins, 1995). However, the epidemiological significance of these sources
of inoculum vary, depending on environment and cropping system.
The development of a BFB epidemic under field conditions is highly dependent
on environmental factors, and conditions of high relative humidity and rainfall are
critical. Bacterial cells are splash-dispersed by wind-driven rain or over head
irrigation and when cells are deposited on uninfected plants they penetrate into
intercellular sub-epidermal spaces by swimming through open stomata or leaf
wounds. Once in the intercellular spaces they multiply rapidly resulting in water-
soaking and the promulgation of secondary inoculum. The bacterium does not move
systemically through the plant and at anthesis, bacteria that are deposited onto the
surfaces of immature ovaries can penetrate through open stomata to initiate fruit
infection. This infection window exists for approximately three weeks after anthesis,
after which the deposition of waxes on the fruit surface prevents stomatal
penetration (Frankle, Hopkins & Stall, 1993). While fruit infection occurs at
anthesis, symptoms do not usually develop until harvest maturity. The rapid onset of
symptoms at harvest is dramatic and can lead to 50-100% yield loss. Because fruit
infections occur early in the season, post-infection chemical treatments are usually
ineffective. At the end of the season, debris or seeds from rotten fruit may allow the
pathogen to survive until the next cycle.
2.5. Trends in Commercial Cucurbit Production

Modern cucurbit fruit production involves practices that significantly improve
production efficiency and fruit quality. These include off shore production of hybrid
diploid and triploid seeds and the use of transplants and grafting to improve
resistance to Fusarium wilt. Unfortunately, these practices predispose cucurbits to
BFB and understanding how these production strategies influence disease
development may inform the development of effective IPM strategies (Fig. 2).
2.5.1. Hybrid Watermelon Cultivars

Traditionally, watermelon seeds were inbred lines produced by open pollination
of bulk-increased breeder seed. However, since the 1970s most commercial
watermelon seeds have been diploid and triploid (seedless) hybrids. Hybrid seed
production requires manual crossing of two parental lines which is labor and time
intensive. To take advantage of low labor costs and to avoid environmental
conditions that favor BFB development, commercial seed production has shifted to
198 R. R. WALCOTT
arid and semi-arid regions in Asia, Central and South America (including western
China, Mexico, Chile, India). It is possible that the shift in cucurbit seed production
to off shore sites has at least in part been responsible for the increase in BFB
outbreaks. However, there is no scientific evidence that A. avenae subsp. citrulli is
indigenous to these regions. In fact, there is debate as to whether the pathogen was
introduced into these regions on commercial stock seed.
Figure 2. Relatedness of seed, seedling and fruit production in commercial watermelon

production, and the potential for introducing Acidovorax avenae subsp. citrulli at each stage.
Black arrows represent potential sources of inoculum.
To further enhance the production of pathogen-free seeds, stock planting seeds

are tested for A. avenae subsp. citrulli and seed fields are located in regions with no
commercial fruit production, and in which strict crop and disease management
strategies including optimum fertilization, weed, insect and disease control are
employed. Additionally, seed fields are inspected for visual foliar and fruit
symptoms by regulatory agencies (e.g. the California Crop Improvement Asso-
ciation and local governmental agencies). Inspections usually occur at transplanting
and at harvest maturity, and seeds from fields with no visible fruit or foliar
symptoms are certified as BFB-free (Sundstrom, Guzman & Stewart, 2003). While
this is certification scheme is successfully employed for other crops, the fact that A.
avenae subsp. citrulli-infected seeds could be produced in symptomless fruits makes
this practice ineffective (Hopkins et al., 1996; Walcott, Gitaitis & Castro, 2003).
2.5.2. Seedling Production
Despite the increased popularity of hybrid watermelons, one distinctive drawback is

poor germination under standard conditions (Grange et al., 2003). To ensure
desirable plant stands and to improve efficiency, cucurbit production has
predominantly migrated to transplanting. The need for commercial sources of
seedlings has spawned a cucurbit transplant industry that is now a critical part of
cucurbit production systems globally. Seedlings are produced under greenhouse
conditions that promote rapid development and subsequently they are sold to
growers for commercial fruit production. With regard to BFB development, the
transplant house is important because one infested seedlot could contaminate an
entire greenhouse, and the seedlings can then introduce BFB into fields across a
wide geographic region.
3. INTEGRATED MANAGEMENT
Because commercial cucurbit production is a multinational system involving seeds
produced in remote locations and regional transplant producers (Fig. 2), there are
many opportunities for the introduction of A. avenae subsp. citrulli inoculum. In the
absence of commercial cultivars with effective BFB resistance or effective
antibacterial chemicals, a multifaceted integrated approach is needed to manage BFB.
These strategies can be organized into avoidance, exclusion, protection,
eradication and resistance. Additionally, since it is widely accepted that seeds
represent the most important source of primary inoculum, significant efforts are
directed at providing clean seeds for commercial cucurbit production.
3.1. Avoidance
Since cucurbit seeds are the most important primary inoculum source for BFB, the
production of pathogen-free seed is important for disease management. Hence, the
selection of seed production area is important to avoid A. avenae subsp. citrulli.
However, many factors are considered when determining the location of seed
production fields including the climate and the availability of labor and
infrastructure to allow crop production, harvest and processing (Lovic & Hopkins,
2003). Seed companies must weigh all of these factors when selecting production
sites, but with regards to BFB management seed fields should be located in areas
with warm; semi-arid to arid climates. Additionally, fields should be selected in
areas with no previous cucurbit seed or commercial fruit production. This avoids the
risk of inoculum survival from one season to the next.
Since the center of geographical origin is unknown for A. avenae subsp. citrulli
it is difficult to predict the risk associated with producing seeds in certain regions.
Infested seeds have been produced when clean stock seeds were planted in fields in
which BFB had not previously been observed. This suggests that at least in some
cases the pathogen can survive in epiphytically in the seed production environment.
Ideally, in addition to climate and logistical considerations, historical data
should be collected and ecological surveys for A. avenae subsp. citrulli should be
200 R. R. WALCOTT
conducted prior to selecting seed production areas. Realistically, however, it may be

impossible to have all of this data for each area but in the least, regions with a
history of BFB or a history of producing A. avenae subsp. citrulli infested seeds
should be avoided. Additionally, seed fields should be isolated from other
commercial cucurbit production fields.
3.2. Exclusion
Seeds free of Acidovorax avenae subsp. citrulli are critical for BFB management.
While the seedborne nature of A. avenae subsp. citrulli was recognized early
(Sowell & Schaad, 1979; Webb & Goth, 1965), the epidemiological significance of
seedborne inoculum was not realized until the mid 1990s, when a comprehensive
BFB management strategy based on stringent seed health testing resulted in a
significant reduction in BFB outbreaks. Seed testing remains a critical component of
BFB management and a disease transmission threshold of 1 infested seed in 30,000
is accepted as the threshold of tolerance. Realistically, however, there is zero
tolerance for A. avenae subsp. citrulli in seed and transplant production systems.
3.2.1. Seed Health Testing

A wide array of seed health tests have been developed for A. avenae subsp. citrulli
but only a few have been accepted commercially (Walcott, 2003; Wang & Cheng,
2001). At present, the most widely accepted assay is the greenhouse seedling grow-
out test, by which samples of seeds (n = 10,000 – 30,000 seeds/lot) are planted under
greenhouse conditions favorable for BFB development (>70% relative humidity and
24-35°C) (see http://www.seedhealth.org/VEG.Crops.pdf). Eighteen days after
planting, the seedlings are inspected visually for seedlings expressing BFB
symptoms. This assay is technically simple and indicates the potential for BFB
seedling transmission. On the other hand, it takes 2-3 weeks for completion and it
requires large amounts of environmentally-controlled greenhouse space and
technicians trained to recognize BFB seedling symptoms. Additionally, subsequent
laboratory assays e.g. serological tests, semi-selective media and polymerase chain
reaction (PCR) assays are required to confirm the identity of bacteria recovered from
seedlings. Finally, despite the fact that the assay is routinely employed, Walcott et
al. reported that the seedling grow out assay could detect only 12.5% (1/8) and
37.5% (3/8) of seedlots (n = 10,000 seeds) with 0.01 and 0.1% infested seeds
respectively (Walcott et al., 2006). Despite being the industry standard, few seed
testing agencies have the capacity to routinely conduct this test.
An alternative to the greenhouse grow-out is the sweatbox assay that similarly
relies on the germination and observation of symptoms on seedlings. With the
sweatbox assay, however, seed samples are planted into growth media in transparent
plastic boxes. This does not require large areas of greenhouse space and it is easier
to maintain the relative humidity and temperature optima necessary for symptom
development. While the ability to standardize the environmental conditions is an
improvement over the greenhouse grow-out assay, it results in the proliferation of
saprophytic fungal microflora that may complicate assay evaluation. Hence, it is
necessary to employ fungicides to limit microflora, and suspects must be confirmed by

laboratory assays including serological tests. Like the greenhouse grow-out assay,
limited data are available on the sensitivity and reliability of the sweatbox assay.
An alternative to the seedling grow-out based assays for cucurbit seed health
testing is the polymerase chain reaction (PCR), which is the in vitro amplification of
nucleic acid sequence targets, specific to the pathogen (Saiki et al., 1988). PCR and
real time PCR (an alternative that couples DNA amplification with the release of a
fluorescent signal that can be monitored in real time) (Schaad & Frederick, 2002)
have great potential to improve the sensitivity, specificity and efficiency of seed
health testing. In general, DNA is extracted from seed samples and used as a
template with specific oligonucleotide primers designed to amplify DNA
specifically from A. avenae subsp. citrulli. Several A. avenae subsp. citrulli primers
have been designed with varying levels of specificity (Jones et al., 2001; Minsavage
et al., 1995; Song et al., 2003; Walcott et al., 2006; Walcott & Gitaitis, 2000).
However, the success of PCR as a seed health assay for A. avenae subsp. citrulli has
been limited by the ability to efficiently extract PCR-quality template DNA from
seeds. This is due to the fact that cucurbit seeds contain compounds that inhibit PCR
and lead to false negative results, or significantly reduce the reliability of the assay
(Walcott & Gitaitis, 2000). To circumvent this obstacle several approaches have
been developed including DNA purification, selective enrichment of A. avenae
subsp. citrulli followed by DNA extraction and PCR, seedling PCR and
immunomagnetic separation and PCR.
Enrichment or BIO-PCR relies on semi-selective media to selectively enhance
the growth of A. avenae subsp. citrulli from seed extracts. Samples of seed extracts
are spread onto selective media and incubated for 24-36 h. During this time small
pin-sized A. avenae subsp. citrulli colonies develop, and can be harvested and used
to extract genomic DNA that can be tested by PCR. This approach has the advantage
of being rapid, highly sensitive and detecting viable cells. This latter advantage is
important because PCR is criticized for detecting DNA that might be associated with
non-viable cells that might yield a false-positive result. On the hand BIO-PCR
requires growth of A. avenae subsp. citrulli on a semi-selective medium, and
depending on the seedlot, microflora might limit the pathogen’s ability to grow
leading to false-negative results.
Seedling PCR seeks to combine the seedling grow-out assay with the power of
PCR, using the seedling tissue and the selective growth medium. Seeds to be tested
are germinated similar to the sweatbox assay and after 14 days the seedlings are
harvested, washed and DNA is extracted from the seedling wash and used for PCR.
Despite the potential of the enrichment PCR approaches, limited data are available
on their reliability, and at present they are not used widely for commercial seed
health testing.
Immunomagnetic separation (IMS) and real time PCR (Olsvik et al., 1994) is
another technique that has been developed for A. avenae subsp. citrulli detection in
202 R. R. WALCOTT
seeds. This technique uses small paramagnetic plastic beads conjugated with
A. avenae subsp. citrulli-specific antibodies to selectively capture and concentrate
target cells from seed extracts. Captured cells can then be rinsed to eliminate non-
target cells and PCR inhibitory compounds and after cell lysis by boiling, DNA can
be used for PCR. Walcott and Gitaitis (2000) found that IMS-PCR had a detection
sensitivity of 10 CFU ⋅ ml -1 and could be completed within 48 h. Additionally, IMS-
PCR was applicable to large commercial seedlots (n = 5000-10000 seeds). In a
direct comparison using artificially infested seedlots, IMS-PCR was twice as reliable
in detecting A. avenae subsp. citrulli in seedlots with 1 and 10 infested seeds in
10000 (Walcott et al., 2006).
3.2.2. Seed Treatments

Seed treatment is an efficient and cost effective approach for managing plant
diseases and many physical and chemical treatments are employed commercially.
Since relatively small amounts of chemicals are applied to seeds, there is a reduced
risk of environmental pollution and seed treatment is compatible with IPM. With
phytopathogenic bacteria, however, the results of seed treatments vary widely,
depending on seed type and the location of the pathogen in the seed. While the exact
location of A. avenae subsp. citrulli in watermelon seeds is not known it has been
recovered from the seed coat and embryo (Rane & Latin, 1992). This explains the
ability of the pathogen to survive for more up to 8 years in stored seeds. Because
of A. avenae subsp. citrulli location under the seed coat, it is likely that the
pathogen would be protected from externally applied antimicrobial chemicals.
Accordingly, many seed treatments have been shown to reduce but not eliminate
BFB seedling transmission. Nevertheless, treatments including seed fermentation,
sodium hypochlorite, hydrochloric acid and biological control with antagonistic
microorganisms have been explored.
Hot water treatment is routinely employed for inactivating pathogens in seeds.
Seeds are generally incubated at 50-55°C for 10-30 min and these conditions are
sufficient to kill pathogen propagules without affecting seed physiology. Wall
reported that hot water treatment of infested watermelon seeds eliminated BFB
seedling transmission (Wall, 1989). In contrast, incubation of infested seeds at 50°C
for 20 minutes reduced, but did not eliminate the pathogen (Rane & Latin, 1992).
One explanation for the discrepancy is that the effectiveness of thermotherapy is
dependent of the ability to uniformly expose bacteria to the critical killing
temperature for the prescribed period of time. Depending on the size of the sample
being treated, it is difficult to ensure that all seeds receive the necessary exposure to
the critical killing temperature. Hence, it is difficult to ensure the complete
eradication of seedborne bacteria by thermotherapy. Since even low levels of seed
infestation can lead to severe BFB outbreaks under highly conducive seedling
transplant production conditions, the high level of risk prevents hot water treatments
from being employed commercially.
Seed fermentation is a commonly employed seed production practice by which

freshly harvested seeds are allowed to incubate in fruit juice for 24 – 48 h followed
by rinsing and drying. While the exact mechanisms of antimicrobial activity are
unknown, it is thought that microflora that grow in the anaerobic conditions and
metabolites of fermentation, combine to kill A. avenae subsp. citrulli cells on and in
seeds. Hopkins et al. demonstrated that fermentation in watermelon juice for 24 h
followed by rinsing and drying completely eliminated BFB seedling transmission,
without adversely affecting seed germination (Hopkins et al., 1996). Fermentation
also improved seed germination after 6 months of storage. Based on these results,
fermentation is routinely employed in commercial seed production; however,
fermentation cannot be employed for certain watermelon hybrids and other cucurbits
because of deleterious effect on seed physiology.
Chemical seed treatments with 1% HCl, CaOCl and NaOCl have also been
evaluated for seedborne A. avenae subsp. citrulli. Treatment with 0.5 - 1% NaOCl
or CaOCl for 15 – 20 min reduced, but did not eliminate BFB seedling transmission
(Hopkins et al., 1996; Rane & Latin, 1992). On the other hand, while Hopkins et al.
(1996) reported that seed treatment with 1% HCl for 15 min eliminated BFB
infection, Rane and Latin (1992) found that BFB seedling transmission was reduced,
but not eliminated, by exposure to 1.8% HCl for 5 min. Hopkins recommended that
a combination of fermentation and HCl or CaOCl was the most effective seed
treatment for BFB. However, this treatment might not be applicable for all cucurbit
cultivars and species.
In response to this concern, Hopkins et al. (2003) explored the potential of
peroxyacetic acid as a seed disinfestant at harvest. Peroxyacetic acid is a commonly
used disinfestant in food and clinical industries, and it has been reported to
significantly reduce BFB seedling transmission. Treatment of artificially infested
seeds with 1600 µg ⋅ ml-1 of peroxyacetic acid for 30 minutes followed by drying at
40°C and low humidity for 48h, completely eliminated BFB seedling transmission.
This treatment also reduced survival and seedling transmission of Didymella
bryoniae (causal agent of gummy stem blight), another important fungal seedborne
pathogen of cucurbits. Currently, wet seed treatment with peroxyacetic acid is
employed for watermelon seed production by many of the major vegetable seed
producers. However, its applicability for other cucurbits must be determined.
Biological control seed treatment employs beneficial antagonistic organisms to
limit the ability of plant pathogens to colonize and infect plant tissues, and as such it
is a desirable strategy for managing phytopathogenic diseases. However, to date
there are few examples of commercial seed treatments for vegetable diseases, and
none for BFB. Fessehaie and Walcott (2005) demonstrated that treatment of A.
avenae subsp. citrulli-infested seeds with A. avenae subsp. avenae strain 99-2
reduced BFB seedling transmission by 96.5% and 100% under growth chamber and
greenhouse conditions, respectively. While this was a promising result, there is a
need to find more suitable biocontrol agents since A. avenae subsp. avenae is
pathogenic on maize. However, through extensive screens Oliveira et al. have
reported several Bacillus species with potential to serve as biocontrol seed
treatments for A. avenae subsp. citrulli (Oliveira et al., 2006).
204 R. R. WALCOTT
3.3. Protection
Traditionally, control of BFB in fruit, seedling and seed production systems is
through the preventative application of copper-based chemicals e.g. Kocide
(Hopkins, 1991; 1995). BFB management requires routine bi-weekly applications
of copper, since it is a contact antimicrobial agent. In general, this represents a
highly effective management strategy. However, under conditions of excessive
rainfall, efficacy is reduced. Additionally, there is a risk of copper resistance
development in A. avenae subsp. citrulli populations (Walcott, Fessehaie & Castro,
2004). Antibiotics including kasugamycin and streptomycin have been evaluated for
BFB management but are not currently used on a wide scale.
3.3.1. Biocontrol Blossom Protection to Limit Seed Infection

With regards to biological control for managing BFB under field and greenhouse
conditions, there have been few (if any) reports of successful attempts. However,
Fessehaie demonstrated the feasibility of biocontrol for protecting watermelon
blossoms and thereby preventing seed infection. Five hours after treatment with
bacterial biocontrol agents or Kocide (0.4% w/v), female watermelon blossoms were
inoculated with 108 A. avenae subsp. citrulli (CFU/ml) and seeds were allowed to
mature. For female watermelon blossom protected with A. avenae subsp. avenae
strain 99-2, 13.8% of the seedlots were infested with A. avenae subsp. citrulli,
which was significantly lower than negative control (63%). Blossom protection with
Kocide also reduced seed infection (24.1%) but this treatment caused blossom
abortion and significantly reduced seed germination. On the other hand, blossom
protection with A. avenae subsp. avenae 99-2 significantly improved seed
germination relative to the negative control (0.1M phosphate buffered saline). These
results indicated that blossom protection might be a feasible approach for protecting
blossoms and limiting seed infection during seed production. Since current hybrid
watermelon seed production requires hand pollination, the blossom protection
approach would be compatible with this system, since lyophilized biocontrol strains
could be applied during pollination. Additionally, since female watermelon
blossoms are open for less than 24 h and are covered during hybrid seed production,
the chances for success would be higher than attempting to control BFB at the field
level. Despite these promising results, it is necessary to determine the role of
blossom invasion in seed production environments, and to evaluate the blossom
protection strategy using more suitable biocontrol strains under field conditions.
3.4. Eradication
The transplant house is a critical aspect in cucurbit production with regards to BFB
epidemic development. Hence, efforts to control BFB in this environment can limit
BFB outbreaks in the field. To exclude A. avenae subsp. citrulli from transplant
facilities, only seeds that have been tested and found pathogen-free should be
employed. However, other cultural practices can significantly reduce the risk of
BFB outbreaks.
To limit BFB outbreaks from inoculum sources other than infested seeds, steps
should be taken to limit the survival of A. avenae subsp. citrulli in the greenhouse.
Latin, Tikhonova and Rane (1995) demonstrated that A. avenae subsp. citrulli
survived for up to 63 days on used plastic trays with plant debris, while the
bacterium did not survive on trays treated with 0.5% NaOCl. Additionally, pathogen
spread was significantly increased with overhead irrigation, relative to sub irrigation.
Overhead irrigation leads to splashing and the generation of aerosols that
disseminate bacterial pathogens. This could be significantly reduced by using ebb-
and-flow or flood and drain irrigation. In this system the flats or polystyrene trays in
which the seedlings are grown are placed on a water tight benches that can be filled
and drained with irrigation water. In the absence of ebb and flow systems, irrigation
should be conducted at low water pressure.
To further limit the risk of pathogen dissemination, plants should be handled as
little as possible and gloves should be used. Additionally, decontamination stations
comprised of 70% ethyl alcohol and paper towels should be maintained at each
entrance of each greenhouse and workers should decontaminate tools and equipment
as they move from one house to another. The movement of equipment between
greenhouses should be limited. At the end of the planting cycle, vegetative material
should be removed from the greenhouse and discarded. Planting trays and bench
surfaces should be disinfested with appropriate disinfectants e.g. NaOCl, and
quaternary ammonium salts. To prevent pathogen survival in field conditions all
plant debris should be ploughed under to facilitate rapid decay. Additionally, three
year rotations to non-cucurbit crops should be practiced.
3.5. Resistance
Plant resistance is the single most effective strategy for managing plant diseases. In
addition to cost effectiveness, resistance-based strategies are compatible with other
integrated disease management approaches. Unfortunately, to date there are no
commercial cucurbit cultivars with resistance to BFB. Different levels of resistance
were reported in commercial watermelon cultivars with Garrisonian being immune
and Congo being resistant (Goth & Webb, 1981; Sowell & Schaad, 1979). However,
these findings were contradicted by Hopkins et al. who found that no cultivars were
immune and those previously thought to be resistant were susceptible (Hopkins,
Thompson & Elmstrom, 1993). These author concluded that cultivars with dark
rinds were more resistant to BFB than those with light-colored rinds and that
moderate levels of resistance could be overcome under conditions that were
favorable for BFB epidemic development. Subsequently, two plant introductions out
of 1344 accessions (PI 482279 from Zimbabwe and PI 494817 from Zambia) were
found to be resistant under field and greenhouse conditions. At present, however,
there is a need to introgress this resistance into cucurbits with agronomically
desirable traits (Hopkins & Thompson, 2002).
206 R. R. WALCOTT
4. CONCLUSIONS
The key objectives of IPM are to limit the environmental impact of agriculture and
to reduce the chances of resistance development to pesticides, while maintaining
acceptable levels of productivity. In the absence of a wide array of chemical options,
IPM is necessary not to reduce the environmental impact of commercial cucurbit
production but to provide adequate levels of BFB management. Modern cucurbit
production is now a global activity where seeds are produced in remote locations in
foreign countries, shipped in to localities and grown under greenhouse conditions to
produce seedlings which are then distributed over large distances. This elaborate
production system presents a significant challenge for managing BFB. Integrated
pest management involving the strategies described in this chapter, represent a
holistic multi-tactic strategy for addressing the threat of BFB at all levels of cucurbit
production. At present, this approach is the best option for BFB of cucurbits. Thus
far, BFB outbreaks in the USA have been substantially reduced relative to the early
1990s. However, BFB outbreaks still occur sporadically with devastating economic
consequences.
Integrated pest management across the seed, seedling and fruit production
systems is the only viable option for BFB management. However, the modern
practices of cucurbit production present barriers to effective disease control.
Namely, while it is logical to produce seeds in cool, dry climates, there is very little
known about BFB epidemiology in these areas. Without specific knowledge about
sources of inoculum, survival and spread of the pathogen it is impossible to develop
effective management strategies to limit seed infestation. Additionally, while
transplanting yields many benefits, greenhouses conditions increase the risk of BFB
development and dissemination. This fact is well known; however, there have been
limited studies to understand the spatial spread of BFB in greenhouses or to develop
specific strategies for disease management.
To address the pervasive problem of Fusarium wilt of cucurbits, grafting of
watermelon on to a resistant cucurbit rootstock is becoming a commonly employed
strategy. Unfortunately, it is likely that this practice will further increase the risk of
BFB development, through the creation of wounds and the potential for grafting
implements to spread the bacterium from plant to plant.
Finally, despite the fact that BFB is a significant threat to cucurbit production,
little is known about the genetic determinants of pathogenicity for A. avenae subsp.
citrulli. However, recently, the genomic sequence for AAC00-1 was completed and
this resource might lead to a better understanding of A. avenae subsp. citrulli
biology and pathogenesis, and eventually lead to novel disease management
strategies that are compatible with IPM.
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11
A. OBRADOVIC*, J. B. JONES, B. BALOGH AND M. T. MOMOL
INTEGRATED MANAGEMENT OF TOMATO

BACTERIAL SPOT
Plant Pathology Department, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville 32611, USA
Abstract. Bacterial diseases of plants play an important role in the world’s agriculture by reducing yield
and marketability of particular crops or by limiting their production in areas with environmental
conditions conducive for disease development. Plant pathogenic bacteria present many obstacles in
developing efficient plant protection practices. In spite of technological advances, there is no bactericide
that can be efficiently used for control of plant bacterial diseases. Due to lack of adequate chemical based
bactericides plant pathologists constantly search for alternatives and possibility for their integration with
preventive measures in order to develop sustainable disease control strategy. Tomato bacterial spot
management currently relies on use of pathogen-free seed and transplants, elimination of volunteer
tomato plants, resistant cultivars, and frequent application of a copper-based bactericides. However, these
practices are ineffective in regions where hot and humid weather favor spread of the pathogen and
development of the disease. Novel technologies, such as application of systemic acquired resistance
inducers and use of biocontrol agents integrated with conventional practices, represent new quality in
plant protection and provide increase in efficiency of the disease management.
1. INTRODUCTION
Fungal and bacterial plant pathogens are responsible for serious economic losses,
especially in conditions favorable for disease development. Although fungal
diseases account for many of the disease problems based on their prevalence and the
amount of loss caused in plant production, there are a significant number of bacterial
diseases that are considered extremely destructive and therefore a threat to crops
worldwide. Control of plant diseases is achieved by utilizing several regulatory,
cultural, biological and chemical tactics. However, disease control still remains a
challenge for farmers and crop protection specialists.
Fresh-market tomato is the most valuable vegetable crop in Florida. In recent
years, commercial tomato production in the state reached over 40,000 acres with
gross sales valued at approximately 600 million dollars. Successful management of
diseases has always been a vital necessity in Florida tomato farming. Effective
*
Present address: Plant Pathology Department, Faculty of Agriculture, University of Belgrade, 11080
Belgrade-Zemun, Serbia
211
212 A. OBRADOVIC ET AL.
control of tomato diseases requires a good understanding of the causal agent

biology, the susceptibility of the host plant to the pathogen, and the effect of
environmental factors, such as temperature and humidity, on host-pathogen
interactions.
Control of tomato bacterial spot, caused by Xanthomonas spp., has been a major
challenge for many tomato growers all over the world. This challenge is a direct
result of pathogen variability, mutation or gene transfer in the pathogen when
confronted with plant resistance genes or bactericides, high pathogen multiplication
rate during optimal conditions for disease development, and lack of adequate
chemical-based control measures. Integrated disease management offers solutions
for some of these challenges.
Figure 1. Tomato plants infected by Xanthomonas perforans, showing leaf watersoaking

lesions (A, natural infection), “shothole” lesions (B, artificial infection) and bacterial
streaming from infected tomato leaf tissue (C, cross section, light microscopy). “Scabby”
lesions on tomato fruits infected by Xanthomonas vesicatoria (D).
2. TOMATO PRODUCTION
2.1. Tomato Production Value

Vegetables, including tomato, are a very important component of the human diet and
health. In terms of economic well being of many vegetable producers, tomato is a
major component. The total world tomato production has increased almost three-fold
MANAGEMENT OF BACTERIAL SPOT 213
over the last 30 years. Annual consumption of fresh tomatoes and tomato products
per person is increasing steadily. Market demands disease-free tomato transplants
and seed as well as high quality tomato fruits. This requires the continuous
improvement of production technologies, including disease management practices
that are economically practical, environmentally responsible and socially acceptable.
Technological advances in seed technology, transplant production, cultivar
development, molecular biology, cultural practices (furrow or drip-irrigation,
fertilization, and plasticulture), integrated pest management, and post-harvest
handling have resulted in increased yield and quality, while trying to optimize the
cost of production.
2.2. Cultivation Technologies

Tomatoes are produced as field or greenhouse crops. The majority of field
production is for processing tomatoes. The unique climate of Florida allows for field
production of fresh-market tomatoes. Low tunnel plastic culture in the field helps to
adjust to market windows in some regions. Greenhouse technologies, structures, and
equipment vary from very high-tech and computer-controlled glasshouses to simple
unheated plastic tunnel or row cover tomato culture. For each climatic condition
there are unique challenges related to production and disease or pest management.
2.3. Tomato Bacterial Diseases

There are several bacterial diseases on tomatoes but bacterial spot, speck, canker,
and wilt are the major ones (Jones et al., 1991a). Bacterial spot of tomato, caused by
Xanthomonas spp., is a widespread disease. High temperature and high humidity are
conducive for the disease development. Therefore, the disease is hard to control
especially in subtropical and tropical regions with perennial occurrence of the
pathogen. Under optimal conditions the bacterium causes leaf spotting and severe
defoliation with great reduction in yields. Furthermore, fruit marketability can be
seriously affected due to occurrence of scabby lesions (Fig. 1).
Bacterial speck, caused by Pseudomonas syringae pv. tomato, is also distributed
worldwide. The disease is favored by low temperatures (18-24oC) and high
humidity. All above ground parts can be affected by dark brown to black lesions,
which reduce fruit marketability.
Unlike bacterial spot and speck, occurrence of bacterial canker (Clavibacter
michiganensis subsp. michiganensis) is sporadic but still can be devastating. The
principal symptoms are systemic wilt of the plant and characteristic spots referred to
as “bird’s-eye” spots on infected fruits. The disease seems to be present in cool
weather conditions.
Bacterial wilt is another serious disease of tomato plants growing in warm,
subtropical and tropical regions of the world. The pathogen, Ralstonia
solanacearum, causes rapid plants death with losses reaching more than 50% of the
crop, in naturally infested fields.
3. BACTERIAL SPOT OF TOMATO
3.1. Historical Perspective

Bacterial spot disease of tomato was reported in 1914 in South Africa and was later
described in 1920 as tomato canker by Doidge (1921). Gardner and Kendrick (1921)
described the same disease in 1921 in the United States and referred to it as bacterial
spot. Doidge (1921) designated the causal agent Bacterium vesicatorium while
Gardner and Kendrick (1921; 1923) were prepared to name the organism B. exitiosa.
However, because of Doidge’s prior publication they accepted her nomenclature. At
the time there were only minor differences between the strains, with those described
by Doidge being feebly amylolytic, whereas those isolated from Gardner and
Kendrick (1921) being strongly amylolytic.
In 1957 Sutic identified a bacterial disease of tomato responsible for the “bird’s
eye” symptom on fruit and originally classified it as Pseudomonas gardneri (Sutic,
1957). Although this bacterium was not formally placed into the genus
Xanthomonas, Dye (1962; 1966; Dye, Starr& Stolp, 1964) compared P. gardneri
with a group of xanthomonads, identifying it as a typical member of this group
based on morphological and biochemical tests.
It was not until the 1990s that Stall et al. (1994) and Vauterin et al. (1995)
independently discovered that X. campestris pv. vesicatoria consisted of two
genetically distinct groups. Vauterin et al. (1995) re-classified the xanthomonads
associated with tomato, that had been thus far designated as X. campestris pv.
vesicatoria, into two species, X. vesicatoria and X. axonopodis pv. vesicatoria.
Vauterin et al. (1993) placed the non-amylolytic strains in X. axonopodis pv.
vesicatoria and the strongly amylolytic strains in X. vesicatoria. These two
organisms were placed in separate groups based on phenotypic and genetic tests.
The non-amylolytic and amylolytic strains were placed in groups A and B,
respectively (Stall et al., 1994) (Table 1).
In 1991 tomato race 3 (T3) was identified in Florida and was determined to be a
new Xanthomonas group associated with tomato (Jones et al., 1995). Originally, it
was considered to be a variant of group A strains, but based on phenotypic and
genetic analyses, it was determined to be distinct such that it was placed in group C
(Jones et al., 2000) (Table 1). Jones et al. (2000) determined that the original X.
gardneri strain was genetically very closely related to strains they isolated in Costa
Rica. Based on a number of phenotypic and genetic tests these strains were placed in
Group D (Table 1).
The nomenclature for three of the xanthomonad groups associated with tomato
was recently revised. The A group, which was X. axonopodis pv. vesicatoria, was
elevated to species status and named X. euvesicatoria; the C group, previously
identified as X. axonopodis pv. vesicatoria, was elevated to species status and
named X. perforans; and the D group, which was of uncertain status, was given
species status and named X. gardneri (Jones et al., 2004) (Table 1). The fourth
group, which consists of B group strains, remained in X. vesicatoria.
Table 1. Phenotypic groups for phytopathogenic xanthomonads associated with tomato
Group a Nomenclature b Reacts Specific DNA PFGE Starch Pectate

with protein Homology group
MAbs groups
A1 Xanthomonas 1, 21 32 1 A - -
euvesicatoria kDa
A2 Xanthomonas 1, 21 25 1 A + +
euvesicatoria kDa
B Xanthomonas 8, 15 27 2 B + +
vesicatoria kDa
C Xanthomonas 30 27 1 C + +
perforans kDa
D Xanthomonas 8 27 3 D - -
gardneri kDa
a
Based on work by Jones et al. (2000)
b
Based on Vauterin et al. (1995) and Jones et al. (2004)
3.2. Host-Pathogen Interactions

3.2.1. Host Range
The host range of Xanthomonas spp. associated with tomatoes includes tomato
(Solanum lycopersicum, formerly Lycopersicon esculentum), pepper (Capsicum
annuum), chili pepper (Capsicum rutescens) and Solanum pimpinellifolium
(formerly Lycopersicon pimpinellifolium).
3.2.2. Resistance and Avirulence Genes

Tomato genotypes vary in susceptibility to certain strains of Xanthomonas complex.
Specific tomato genotypes have been very important in characterizing races. Three
sources of resistance to strains of Xanthomonas have been reported in Lycopersicon
species (Astua-Monge, 2000a; 2000b; Jones & Scott, 1986), each resulting in a
hypersensitive reaction to certain strains pathogenic to tomato.
The tomato group A strains (X. euvesicatoria) contain the single avirulence gene,
avrRxv, which induces an avirulent reaction on Hawaii 7998 (Whalen et al., 1993;
Minsavage, Jones & Stall, 1996). The tomato group C strains (X. perforans) contain
avirulence genes, avrXv3 and avrXv4, which induce avirulent reactions in L.
esculentum genotype Hawaii 7981 and L. pinnellii LA716, respectively (Astua-
Monge, 2000a & b; Jones et al., 1995). In tomato, four races have been
differentiated, based on differential reactions in four tomato genotypes (Table 2).
Table 2. Differential reactions of tomato genotypes to xanthomonad races a.
Tomato differentials
Race Bonny Best H 7998 H 7981 LA716
T1 S HR S S
T2 S S S S
T3 S S HR HR
T4 S S S HR
a
S = susceptible; HR = hypersensitive reaction.
3.3. Distribution of Pathogen Groups

Group A (tomato race 1 - T1) and group B (T2) are worldwide in distribution
(Bouzar et al., 1994) (Fig. 2). Both races have been identified in North America,
South America, Europe and Australia. A limited collection from Europe and Africa
revealed mostly T1. In Serbia, strains from groups A, B and D have been identified
(Obradovic et al., 2004b). In the Caribbean islands only group A strains have been
identified, whereas A, B and D strains were detected in Central America (Bouzar et
al., 1994; 1999; Jones et al., 2000). The C group of strains (T3) was originally
identified in Florida (Jones et al., 1995) and has since been identified in Mexico
(Bouzar et al., 1996), Thailand (Jones et al., unpublished), Brazil (Quezado-Duval et
al., 2004) and Canada (Cuppels, personal communication). In 1998 tomato race 4,
which is a variant of T3 strains and is in group C, was identified in Florida
(Minsavage et al., 2003).
3.4. Ecology and Epidemiology

Bacteria hve been shown to survive on seed for years, and in crop residues for weeks
to months, depending on the weather conditions, on volunteer tomato plants present
for at least one season after the original crop, and in association with weeds for short
periods of time. In a study by Jones et al. (1986), weeds including solanaceous
species were found to harbor low populations of the bacterium. Laub and Stall
(1967) determined that Physalis minima and Solanum nigrum were not hosts of X.
campestris pv. vesicatoria. These studies in Florida revealed that the weed species
were unlikely hosts for the bacterium, but that they may serve as short-term
inoculum sources.
Inoculum for primary disease can originate from infested seed, volunteer tomato
plants, crops in established fields in close proximity to transplant production or
newly established fields. The pathogen enters through natural openings including
stomata and hydathodes and through wounds created by wind-blown sand or various
other factors. Infection is favored by high relative humidity and temperatures
between 25 and 28 ºC.
Shifts in tomato race populations have been well documented. In Florida T1 was
the only race identified on tomato until 1991 at which time T3 became established
(Jones et al., 1995). Race T3 has a competitive advantage over T1 strains in Florida
as determined in field studies (Jones et al., 1998). T3 strains have been shown to
produce three bacteriocin-like compounds that are inhibitory to T1 strains (Tudor-
Nelson et al., 2003). Hert et al. (2005) demonstrated that these compounds do
produce strains with a competitive advantage over non-producing sensitive ones.
Figure 2. Geographic distribution of genotypic/phenotypic groups of xanthomonads

pathogenic on pepper and tomato
4. INTEGRATED APPROACH TO BACTERIAL SPOT MANAGEMENT
4.1. Bacterial Spot Control Practices and Recent Trials

Improvements in agricultural cropping systems necessitated a concomitant need for
advances in disease control management. In order to prevent economically
unacceptable losses in yield and quality of agricultural crops, continuous efforts
have been made to identify chemicals and other strategies that could be effectively
used for controlling plant pathogenic bacteria. Tomato bacterial spot is a constant
threat to tomato commercial production (Jones et al., 1991a), but it is especially
severe in Florida and the southeastern USA, where weather conditions (high
temperature, high humidity and rain) favor disease development (Momol et al.,
2002). Thus, there was urgent need for disease management strategies that resulted
in improved control and yield responses.
Chemical control of tomato bacterial spot mostly relied on the application of the
antibiotic streptomycin and copper-based compounds. Antibiotics were introduced
for agricultural use in several countries. The most frequently used antibiotics against
plant bacterial diseases were formulations of streptomycin or streptomycin and
oxytetracycline (McManus et al., 2002). However, soon after widespread application
of antibiotic-based compounds in plant protection, resistant strains of the tomato
bacterial spot pathogen emerged (Minsavage, Canteros & Stall, 1990; Ritchie &
Dittapongpitch, 1991; Thayer & Stall, 1961). In addition, there was a major concern
in many countries that antibiotic application and release in the environment might
cause natural resistance in many bacterial species rendering useless, for medical
treatments, not only these but also other related antibiotics.
The use of copper-based compounds in agriculture started in early 1800’s.
Copper-containing bactericides proved to be an effective preventive treatment
against many bacterial diseases, mostly leaf spots and blights. However, efficacy of
copper bactericides for control of tomato bacterial spot was compromised by
reduced sensitivity of the bacterium as a result of the excessive application of these
chemicals. Copper-tolerant strains became quite prevalent in the 1980s (Jones et al.,
1991b; Marco & Stall, 1983; Martin, Hamilton & Kopittke, 2004; Ritchie &
Dittapongpitch, 1991). Although the combination of copper bactericides and
ethylene-bis-dithiocarbamates resulted in improved disease control, the combination
remained ineffective when weather conditions were optimal for the disease
development (Jones & Jones, 1985).
Recently, a novel class of chemicals called “plant activators” has been
introduced in disease management programs (Louws et al., 2001; Qui et al., 1997).
One of them, acibenzolar-S-methyl (Syngenta Crop Protection), showed excellent
potential for control of bacterial spot of tomato (Louws et al., 2001; Obradovic et al.,
2004a, 2005). However, some results showed adverse effects of this compound on
tomato growth and yield (Louws et al., 2001).
Foliar applications of ammonium lignosulfonate (ALS), a product derived from
the wood pulping process, and the fertilizer potassium phosphate (KP) were tested
for their ability to control the disease under both greenhouse and field conditions
(Abbasi et al., 2002). A three-year field study demonstrated that ALS and KP
significantly reduced bacterial spot disease severity on tomato and pepper foliage
and fruits, compared to untreated control. However, further studies of optimal spray
intervals, rates, and adjuvants are needed.
Weekly sprays of neem oil and fish emulsion reduced disease severity on the
foliage of inoculated tomato and pepper plants under both greenhouse and field
conditions in two consecutive seasons (Abbasi, Cuppels & Lazarovits, 2003). The
disease incidence on the fruit of these plants was reduced but the effect was not
always statistically significant. These results suggested that tested products may
enhance efficiency of bacterial spot management programs.
Al-Dahmani et al. (2003) investigated the effects of foliar sprays with compost
water extracts (compost extracts) on severity of bacterial spot of tomato. Efficacy of
the compost extracts ranged from being effective on tomato seedlings in greenhouse
bioassays, to marginally effective on fruit and ineffective in controlling foliar
symptoms in the field under high disease pressure. Even though some degree of
efficacy of compost extracts was observed, it was not comparable to the effect of a
mixture of copper hydroxide and chlorothalonil (Al-Dahmani et al., 2003).
Besides the continuous search for an effective chemical treatment, extensive
research has focused on identifying biological control agents for use in plant
protection. Among the limited number of biological agents commercially available
for the control of bacterial diseases, the most encouraging results for control of
bacterial spot on tomato were obtained using host specific phages (Balogh et al.,
2003; Flaherty et al., 2000; Obradovic et al., 2004a). Application of phage mixture,
prepared according to the phage host range and specificity to predominant race,
reinforces the importance of timely and accurate diagnosis of the disease and correct
identification of the pathogen.
4.2. Integrated Strategies

In contrast to the search for efficient antifungal natural products and analogues, the
task to find bactericides suitable for crop protection turned out to be exceedingly
difficult. The lack of a bactericidal “silver bullet”, comparable to synthetic
fungicides in control of plant pathogenic fungi, has continually forced plant
pathologists to search for non-pesticide control/management strategies for
controlling plant bacterial diseases. The most frequently implemented approaches
were to use pathogen-free seed or planting material, genetic host resistance, cultural
and sanitation practices, and biological control agents. However, none of the above
non-chemical treatments have been able to stand alone and provide efficient control.
Therefore, tomato bacterial spot control is currently based on an integrated
approach by combining proper cultivation practices, plant resistance, bactericides,
plant activators and biological control agents (Obradovic et al., 2004a). A
combination of practices, such as the use of pathogen-free seed and transplants,
elimination of volunteer tomato plants, resistant cultivars, and frequent application
of a copper/mancozeb mixture (Momol & Pernezny, 2005) reduces the damage from
tomato bacterial spot. However, continuous research efforts have been made in order
to develop new control methods, which could increase effectiveness and
sustainability of present disease management strategies.
In 1989, Jackson developed a method for selecting bacteriophage strains based
on their host specificity (h-mutant phages) (Jackson, 1989). Based on this
technology, it was necessary to determine the efficacy of h-mutant bacteriophage
mixtures for control of tomato bacterial spot. The initial trials were conducted on
overhead-irrigated tomato transplants and resulted in reduced disease incidence on
plants irrigated with water containing phages specific to the bacterial spot pathogen
(Somodi, Jones & Jackson, 1997). In greenhouse and field experiments, treatment of
plants with a mixture containing four h-mutant phages provided effective control of
two predominant tomato races of X. campestris pv. vesicatoria (Flaherty et al.,
2000). However, these experiments showed that phage efficacy was greatly
dependent on their susceptibility to environmental conditions (desiccation, UV light)
and ability to maintain their population on plant surfaces. After investigating various
treatment combinations it has been shown that a mixture of phages formulated with
powdered skim milk and sucrose (Balogh et al., 2003), applied close to sunset
(Balogh et al., 2003; Flaherty et al., 2000) resulted in significantly less disease on
plants compared to the standard copper treatment and untreated control.
Besides bacteriophages, several other alternatives associated with a reduction in
severity of tomato bacterial diseases were investigated. They included activation of
natural plant defense mechanisms by systemic acquired resistance (SAR) inducers
(Louws et al., 2001; Qui et al., 1997), plant growth-promoting rhizobacteria (PGPR)
(Ji et al., 2006), and application of antagonistic bacteria (Byrne et al., 2005; Wilson
et al., 2002). However, many of the treatments resulted in control very often not
comparable to conventional control practices. In an effort to develop more
sustainable strategies for reducing bacterial spot severity on tomato, various
combinations of biocontrol agents, including strains of PGPR, bacterial antagonists,
unformulated phages and SAR inducers (harpin, acibenzolar-S-methyl) were
investigated in greenhouse experiments (Obradovic et al., 2005). The intention was
to integrate some of these practices, optimizing their benefits in control of tomato
bacterial spot in the greenhouse, aiming at designing an integrated management
strategy for disease control in commercial tomato field productions.
Although phage effectiveness was demonstrated in the field in a previous study
(Flaherty et al., 2000), greenhouse experiments showed inconsistent performance of
the phage treatment for disease control. A single application of unformulated phages
used in this study probably contributed to this inconsistency. However, phage
treatment combined with application of SAR-inducer compounds improved disease
control compared to the application of SAR inducers alone, indicating a combination
of treatments with high potential for integrated disease management program
(Obradovic et al., 2005).
In these experiments the harpin-phage combination significantly reduced disease
severity compared to harpin alone. The other SAR-inducer compound (acibenzolar-
S-methyl – ASM) effectively controlled bacterial spot under greenhouse conditions.
However, plants treated with ASM developed a strong defense response resulting in
an HR-like reaction when challenged by high concentrations of the pathogen.
Application of phage in combination with ASM resulted in elimination of HR-type
lesions. It was also shown in that study that phage application decreased the
pathogen population on the leaf surface and consecutively reduced its ingress and
intensity of plant defense reaction induced by ASM (Obradovic et al., 2005).
After identifying several combinations of treatments that effectively controlled
tomato bacterial spot in the greenhouse experiments, these combinations of SAR
inducers and host-specific phages were tested in field trials carried out in north and
central Florida. During three consecutive seasons, application of phages constantly
provided a significant reduction in bacterial spot severity compared with the
untreated control. In addition, phage-treated plants produced significantly more
marketable fruit than plants not receiving the phages. An improved success of the
phages, in all likelihood, was a consequence of mixing them with powdered skim
milk and sucrose as described by Balogh et al. (2003), providing greater stability
and prolonged activity of formulated phages on leaf surfaces. Although copper-
sensitive strains were used in this study, which favored more effective control of
bacterial spot with copper bactericides, phages applied twice a week were either
more effective or equally effective compared with the standard copper-mancozeb
treatment. Application of ASM significantly reduced disease severity compared to
untreated control. However, integration of ASM and phage treatments provided an
additional reduction in disease pressure and resulted in more efficient foliar disease
control than ASM, phage, or copper-mancozeb alone (Obradovic et al., 2004a).
The fact that ASM can trigger a natural defense response against other tomato
diseases (Benhamou & Belanger, 1998, Momol et al., 2003) increases the potential
benefit of this compound in integrated disease management and offers potential for
controlling multiple bacterial diseases. Both phages and ASM have unique modes of
action, targeting the pathogen only, not affecting beneficial microflora and not
overlapping with any of conventional practices that are in use at present. When they
are applied as recommended, they represent no threat for humans and environment.
Being compatible with other treatments, they are suitable for integration in complex
tomato disease and pest management programs.
Integrated application of phages, ASM and other practices is currently widely
used in greenhouses and production fields in Florida as a part of a standard
integrated management strategy for tomato bacterial spot control.
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Whalen, M. C., Wang, J-F, Garland, F. M., Heiskell, M. E., Dahlbeck, D., Minsavage, G. V., et al.
(1993). Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance
on tomato line Hawaii 7998. Molecular Plant-Microbe Interactions, 6, 616-627.
Wilson, M., Campbell, H. L., Ji, P., Jones, J. B., & Cuppels, D. A. (2002). Biological control of bacterial
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1284-1292.
12
GIOVANNI BUBICI AND MATTEO CIRULLI
INTEGRATED MANAGEMENT OF VERTICILLIUM

WILT OF TOMATO
Dipartimento di Biologia e Patologia Vegetale,
Università degli Studi di Bari, Bari, Italy
Abstract. Verticillium wilts of tomato, caused either by Verticillium dahliae or V. albo-atrum, and their
control are revised. Introgression of the single dominant gene Ve in all the commercial tomato cultivars
have reduced the importance of the disease. However, the race 2 of V. dahliae breaks the Ve resistance.
Once a pathogen-free field is not available or an infected plant material is accidentally used, an integrated
approach is currently the best way to limit the damage caused by Verticillium wilt. Here, several control
measures including selecting field criteria, heat treatments, solarization, sanitation, tillage, use of plant
residues, weed control, resistant rootstocks, cultivars, fertilization, irrigation, chemical treatments and use
of microbial antagonists are revised.
1. INTRODUCTION
Verticillium wilt of tomato is caused either by Verticillium dahliae Kleb. or V. albo-
atrum Reinke & Berth. These species are closely related such that in the past there
has been some confusion on their description or identification in many reports. Like
all species of the genus Verticillium (order Hyphomycetes), V. dahliae and V. albo-
atrum produce conidia in moist droplets from the tips of phialides arranged in
verticils on conidiophores (Fig. 1A). However, the two species are distinguished by
some fundamental characters. Basically, V. dahliae produces subspherical to
elongate melanised true microsclerotia, while V. albo-atrum produces resting
mycelium consisting of dark-thick walled hyphae. Conidiophores structure, and
conidiophores and conidia size are also diagnostic elements. No sexual state is
known for both V. dahliae and V. albo-atrum. Microsclerotia (Wilhelm, 1955) or
resting mycelium (Luck, 1954) are responsible for the long perennation of the fungi
in soil (Fig. 1B). The long survival together with the wide range of hosts and the
wide spread in the world represent the major problems for their control.
Verticillium dahliae is present between 60°N and 50°S latitude, while V. albo-
atrum is present at the same latitudes but it is excluded between 20°N and 20°S
latitude, demonstrating that the latter species is adapted to cooler regions (Pegg,
1984). However, V. dahliae is the most common species causing crop damage of
high economical importance.
225
226 G. BUBICI AND M. CIRULLI
A review of the plant host list of Verticillium spp. was recently made by Pegg
and Brady (2002). More than 400 species of trees, shrubs, vegetables, ornamentals,
weeds and other herbaceous plants belonging to 76 botanical families are included in
such list.
Figure 1. Conidiophore bearing conidia heads (A), and microsclerotia (B)

of Verticillium dahliae.
The most widespread pathogenic species on tomato is V. dahliae. The pathogen

penetrates actively through the root tips or through wounds on roots. Once
penetrated, the developing mycelium colonizes the cortex and then the xylem, thus
leading to a systemic infection (Huisman & Gerik, 1989). The host plant reacts to
the pathogen by producing callose deposits in the paravascular parenchyma, and gels
and tyloses in the vessels. Infected vessels are infused by pre-formed phenolics
which are responsible for vascular browning after oxidation by polyphenol oxidases.
Presence of the fungal structures, gels and tyloses in the xylem cause occlusions
which slow down the normal water stream. Also, fungal lytic enzymes, growth
substances and phytotoxins are involved in the pathogenesis (Beckman, 1981).
On tomato, symptoms occur as wilting of basal leaves beginning at a leaflet
margin, later developing into yellow and then brown V-shaped patterns. As the
MANAGEMENT OF VERTICILLIUM WILT 227
fungus progresses within the plant, basal and upper leaves turn yellow and dry up,
the shoot tip become wilted and diseased plants grow dramatically stunted (Fig. 2).
Vascular browning can be observed on sections of the stem, from near the crown to
the upper part of the plant. Fruit size and, consequently, yields are reduced.
Symptoms of Verticillium wilt are similar to those of Fusarium wilt, but progress
slower and usually do not result in death as for Fusarium wilt.
Verticillium wilt in susceptible cultivars causes dramatic yield losses rendering
economically unfeasible the tomato cultivation in soils harbouring high
concentrations of the pathogen propagules. A 100% incidence of Verticillium wilt
was obtained on tomato cv. Bonny Best grown in soil artificially infested by at least
200 microsclerotia of V. dahliae per gram of soil (Conroy et al., 1972). Several
reports also confirm a synergistic enhancement of Verticillium spp. infections by
different nematode genera and species (Pegg & Brady, 2002).
Figure 2. Symptoms of Verticillium wilt on tomato in the field (A) and greenhouse (B).
Since the first tomato cultivars resistant to Verticillium wilt were developed by
Blood in 1925 using the wild Peruvian cultivar Peru Wild of Lycopersicon
pimpinellifolium (Bryan, 1925), the importance of this disease for tomato crop has
been somewhat reduced (Walter, 1967). It has been found that such resistance was
conferred by a single dominant gene denominated Ve (Shapovalov & Lesley, 1940).
Subsequently, the dominance of the Ve gene was confirmed by Schaible, Cannon
and Waddoups (1951), Cirulli and Renzoni (1971), Kosuge, Iijima and Ida (1977),
Matta et al. (1980) and Petrovskaya (1985). In addition to the Ve resistance, Walter
(1967) noted a multigenic resistance.
However, a new pathotype (race 2) of V. dahliae, breaking the resistance
conferred by the Ve gene, occurred in Wisconsin in 1957 (Nachmias et al., 1987).
Later, race 2 was found in Ohio (Alexander, 1962), France (Laterrot & Pécaut,
1966), Italy (Cirulli, 1969), Greece (Tjamos, 1980), Queensland, Australia (O’Brien
and Hutton, 1981), Morocco (Besri, Zrouri & Beye, 1984), The Netherlands
(Paternotte & Van Kesteren, 1993) and Tunisia (Daami Remadi et al., 2006).
Dobinson, Tenuta & Lazarovitis (1996) claimed that V. dahliae race 2 was
establishing as the predominant race in Essex County, Ontario. V. dahliae is known

to have a low level of specialization towards plant species (Matta et al., 1980),
though strains with a higher specialization are known to infect Mentha piperita
(Nelson, 1950). In tomato, no varietal specialization is known for V. albo-atrum.
2. INTEGRATED CONTROL
Verticillium wilts are generally controlled by a combination of measures including:
use of resistant cultivars or rootstocks, reduction of soil inoculum, actions limiting
pathogen propagules spread, and manipulation of epidemiological factors, in order
to reduce severity and to delay the progress of the disease.
2.1. Selecting Soil for Cultivation

It is known that V. dahliae prefers neutral or alkaline soils (pH 6-9), rarely acid ones
(Bell, 1989). At pH below 5.5, growth, microsclerotia production and survival are
inhibited. In USA, Verticillium wilt of cotton caused by V. dahliae has been
reported as more severe on sandy loam, clay loam and organic soils (Rudolph,
1931). In acidic soils, limed to increase pH, tomato wilt severity increased (Jones,
Overman & Geraldson, 1971).
The choice of soil for planting tomato should be done carefully, taking into
account also the field cropping history. Plant species such as eggplant, cotton and, to
some extent, pepper are generally susceptible to Verticillium wilt, therefore they
could be used as indicators of the pathogen presence in soil. If symptoms of the
disease are observed on such crops in previous years, the pathogen is very likely still
present at high concentrations in the field. Microsclerotia of V. dahliae can survive
for many years in soil, also when it is cultivated with non-host plants. In addition,
the presence of the disease on surrounding crops should be considered: the pathogen
might be carried over to the uninfested fields by tillage devices and plant debris or
soil particles spread by wind. Fields cultivated for many years with small grains or
corns should be preferred for planting tomato, because such crops are non-host of V.
dahliae. They hence contribute to reduce the inoculum in soil, though this effect is
not enough to eradicate the pathogen in the field, because it can also survive on
many weeds.
Some chemical characteristics of soil are known to affect Verticillium wilt. For
example, low calcium level along with low soil pH may result in an increase of the
disease severity (Hubbeling & Chaudhary, 1969).
The characteristics of V. dahliae to survive for long periods in the soil by
means of resting structures, in combination with a wide host range, makes crop
rotation an insufficient control measure, especially if used alone. However, when
rotation is combined with other control measures, it may help to slow down the
build up of the pathogen inoculum in soil.
Rotation crops must be chosen taking into account both agronomic and
economic aspects. Growers are generally reluctant to adopt long rotations because of
economic reasons. Planting tomatoes as well as other solanaceous crops in the same
field year after year must be avoided. It has been widely demonstrated that V.
dahliae inoculum increases in the soil following even a 1-year cultivation of a
susceptible crop (Huisman & Ashworth, 1976; Xiao et al., 1998). For tomato, at
least a three-year rotation should be adopted and small grains, corn, soybean,
grasses, sweet clover, legumes, crucifers, sugar beet, alfalfa (Huisman & Ashworth,
1976; El-Zik, 1985) or rice (Milev & Nechev, 1973; Butterfield, DeVay & Garber,
1978) should be preferred. Pea, clover, barley, rye and maize have been reported as
the most effective crops in order to reduce V. dahliae inoculum, because no root
penetration by the pathogen was observed (Sidorova, 1974). The incidence of wilt
on tomato was greatly reduced by previous cropping with rice (Milev & Nechev,
1973; Butterfield, DeVay & Garber, 1978). Also, rice was successfully used as a
rotating crop with cotton (Pullman & DeVay, 1981).
Although longer non-host crop rotations are ideal, they often are not
economically feasible, and for this reason they are not easily accepted in local
situations by growers. A rotation of lesser duration may be still beneficial, but with a
lesser degree. Rotating to a non-host crop can significantly reduce V. dahliae
populations in the field. However, the pathogen may survive, multiply and, thus,
persist in soil. In cauliflower, broccoli proved to be a valid candidate for use in
rotation: after two broccoli crops with final residues incorporation to soil, a 94%
reduction in the number of microsclerotia was reached (Xiao et al., 1998).
Summer weed-free fallow and soil aeration reduce the amount of V. albo-atrum
resting mycelia and conidia. However, microsclerotia are much more durable
(DeVay et al., 1974). Wilhelm (1955) recorded V. dahliae symptoms on field test
tomato plants after 14 years in the absence of a tomato crop, though at a very low
incidence. Conversely, Ben-Yephet and Szmulewich (1985) observed that no
microsclerotia survived in soil transferred to the laboratory after 5 years, although
4% of the field population survived after a 7-year crop rotation.
2.2. Heat
Nelson and Wilhelm (1958) found that the thermal death point for hyphae and
conidia of V. dahliae in soil was a minimum of 10 min at 50 °C. In an early study,
Bollen (1969) found 30 min at 57.5 °C for V. dahliae and 52.5 °C for V. albo-atrum.
Subsequently, Bollen (1985) found different values: 45-47.5 °C for potato and
tomato strains and 40-45 °C for a sugarbeet strain, which were in agreement with
Pullman, DeVay & Garber (1981). Katan (1981) reported 50 °C for 23 or 27 min,
for two V. dahliae strains.
2.3. Solarization
Solarization is the procedure aimed at increasing soil temperature by means of
ground cover with polyethylene or PVC sheets to trap solar radiation into the soil. In
summer period in temperate regions, temperature of soilarized soil can reach 45-55
°C at 5 cm and 35-45 °C at 15-20 cm below the surface. This technique was
pioneered by Katan et al. (1975) and has been proved to be effective in reducing
V. dahliae, other soil-borne pathogens, nematodes and weed seeds. A successful

application of this technique was obtained in Crete on greenhouse-grown tomato:
after 10 weeks of solarization during June-August, no V. dahliae propagules and no
infected plants were found in the solarized soil, and yields increased by 112.4%
compared with control (Bourbos & Skoudridakis, 1996). Ioannou (1999) proved
solarization to be effective in controlling V. dahliae, other soil-borne pathogens and
several V. dahliae non-host weeds. Also in Italy, solarization has proved to be
effective against tomato Verticillium wilt (Aloj & Noviello, 1982; Minuto et al.,
2006). However, sometimes soil solarization has provided inconsistent results
(Porter & Merriman, 1985).
Evidence for the effect of mulching with black plastic sheets on Verticillium
wilt is limited. Nevertheless, it was observed that tomato glasshouse plants under
black mulching developed wilt symptoms earlier than on non-mulched plants
(Basoccu & Garibaldi, 1974). More recently, this effect was observed also for
eggplant (Elmer & Ferrandino, 1991).
Solarization treatment acts against pathogens directly by heat and indirectly by
changes in chemical and biological soil properties. Solarization temperatures and
conditions seem to increase concentrations of ammonium, nitrate, phosphorus,
potassium, calcium and magnesium in the soil (Cartia, 1989; Stapleton, DeVay &
Lear, 1991). In addition, solarization favours thermophilic and thermotolerant
microbial antagonists such as Trichoderma, Talaromyces flavus, lytic bacteria (i.e.:
Bacillus spp.) (Elad, Katan & Chet, 1980; Tjamos & Paplomatas, 1987), Aspergillus
terreus (Tjamos & Skretis, 1990) and actinomycetes (Wadi, 1999). Also, fluorescent
pseudomonads play an important role as antagonists: they are sensitive to heat but
able to rapidly re-colonize the soil after solarization treatment (Wadi, 1999).
Effectiveness of solarization is strongly correlated to the temperature reached in
the soil and consequently to the solar radiation. For this reason, it is more practicable
in hot or warm countries than in cool ones, and it is more effective in greenhouses
than in the field. Conditioning factors in using this technique are the long period of
non cultivation, costs and disposal of plastic films.
2.4. Sanitation
Sanitation is important in preventing the introduction of the pathogen propagules
Verticillium-free soil. Consequently, implements and equipment used to prepare the
soil for planting, cultivation or other operations should be sanitized before using to
prevent movement of infested soil or infected plant debris.
2.5. Tillage
Propagules of V. dahliae are generally present in the top 30 cm of soil, prevaling in
the top 10 cm. Ploughing redistributes inoculum vertically while deep-rip tillage do
not (Taylor, Pasche & Gudmestad, 2005). Deep ploughing and even complete soil
inversion can be effective in reducing the disease losses. Conversely, during the
growing season, deep cultivation and root pruning can enhance wilt.
2.6. Plant Residues

The reduction of V. dahliae microsclerotia in soil after burial of plant debris has
been proved in several crops. In cauliflower, by incorporating fresh or dry broccoli
residues, microsclerotia were reduced at 30 °C or below, and eliminated at 45 °C
(Subbarao & Hubbard, 1996). In another study on cauliflower, two broccoli crops
and residue incorporation in soil caused a 94% reduction in the soil density of
microsclerotia (Xiao et al., 1998). On potato, the use of Brassicaceae green manure
reduced pathogen propagules and Verticillium wilt incidence (Davis et al., 1996).
Eradicative effectiveness of a number of organic amendments from various
botanical species on microsclerotia of defoliating and non-defoliating V. dahliae
pathotypes was investigated by López-Escudero, Mwanza and Blanco-López (2007).
In such study, Lavandula stoechas, Thymus mastichina and especially Diplotaxis
virgata debris proved highly effective in reducing the viability of microsclerotia
over time, and the non-defoliating pathotype was more markedly affected than the
defoliating one. In cotton, Verticillium wilt development was completely prevented
by all the organic substrates tested in soil infested with the non-defoliating
pathotype, and by Cistus albidus and L. stoechas treatments in soils infested with the
defoliating pathotype.
2.7. Weed Control

Many weed species, particularly broadleaf ones, may serve as alternate hosts for
V. dahliae. Moreover, many weeds may harbour Verticillium in their vascular
system without showing detectable symptoms, hence acting as inoculum reservoirs.
Development of microsclerotia in senescent tissues contributes to raise the inoculum
level in the field. An effective weed control program should hence contribute to
slow down the inoculum increase over time. In weed control, field selection and
crop rotation play an important role. Particularly, fields with weed species closely
related to tomato, such as nightshades, should be avoided because they may allow
V. dahliae inoculum to increase, and at the same time they are troublesome to
control. Maize can be included advantageously in rotation cycles with tomatoes,
because several herbicides, not selective for tomato crops, are available. Some maize
herbicides control weeds such as nightshades, that are tolerant to tomato herbicides.
Chemical or mechanical control of weeds is mostly important in the first stages
of tomato growth in order to minimize competitions and limit V. dahliae inoculum
renewing.
2.8. Resistant Rootstocks

Rootstocks resistant to Verticillium wilt are available, however they are not
commonly used in tomato. In the past, cost of resistant rootstocks was found
comparable to that for soil fumigation (Ginoux, 1974).
2.9. Cultivars
The single dominant Ve gene has been used for a long time in breeding tomato
cultivars. The Ve gene confers resistance to V. dahliae race 1 and to tomato and hop
strains of V. albo-atrum (Pegg & Dixon, 1969). In 1982, Okie and Gardner (1982)
found that F1 hybrids heterozygous for the Ve gene were less resistant than
homozygous F1 hybrids, suggesting the incomplete dominance of the Ve gene. Also,
it has been reported that Ve resistance is always associated with a slight colonization
by the pathogen.
By means of molecular techniques, it has been found that the Ve gene is located
on the short arm of the chromosome 9 (Diwan et al., 1999) and encodes a class of
cell surface-like receptors (Kawchuk et al., 2001).
No resistance comparable to that of the Ve gene has been found against the
recent new race of the pathogen. A field resistance to race 2 of V. dahliae was
reported by Hubbeling, Alexander and Cirulli (1971) but it appeared incomplete and
depended on soil type and pH. Okie and Gardner (1982) found that a tolerance to
race 2 was conferred by 2 or 3 recessive genes.
Tabaeizadeh et al. (1999) introduced the acidic endochitinase gene pcht 28
deriving from L. chilense into L. esculentum via Agrobacterium-mediated
transformation. Transformed plants produced a high level of constitutive chitinase
enzyme and possessed a high level of tolerance against both races of V. dahliae.
Robison et al. (2001) found that significant reductions in Verticillium wilt symptoms
on tomato could be obtained by preventing disease-related ethylene production in
transgenic tomato plants expressing the bacterial deaminase l-aminocyclopropane-l-
carboxylic acid (ACC), which cleaves the ethylene biosynthetic precursor ACC in
plants.
Gold and Robb (1995) stated that polygenic partial resistance (tolerance) was
the only defence available against V. dahliae race 2. Recently, a dominant resistance
to race 2 coming from cv. Veda was introgressed into open pollinated processing
tomato cultivars, which can be developed into hybrids by commercial seed
companies (Stamova, 2006).
2.10. Fertilization
The generalized understanding is that low nitrogen regimes regardless of nitrogen
type are associated with decreased wilt severity and incidence. High levels of
nitrogen have not increased (Roberts, 1943; Jones & Overman, 1986) or even have
reduced wilt susceptibility in tomato (Wilhelm, 1951).
The use of ammonium sulphate, bone or fish meal reduced the V. albo-atrum
inoculum potential in tomato soil. Reduced content of nitrate in soil resulted in a
depletion of soluble carbohydrates in the roots and, consequently, in an induction of
resistance of tomato plants to Verticillium wilt (Roberts, 1944).
Phosphate alone may have little influence on Verticillium wilts, and this was
demonstrated also in tomato (Roberts, 1943). The interaction of phosphate with
other mineral elements seems to produce a more significant effect. In cotton, for
example, when a high phosphate level was combined with a high nitrogen level, wilt
severity increased (Longenecker & Hefner, 1961). When potassium is deficient,

application of phosphate and low nitrogen level increased wilt (Presley & Dick,
1951).
Potassium nutrition has been known to affect wilt development. Pegg (1985)
described an increase in divalent and polyvalent cations, but not monovalent, in
leaves of tomato infected with V. albo-atrum, while Krikun, Barash and Nachmias
(1990) reported potassium deficiency in V. dahliae infected tomato, potato,
aubergine, groundnut and rapeseed.
Several studies have demonstrated that trace elements can diminish the effect of
V. dahliae. On cotton, cobalt, manganese, zinc, copper and molybdenum seemed to
have a beneficial effect. Dutta and Bremmer (1981) found that dipping tomato
seedlings in a solution containing copper, boron and manganese prior to inoculation
with V. albo-atrum significantly reduced infection. Selvaraj (1974) found an
increased resistance to V. dahliae in tomato by augmenting calcium levels.
2.11. Irrigation
Irrigation has been reported to affect severity and incidence of Verticillium wilt in
several crops, but the effect of water on the disease progress depends very much on
the soil type and ambient climate. Generally, wilt worsens by increasing watering
amounts and frequency. Possible explanations of the relationship between water and
wilt severity and incidence relate to the microsclerotia survival and germination.
Karaca et al. (1971) reported that the decreasing of soil temperature following
watering favoured infections, whereas Farley et al., (1971) demonstrated that
microsclerotia germinated and sporulated repeatedly by periodic wetting and drying
cycles.
In other crops, irrigation has been reported to favor Verticillium wilt. On cotton,
wilt increased with moisture increasing (El-Zik, 1985), and on potato it was higher
in furrow-irrigated than in sprinkler-irrigated fields (Davis & Everson, 1986). Wilt
was more severe under excessive than moderate or deficit irrigation regimes
(Cappaert et al., 1992; 1994). On cauliflower, wilt was higher in excessive and
moderate irrigation regimes than in deficit one, and no difference was detected
between furrow and subsurface drip irrigation methods (Xiao et al., 1998; Xiao &
Subbarao, 2000). Reducing the amount of water and the frequency of irrigations
helps to lower incidence of wilt damage on the crop, however these tactics must be
carefully adopted because, at the same time, they can greatly compromise crop yield.
Ioannou et al. (1977) demonstrated that the production of microsclerotia in
infected tomato stems buried in non-sterilized soil was maximal at a water potential
of -32 bars at 24 °C, while in saturated soil and at -100 bars water potential,
microsclerotia production was hardly inhibited.
Soil flooding has been reported to exert a control against V. dahliae. Ioannou
et al. (1977) observed that equal number of microsclerotia was produced in tomato
debris under no-irrigation or one 12h-irrigation conditions. Under flooding,
especially when protracted for 20 or 40 days, no or a very few microsclerotia were
produced because of decreased O2 and increased CO2 concentrations. Nevertheless,
40-day flooding did not reduce the numbers of viable microsclerotia, suggesting that
short-term flooding is ineffective for the control of tomato Verticillium wilt, either
by its effect on production or, particularly, on survival of microsclerotia. On the
contrary, some authors have found that 6-week flooding killed microsclerotia as did
3-week anaerobic conditions under N2 in laboratory (Nadakavukaren, 1960;
Menzies, 1962). Soil flooding prior to cotton crop eradicated V. dahliae as well as
rotation with paddy rice (Pullman & DeVay, 1981).
Irrigation with saline water has been reported to enhance V. dahliae infections
in tomato (Besri, 1981; Kaufman et al., 1990). High salt levels in irrigation water
caused a 100% breakdown of resistance in Ve tomato cultivars resistant to V. dahliae
race 1. It was also claimed that susceptibility of tomato cultivars resistant to race 1
increased with increasing soil salinity (Besri, 1990).
2.12. Chemicals
In the past, many studies have been made on chemical control of Verticillium wilts.
Best results were obtained with fumigants such as methyl bromide, chloropicrin and
methyilisothiocyanate, because of their wide range efficacy on soil-borne pathogens,
nematodes and weed seeds. Soil fumigation needs ground cover with polyethylene
tarp to maintain effective fumigant concentrations into the soil and hence it may also
be combined with a solarization treatment.
Methyl bromide, alone or in combination with chloropicrin, proved to be
effective against tomato Verticillium wilt (Matta, 1976; Noling, 1987). Although
methyl bromide is effective in controlling V. dahliae, live pathogen below the depth
of effective fumigation can re-infest the upper soil layer (Bourbos, 1986).
Chloropicrin (30 or 40 g m-2) provided a satisfactory and consistent control of
tomato Verticillium wilt especially when applied by means of a drip irrigation
system. However, it was less effective than methyl bromide (60 g m-2) using the
shank injection application method (Gullino et al., 2002). Dimethyl disulfide (80 g
m-2) was found to be effective against V. dahliae on strawberry (Fritsch, 2005).
A mixture of dichloropropene + methylisothiocyanate allowed a satisfactory
disease control (Moens & Ben Aicha, 1986). Metam sodium reduced levels of V.
dahliae inoculum in potato field experiments by 60% to 80%, and shank injection
was more effective than chemigation. However, in some experiments, results
obtained with methyilisothiocyanate compounds such as metam sodium (Vapam®)
(Overman, 1982) and dazomet (Gindrat, Vallotton & Neury, 1973) have been less
encouraging.
Since 1968, systemic fungicides, particularly benzimidazoles (i.e. carbendazim,
benomyl, thiabendazole and thiophanate-methyl) have been studied for controlling
Verticillium wilts on several crops, including tomato, by means of foliar spray, soil
drenching, stem injection (on trees) or soil application of granular preparations. The
sensitivity of V. dahliae to benzimidazoles has been shown, but good wilt control
has been obtained especially with high dosages, which sometimes combined with
some phytotoxic effects (Fuchs, 1977; Matta & Garibaldi, 1970; Garibaldi &
Lamonarca, 1974; Garibaldi, 1976; Formigoni, Guidi & Tumino, 1973).

Encouraging results obtained in a pot experiment by soil drench with the strobilurin
azoxystrobin against Verticillium wilt on eggplant, however, could not be confirmed
in a field trial (Bubici et al., 2006).
Some fundamental problems have limited the use of systemic fungicides. Low
water solubility of such fungicides prevents soil leaching deeper than 100 mm,
leaving deep roots open to attack of V. dahliae. The acropetal movement of
chemicals through the transpiration stream causes a rapid accumulation in leaves, far
from the site where their action is required, and a deprivation in deep roots
harbouring the pathogen (Talboys, 1984). Water solubility of benzimidazoles can be
increased by pH decrease, leading to a better movement in the plant water stream.
For example, Buchenauer and Erwin (1971, 1972), working with carbendazim-HCl
and thiabendazole-HCl applied by foliar spray on cotton, obtained a good control of
Verticillium wilt and observed a movement of fungicides toward the untreated upper
leaves, while benomyl did not control the disease and had not translocated.
However, benomyl-tolerant Verticillium strains have been obtained easily in
laboratory and have been isolated from a commercial tomato crop (Talboys & Frick,
1974; Locke & Thorpe, 1976).
Chemical control by non-fungicide compounds has been attempted. Grinstein,
Katan and Eshel (1976) have shown that the herbicides nitralin and trifluralin added
to soil at the dose of 1 mg g -1 reduced Verticillium wilt of tomato by 97%. Pre-
inoculation treatment of tomato plants with trifluralin and other dinitroaniline
herbicides stimulated the synthesis of a water-soluble phytoalexin so that
Verticillium wilt severity on susceptible herbicide-treated plants and resistant ones
was similar (Grinstein et al., 1981).
Among non-fungicide compounds, catechol has been reported to reduce the
incidence of tomato Verticillium wilt when applied to the soil (Chet et al., 1978).
Finally, tomato wilt has been successfully controlled by means of plant growth
retardants such as chlorocholinechloride (Sinha & Wood, 1967; Buchenauer, 1971)
and pydanon (Buchenauer & Erwin, 1976), and by means of hormones such as 2-
napthyloxy acetic acid and gibberellic acid (Dutta, 1981).
2.13. Microbial Antagonists

Several bacteria species have been found to act as antagonists of Verticillium spp.
After a screening of more than 5,000 oilseed-rape-rhizosphere bacteria for
antifungal activity against V. dahliae var. longisporum, Berg and Ballin (1994) and
Berg (1997) found that the majority of antagonists were pseudomonads
(Pseudomonas aurofaciens, P. chlororaphis, P. fluorescens and P. putida), and with
a minor incidence isolates of Bacillus cereus, B. subtilis and other species.
Some pseudomonads and other IAA- and gibberellin-producing bacteria have
been known to promote plant growth. Sharma and Nowak (1998) studied in vitro
and in vivo the effect of a plant-growth-promoting pseudomonad strain on a wilt-
susceptible tomato. Severity reduction, delayed wilt symptoms and stimulated plant
growth were observed in vitro, especially at low inoculum density, while only wilt
severity reduction was observed in vivo. After a screening of endorhizosphere

bacteria obtained from root tips of tomato plants grown in solarized soils, Tjamos
et al. (2004) found one isolate of Paenibacillus alvei and one of Bacillus
amyloliquefaciens which were effective against V. dahliae in vitro, as well as on
eggplant in the greenhouse and on potato in the field. These antagonists promoted
plant growth and had a chitinolytic activity. Though they are rhizosphere
inhabitants, they preferentially colonized the endorhizosphere of tomato and
eggplant. In such study, the authors failed to find effective antagonistic fluorescent
pseudomonads, though they were also isolated from root tips of tomato plants.
Moreover, streptomycetes are known as the most important antibiotic-producing
microorganisms. El-Abyad et al. (1993, 1996) observed the efficacy of Streptomyces
pulcher, S. canescens and S. citreofluorescens against V. albo-atrum in vitro and in
vivo, after coating tomato seeds with spores of the antagonists. Streptomyces
plicatus, a chitinase producer, markedly protected tomato plants against Verticillium
and Fusarium wilt, as well as against stem canker, when applied to the roots before
transplanting under greenhouse conditions (Abd Allah, 2001). Inconstant results has
been obtained with a commercial formulation of S. griseoviridis (Mycostop®,
Bioplanet, Italy) against tomato Verticillium wilt in two field trials (Minuto et al.,
2006). Positive results obtained with Streptomyces spp. against V. dahliae in vitro
and against Verticillium wilt on pot-grown eggplants in greenhouse (Bubici, 2005)
need a further confirmation through field trials.
Among the fungal antagonists of Verticillium spp., the three genera
Gliocladium, Trichoderma and Talaromyces have been considered as the most
effective. In an early study, Trichoderma viride was found as more abundant in the
rhizosphere of tomato resistant cultivars than in that of susceptible ones (Subbarao
& Bailey, 1961). Later, T. viridae was used effectively on tomato against
Verticillium wilt by Sportelli, Nipoti and D'Ercole (1983), and together with T.
harzianum and T. koningii by D'Ercole and Nipoti (1986). A partial control of
artichoke Verticillium wilt in the field was obtained by soil application of olive husk
compost colonized by T. viride (Colella et al., 2001). Zeise (1997) obtained a good
control of Verticillium wilt of tomato by inoculating T. flavus into the soil, before
transplanting or sowing.
Pre-transplanting treatments with Penicillium oxalicum conidia (6 ⋅ 10 6 conidia
-1
g of seedbed substrate) resulted effective in controlling tomato Verticillium wilt,
under greenhouse and field conditions (Larena et al., 2003).
The protective effect against tomato Verticillium wilt of pre-inoculation with
avirulent fungi has been also investigated. A good protection was provided by an
avirulent strain of V. albo-atrum, which was more effective than avirulent strains of
Fusarium oxysporum f. sp. dianthi and F. oxysporum f. sp. lycopersici (Matta &
Garibaldi, 1977). At present, although many researches have been carried out,
biological control of Verticillium wilt still remains at an experimental stage.
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13
CEZARINA KORA1, MARY RUTH MCDONALD 2 AND

GREG J. BOLAND 3
NEW PROGRESS IN THE INTEGRATED

MANAGEMENT OF SCLEROTINIA ROT OF CARROT
1
Pest Management Centre, Agriculture and Agri-Food Canada, 960 Carling
Ave., Ottawa, ON, K1A 0C6, Canada
2
Department of Plant Agriculture
and
3
Department of Environmental Biology,
University of Guelph, Canada
Abstract. Sclerotinia rot, caused by the pathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary, is an
economically important disease of carrot (Daucus carota L.) occurring in the field and storage. This review
describes a range of control methods for Sclerotinia rot of carrot, emphasizing emerging strategies supported
by new information on the etiology and epidemiology of the disease. Prospects and recommendations are
outlined for integrating current and emerging control methods to attain sustainable management of the
disease. The primary strategy to managing Sclerotinia rot is the integration of methods that reduce within-field
sources of inoculum, suppress the development of S. sclerotiorum, and reduce the infection rate in the field
and storage. The integrated strategy recommended in this review aims at achieving disease suppression
through sanitation of soil and equipment, monitoring the crop development and microclimate, modifying the
microclimate through canopy manipulation, predicting the disease, and timing the application of disease
control practices as required. Breeding carrot cultivars for an upright and compact top growth may offer
important contributions to the sustainable management of Sclerotinia rot.
1. INTRODUCTION
Carrot is a commercially important root vegetable cultivated for human consumption for
over ten centuries and valued for its dietary and health benefits (Simon 1990; Rubatzky,
Quiros & Simon, 1999). The world production of carrots has almost tripled over the last
three decades to meet increasing consumer demand. In 2003, carrots were grown in
approximately 1 million hectares in 108 countries with an annual production of 23.3
million metric tons, representing 3.4% of the total world root and tuber production
243
244 C. KORA ET AL.
(Anonymous, 2004a). Carrot is a cool-season vegetable that is cultivated in temperate

and subtropical regions of the world (Rubatzky, Quiros & Simon, 1999). The viability
of the carrot industry depends upon the year-round availability of quality carrots for the
continuous supply of fresh and processing markets. Therefore, storage of fresh carrots
outside the normal harvesting season is important.
Several methods of storing carrots include indoor common storage using ventilated
ambient air, controlled temperature refrigerated storage, in the ground covered with
earth or polyethylene sheeting and straw, and in field clamps alternated with layers of
sand or earth (Geeson, Browne & Everson, 1988; Rubatzky, Quiros & Simon, 1999;
Cheah & Brash, 2001). Much effort is directed toward improving cropping, handling,
and storage conditions to optimize shelf life and reduce spoilage of carrots. However,
crop losses due to storage rot diseases continue to be prevalent and a primary challenge
to carrot production (Cheah & Brash 2001; Stack et al., 2002; Molloy, Cheah &
Koolaard, 2004).
Sclerotinia rot of carrot (SRC) is a common disease in many regions of the world
where carrot is cultivated (Rubatzky, Quiros & Simon, 1999; Cheah & Brash, 2001;
Kora, McDonald & Boland, 2003). The disease affects both the foliage and root
portions of the plant and occurs in carrot crops in the field and storage. However, crop
losses are particularly prevalent during long-term (e.g., up to 8 months) storage of carrot
which is a common practice in many temperate growing regions. Crop losses of 50 to
100% attributed to SRC have been reported in cold storage (Anonymous, 1970;
Finlayson, Rimmer & Pritchard, 1989) and during long distance transport (Rader, 1952).
Currently available management practices often do not provide effective control of the
disease in commercial crops. The bi-cyclic nature of epidemics, with distinct pre- and
post-harvest stages developing in the field and storage (Kora, McDonald & Boland,
2003), further complicates the management of SRC. Recent epidemiological studies,
however, have elucidated important relationships among the crop, pathogen, and
environmental factors that influence the development and management of SRC (Couper,
2001; Kora, 2003). The objectives of this review are to summarize control methods that
have achieved satisfactory results at the level of research and those that have gained
commercial application, to highlight progress in the development of new control
methods in relation to epidemiological findings, and provide recommendations on
integrating these methods for improved SRC management.
2. THE DISEASE
2.1. Damage and Symptoms

The damage caused by SRC is usually sporadic in time and space, but outbreaks are
common and losses can be serious. Carrots are particularly susceptible to S.
sclerotiorum late in the growing season and during storage. In the field, foliar infections
can reduce yield by weakening the tops, which subsequently break off during
mechanical harvest and leave the roots unharvested (McDonald, 1994). Initial symptoms
appear on leaves and petioles as individual water-soaked, olive-green lesions associated
with collapsed tissues. As disease progresses, lesions expand over the entire leaf,
SCLEROTINIA MANAGEMENT ON CARROT 245
petiole, and the rosette with infected tissues becoming covered by white mycelial mats.
Eventually, characteristic black sclerotia (2 to 20 mm) form, embedded in the mycelium
covering infected tissues. Occasionally, the entire top may collapse and rot symptoms
may be evident on the crown. However, symptomatic diseased crowns and roots are
rarely visible at harvest.
Disease in storage develops mainly from infected roots introduced from the field and
can substantially reduce marketable yield due to extensive decay. Symptoms on infected
roots appear as soft, watery rot lesions, characterized by a rapidly spreading white
mycelium and superficial black sclerotia. Mycelium arising from an infected carrot can
spread to adjacent roots forming enlarging pockets of infection (Snowdon, 1992;
McDonald, 1994). Colonized carrots are usually held together in large clumps by the
extensive mycelial growth. In addition, secondary pathogens may gain entrance into
infected areas and contribute to further disintegration of the roots.
2.2. Causal Organism

Sclerotinia sclerotiorum is a necrotrophic soilborne fungus pathogenic to at least 408
species of herbaceous plants (Boland & Hall, 1994). The fungus is distributed
worldwide, but it is more common in temperate and subtropical regions possessing cool
and wet seasons (Purdy, 1979; Willets & Wong, 1980). Sclerotinia sclerotiorum can
cause severe outbreaks and considerable losses of yield and quality in a number of
economically valuable crops, including bean (Phaseolus vulgaris L.), soybean (Glycine
max L.), rapeseed (canola) (Brassica napus L. and B. rapa L.), and several vegetables.
Widespread and severe epidemics are facilitated by the ability of the fungus to
reproduce sexually by production of apothecia and air-borne ascospores, and asexually,
by production of sclerotia.
The fungus can spread and establish itself across fields and regions through wind-
dispersed ascospores and sclerotia transported through farming operations such as
tillage, irrigation, manure fertilization and infested seeds (Schwartz & Steadman, 1978;
Adams & Ayres, 1979; Steadman, 1979). Sclerotia that develop on diseased plant
tissues may dislodge and persist in soil for at least 7 years (Cook, Steadman & Boosalis,
1975; Adams & Ayres, 1979; Ben-Yephet, Genizi & Siti, 1993), although up to 93%
decline in the viability of sclerotia within the first year of burial in soil has been reported
(Merriman et al., 1979; Alexander & Stewart, 1994; Kora, 2003). Despite in small
numbers, surviving sclerotia appear sufficient to maintain effective levels of inoculum
and initiate important epidemics. Therefore, adequate prevention or management of
diseases caused by S. sclerotiorum is important to minimize the increase of inoculum in
soil and severity of epidemics in subsequent years.
Sclerotinia sclerotiorum spends about 90% of its life cycle in soil as dormant
sclerotia produced on diseased tissues during previous epidemics. Sclerotia are
susceptible to prolonged periods of high temperatures and flooding, sequential drying
and wetting, solar radiation and biological (e.g., by mycoparasitic microbes and
mycophagous animals) degradation (Adams & Ayres, 1979). Under suitable
environmental conditions, viable mature sclerotia can germinate myceliogenically to
246 C. KORA ET AL.
produce vegetative hyphae, or carpogenically to produce apothecia. A period of

preconditioning (e.g., chilling) is usually required to relieve constitutive dormancy,
before sclerotia can germinate carpogenically (Coley-Smith & Cooke, 1971; Phillips,
1987; Dillard, Ludwig & Hunter, 1995).
In the field, development of apothecia followed 10 days or more of continuous soil
moisture near field capacity (0 to -0.3 bars), soil temperatures of 4 to 20°C, and a closed
crop canopy over the soil (Schwartz & Steadman, 1978; Abawi & Grogan 1979; Morrall
& Dueck, 1982). An apothecium can release ca. 2 x 106 ascospores over the period of 5
to 10 days that it remains functional in the field (Schwartz & Steadman, 1978;
Steadman, 1983). Ascospores can initiate infection when in contact with susceptible
tissues, such as senescing flower petals, and in the presence of free moisture (Abawi &
Grogan, 1979; Lumsden, 1979). Mycelium arising from infected senescing tissues can
then infect adjacent healthy tissues of the host.
2.3. Etiology and Epidemiology

Knowledge of the life cycle of S. sclerotiorum in relation to carrot development is key
to understand the epidemiology and management of SRC. Several phenological and
architectural attributes of carrot crops have been identified that influence the
development of the pathogen and the disease (Kora, 2003).
1) Carrot is a biennial plant and flowers are absent in crops that are grown for root
production. Consequently, unlike initial petal infestation in flowering plants (McLean,
1958; Abawi & Grogan, 1979; Atallah & Johnson, 2004), in carrots, the exogenous
nutrient source for germinating ascospores during the saprophytic growth of the fungus
is provided by senescing leaves and petioles. The presence of senescing leaves is a
prerequisite for infection of carrots by S. sclerotiorum and determines the susceptible
stage of the crop (Geary, 1978). However, the contact of senescing leaves with soil after
they collapse is the most important phenological event for initiation of disease (Kora et
al., 2005b). This feature emphasizes the importance of plant-to-soil contact in the
epidemiology of SRC.
2) The susceptible stage of carrots crops is relatively long, extending from the
collapse of the first senescing leaves on the soil until harvest. Once initiated,
accumulation of collapsed senescing leaves as well as lodging healthy leaves in the
furrow progresses until harvest due to aging, the growth pattern of carrot foliage, and
other foliar diseases. This results in the continuous increase of potential infection sites
and plant-to-plant contacts which promote the secondary spread of the disease.
3) Apothecia and (or) ascospores are usually present in the crop when carrots are
susceptible to S. sclerotiorum (Geary, 1978; Couper, 2001; Kora et al., 2005b)
representing an important event for the development of SRC. Apothecia usually appear
following the development of a closed carrot canopy (Couper, 2001; Kora et al., 2005b)
as reported for a number of other crops (Schwartz & Steadman, 1978; Morrall & Dueck,
1982; Boland & Hall, 1987a; 1988; Atallah & Johnson, 2004). Senescing leaves also
start to collapse concurrently with, or shortly after, closure of the carrot canopy. This
feature emphasizes the significance of canopy development and within-field apothecia

in the epidemiology of SRC.
4) Preharvest epidemics of SRC can lead to the development of a secondary,
postharvest cycle of disease when harvested roots are designated for storage or long
distance transportation. Every infected carrot root introduced from the field has the
potential to develop into a source of inoculum and initiate new epidemics in storage. An
example of these consecutive cycles of disease is illustrated in Fig. 1.
2.3.1. Preharvest Epidemics

Ascospores originating from apothecia within the crop appear to be the most important
primary inoculum to initiate foliar infection and significant epidemics of SRC in the
field (Geary, 1978; Couper, 2001; Kora, McDonald & Boland, 2005b). Airborne
ascospores from apothecia in surrounding fields can contribute to development of
disease when inoculum coincides with the susceptible stage of the crop (Geary, 1978;
Kora, McDonald & Boland, 2005b). Mycelium originating from germinating sclerotia
on or near the soil surface can also initiate epidemics of SRC through direct
colonization of leaf and stem tissues located in close proximity (e.g., up to 1 cm) or in
contact with the sclerotia (Finlayson, Rimmer & Pritchard, 1989). Root infection results
from infected foliage and occurs via the crown; direct infection of carrot roots by
mycelium arising from sclerotia in soil has not been observed (Geary, 1978; Lewis &
Garrod, 1983; Finlayson, Rimmer & Pritchard, 1989).
Figure 1. Pre and postharvest cycles of Sclerotinia rot of carrot caused by Sclerotinia
sclerotiorum in a cropping system in temperate regions (adapted from Kora, McDonald &
Boland, 2003).
248 C. KORA ET AL.
Typically, production of apothecia and ascospores in infested fields requires a

combination of favorable moisture, temperature, and crop development conditions over
critical duration periods. In aerobiological studies conducted in summer-grown carrots
crops (e.g., May to October), apothecia developed after the canopy closed, following 7
to 11 days of soil matric potentials of -0.1 to -0.4 bars and soil temperatures of 14 to
23°C (Kora, McDonald & Boland, 2005b).
The closed carrot canopy can promote the development of apothecia by buffering
fluctuating temperature and moisture (Kora, McDonald & Boland, 2005a) and by
obstructing direct radiation and aeration as reported for other crops (Schwartz &
Steadman, 1978; Abawi & Grogan, 1979; Weiss et al., 1980). Similarly, 7 to 12 days of
soil matric potentials of -0.1 to -0.3 bars and air temperatures of 15 to 21°C appeared
necessary before consistent and increasing numbers of ascospores could be detected in
carrot crops (Kora, McDonald & Boland, 2005b). In these studies, apothecia and
ascospores were detected for periods ranging from 1 to 7 weeks during the last 13
weeks before harvest (e.g., August to October and July to October, respectively), with
peak numbers observed mainly when plants were in their susceptible stage (i.e. had ≥ 3
collapsed senescing leaves per plant).
Foliar epidemics of SRC require the presence of senescing leaves, inoculum, and
free surface moisture to occur. These factors usually persist under the closed carrot
canopy until harvest, unless excessively dry periods prevail. In summer-grown carrots,
disease first appeared 5 to 9 weeks before harvest (i.e., from mid-August to mid-
September) following the closure and lodging of carrot canopy, collapse of senescing
leaves on the soil, appearance of ascospores in the crop, and prolonged periods of
surface wetness on lower leaves (Kora, McDonald & Boland, 2005b). Symptoms
appeared on senescing leaves that were in contact with moist soil and on leaves in
contact with diseased tissues, but not on senescing, or healthy leaves in an upright
position.
Longer wetness periods and obstruction of direct light penetration on bottom
senescing leaves under the closed canopy provide favorable conditions for survival of
ascospores and for infection to occur (Caesar & Pearson, 1983; Deshpande et al., 1995).
Mycelium arising from lesions on leaves can advance basipetally, through the petiole
toward the crown, from where it enters the root. In addition, mycelia originating from
diseased leaves colonize touching senescing leaves, foliar debris lying on the soil, and
healthy leaves of neighboring plants resulting in secondary spread of the disease.
Air temperatures of 12 to 18°C and periods of 12 to 24 h/day leaf wetness duration
appear to be optimum conditions for infection of carrots (Kora, 2003). Typically, if
conditions in carrot crops are adequate for apothecia and ascospore production, and
older leaves are collapsed on the soil, leaf wetness and temperature are unlikely to be
limiting for infection to occur. Moisture from soil and dew under the dense canopy are
most likely sufficient for disease development. Higher disease incidence was usually
associated with carrot crops that had earlier senescence, higher numbers of senescing
and damaged leaves accumulated on soil, a vigorous and severely lodged canopy, and
longer periods of inoculum availability during the susceptible stage (Geary, 1978;
Couper, 2001; Kora, McDonald & Boland, 2005b).
2.3.2. Postharvest Epidemics

Disease of carrots in storage is a direct consequence of foliar and crown infection in
the field although quantitative relationships between the two epidemic cycles have not
been observed (Geary, 1978; Lewis & Garrod, 1983; Finlayson, Rimmer & Pritchard,
1989). Infections in storage are initiated mainly by mycelium arising from the crowns of
diseased roots introduced from the field. Infested or infected foliar debris adhering to
harvested roots represent an additional source of contamination for carrots in storage
(Geary, 1978). Mycelium of S. sclerotiorum persisting on the surface of infested
wooden bins used for storage can also initiate new infections on stored carrots (Rader,
1952; Mukula, 1957; Subbarao, 2002). However, this source of inoculum is unlikely to
be important if propagules on bins previously used to store diseased crops occur at low
frequency (Kora, 2003). A colonized root can provide sufficient nutrient reserves for the
fungus to spread rapidly toward adjacent roots. The presence of wounded tissue, caused
by mechanical harvesting or handling, can increase susceptibility of carrots to SRC and
encourage secondary infections in storage, but it is not essential (Geary, 1978).
Disease on carrot roots in storage can develop at temperatures as low as 0°C, but
progresses faster when the temperature of surrounding air increases. The optimum
temperature for disease development in storage is 13 to 18°C (McDonald, 1994). Free
moisture on the root surface also promotes initiation of infection. Once infection is
established, infected tissues usually provide sufficient moisture for further disease
development. High disease incidence can occur during long term storage (e.g., 38%
after 6 months at 1°C) when disease in the field progresses rapidly and is associated
with continuous periods of high soil moisture and leaf wetness, particularly close to
harvest (Kora, McDonald & Boland, 2005b). Postharvest epidemics occur sporadically
in that preharvest epidemics do not always result in epidemics in storage; however,
there appears to be a connection between high disease incidence at harvest and high
disease severity in storage (Kora, McDonald & Boland, 2005b).
3. DISEASE MANAGEMENT
The sporadic occurrence and widespread distribution of epidemics, and long-term
persistence of sclerotia in soil, have prevented the consistent and economical
management of most diseases caused by S. sclerotiorum, including SRC. Most
management strategies in the past have relied on chemical control and cultural practices
directed at reducing the population, or inhibiting the development, of sclerotia in soil
and protecting the crop from infection. The use of tolerant or resistant cultivars and
microbial antagonists has been sporadic. However, chemical control is considered
cost-effective only on selected high-value crops and cultural practices have had variable
results (Steadman, 1979). The limited availability of effective fungicides, development
of pathogen resistance, and increasing concerns of chemical use on human and
environmental health have encouraged further investigation of alternative control
methods. Of 61 articles on the management of Sclerotinia diseases published in the last
5 years (e.g., 2000-2004), 17 studies emphasized host resistance, 15 biological control,
14 disease epidemiology and forecasting, 11 cultural methods, and 4 chemical control.
250 C. KORA ET AL.
3.1. Field Practices

3.1.1. Cultural Control
Cultural practices such as flooding (Moore, 1949), deep burial of crop refuse in soil
(Merriman et al.,1979), reduced irrigation frequencies (Steadman, 1983; Ferraz et al.,
1999), weed management (Dillard & Hunter, 1986), soil solarization (Ben-Yephet,
1988; Phillips, 1990), and grass mulching of soil (Ferraz et al., 1999) have been shown
to reduce populations of sclerotia in soil, prevent carpogenic germination, or decrease
disease in several crops. Subsurface drip irrigation, instead of surface irrigation, can
provide good control of apothecia production by maintaining dry soil conditions in the
top 5 to 8 cm of the planting bed (Subbarao, 2002; Davis, 2004).
High-temperature steam sterilization of soil (e.g., up to 100°C) prior to seeding can
substantially reduce the number of viable sclerotia (e.g., in carrot crops), especially
when sclerotia are partially hydrated and located near the soil surface (Couper, 2001).
Steaming of soil for 15 min at soil matric potentials of ≥-145 kPa was suggested as an
effective treatment to suppress apothecia production from sclerotia within a 60 mm
depth from soil surface. However, laboratory studies suggested that steaming can also
provide complete control of imbibed sclerotia of S. sclerotiorum at lower temperatures
and shorter durations (e.g., 60°C for 3 min) (Van Loenen et al., 2003). Soils amended
with calcium cyanamide (e.g., the fertilizer product Perlka) (Huang & Sun, 1991) or
fermented agricultural wastes (Huang et al.,1997) also effectively suppressed the
development of sclerotia in soil. In general, adoption of good field practices, such as
growing carrots in well drained soils, and using hygienic handling procedures that
confine and reduce field infestation, are important to ensure proper crop health.
Crop rotation with plants that are not susceptible to S. sclerotiorum (e.g., nonhosts) has
been long advocated for managing diseases caused by S. sclerotiorum (Steadman, 1979),
despite the long-term survival of sclerotia and the wide host range of the pathogen.
Rotation (e.g., up to 3 or 4 years) has not been always successful in reducing the number
of sclerotia in soil (Schwartz & Steadman, 1978; Williams & Stelfox, 1980), or disease
(Morrall & Dueck, 1982). However, new evidence confirms the viability of this control
option. Two-year rotations with corn or wheat reduced the numbers of apothecia in
soybean crops by up to 75% compared to continuous cropping of soybean (Gracia-Garza
et al., 2002). Rotation with broccoli (Brassica oleracea L.) and incorporating broccoli
residues in soil also can reduce the number of sclerotia and the incidence of the diseases
caused by Sclerotinia spp. (e.g., S. minor Jagger) (Subbarao, 1998; 2002).
Brassica crops containing high levels of glucosinolates with antifungal properties,
offer a biological alternative (e.g., biofumigation) to chemical fumigation of soil
(Warton, Matthiessen & Shackleton, 2001). This rotation could be particularly
beneficial for carrots because carrot is susceptible to S. minor (Melzer, Smith & Boland,
1997) and concurrent infection of carrot crops by S. sclerotiorum and S. minor has been
reported (Kora, Boland & McDonald, 2002). Currently, a 2- to 3-year rotation of carrots
with beets, onion, spinach, cereals, and corn is among the few strategies available to
carrot growers in Canada to manage SRC (Anonymous 2001; 2004b), but this is not
always effective in reducing the disease.
Management of soil and crop residues has been shown to be a useful approach in
reducing inoculum and diseases caused by S. sclerotiorum. No-till systems reduced the
number of apothecia in soybean crops, especially when chopped residues of previous

crops were returned to the soil surface (Gracia-Garza et al., 2002). Similarly, direct
drilling of lupin into cereal residue reduced the incidence and severity of Sclerotinia
stem rot and increased biomass of lupin at harvest (Simpfendorfer et al., 2004).
Moreover, higher populations of microorganisms in the upper layer of soils with no
tillage and (or) mulching compared to conventional tillage (Doran, 1980a; 1980b) may
contribute to a higher degradation rate of sclerotia.
Mulching can act as a physical barrier preventing apothecia from reaching the soil
surface or obstructing the discharge of ascospores and, therefore, may reduce the
disease severity (Ferraz et al., 1999; Gracia-Garza et al., 2002). Evidence of the effects
of the methods described above is not available for SRC, but these studies suggest that
similar methods may also be effective to manage inoculum in carrot crops. However,
methods that target inoculum in the field need to be integrated with other management
tools for enhanced efficacy, especially when out-of-field sources of ascospores are
important for initiating epidemics (Abawi & Grogan, 1979; Morrall & Dueck, 1982;
Steadman, 1983; Wegulo et al.,2000).
Products or cultural practices that affect the canopy architecture and phenology of a
carrot crop could indirectly influence the emergence of apothecia and subsequent
development of SRC. Reducing crop density through increased plant spacing and
cultivation in raised beds can lower soil moisture and enhance air circulation within the
canopy and, thus benefit disease control (Rubatzky, Quiros & Simon, 1999).
Application of low rates of nitrogen (N), such as 6 kg a.i. (active ingredient) N ⋅ ha -1,
was associated with significant reduction of canopy size and density, less lodging, and
lower levels of SRC compared to 60 kg a.i. N ⋅ ha-1, with negligible effects on yield
(Couper, 2001). By promoting excessive foliar growth, high N inputs may increase
shading and accelerate the senescence of lower leaves. The development of full,
vigorous, and heavily-lodged canopies was always associated with higher incidence of
SRC (Geary, 1978; Couper, 2001; Kora, McDonald & Boland, 2005b). Therefore,
balanced N applications that meet crop requirements based on soil type and fertility
analyses should be promoted especially for fields with high disease risk. Growers in the
UK use carrot varieties with stocky, upright, and less lush foliage (e.g., cv. Bolero)
and lower N inputs to reduce canopy size and predisposition of plants to disease
(McQuilken M., personal communication).
Recently, we tested the lateral clipping of carrot canopies as a cultural method
designed to manipulate the architectural crop attributes and render the microclimate less
conducive for development of epidemics. Lateral clipping (also known as side trimming) is
recommended (Subbarao, 2002; Davis 2004) and practiced commercially in California
(Davis, M., personal communication) and Washington State (Kora, McDonald & Boland,
2003) to control SRC. Reducing the canopy width by ca. 20% on both sides of the carrot
bed increased maximum air and soil temperatures and decreased relative humidity within
the crop, reduced the number of apothecia by up to 76%, and completely controlled SRC
where it occurred (Kora, McDonald & Boland, 2005a). Clipping removed overlapping
leaves above the furrow and senescing leaves lying on the soil surface, and reduced
lodging to zero without affecting fresh foliage and root weights of carrots at harvest. By
252 C. KORA ET AL.
removing senescing and lodging leaves, clipping reduced potential infection sites and
minimized plant-to-soil and plant-to-plant contacts. Extensive trials in Prince Edward
Island province of Canada, demonstrated that trimming carrot canopies sideways at row
closure reduced the incidence of SRC in commercial fields and storages by up to 80%
(Sanderson & Peters, 2008). It is anticipated that this technology will soon become a
standard practice, as more commercial trimmers are being constructed and adapted for use
in various carrot production regions.
Crop attributes such as denser, taller, and lodged canopies have been associated with
cooler and moister microclimates, greater numbers of apothecia, and higher incidence of
Sclerotinia diseases in several crops (Blad, Steadman & Weiss, 1978; Weiss et al.,
1980; Caesar & Pearson, 1983; Fuller, Steadman & Coyne, 1984; Boland & Hall,
1987b; Turkington & Morall, 1990; Deshpande et al., 1995; Jurke & Fernando, 2002;
2003). Consequently, several genetic and cultural methods that manipulate the canopy
architecture (Blad, Steadman & Weiss, 1978; Weiss et al., 1980; Fuller, Steadman &
Coyne, 1984; Boland & Hall, 1987b; Pratt, 1991; Saindon et al., 1993; Butzler, Bailey
& Beute, 1998) were used to modify the microclimate and maximize avoidance of
diseases caused by Sclerotinia spp. These studies suggest that architecturally-based
disease avoidance is important for improving the management of diseases caused by
Sclerotinia spp., including SRC.
Preharvest cultural practices that influence postharvest quality of carrot can reduce
its susceptibility to SRC during long-term storage. Balanced fertilization programs that
provide adequate potassium and avoid excess nitrogen (Shibairo, Upadhyaya &
Toivonen, 1998b), and prevention of water stress (Shibairo, Upadhyaya & Toivonen,
1998a) can increase membrane integrity of carrot roots and reduce moisture loss during
storage. Supplemental applications of calcium nitrate (e.g., 10 days before harvest) may
enhance the quality of carrot roots during storage (Lee et al., 2000), but did not affect
the incidence of SRC in storage (Stack et al., 2002).
Timing of harvest may affect the susceptibility of carrot to storage rot diseases
through the effect of age and physiological state of the root, and weather conditions.
The recommended harvest time for optimum storability is when carrots are physically
and biochemically mature (e.g., have reached their full size and maximum carotene and
carbohydrate concentrations) (Phan & Hsu, 1973, Cheah & Brash, 2001). Mature
carrots store better, probably because of lower growing and respiration rates. Longer
carrot growing periods, later harvests, higher sucrose-glucose ratios in roots at harvest,
and dry weather conditions during harvest were associated with reduced losses from
SRC in storage (Mukula, 1957; Suojala & Pessala, 1999). A harvest indicator or model
based on root maturity is not yet available (Cheah & Brash, 2001), but it would be a
useful tool in order to determine the overall storability of carrot cultivars.
3.1.2. Host Resistance

Carrot genotypes that possess qualitative germplasm resistance to S. sclerotiorum are
not available (Subbarao, 2002). However, differences among carrot cultivars in the level
of root disease during storage have been observed (Finlayson, Pritchard & Rimmer,
1989). The differences in susceptibility of these carrot cultivars to S. sclerotiorum was
related to variations in the integrity and permeability of root cell membranes, with cv.
Dess Dan being more resistant than cv. Six Pak II.
The varying response of carrot cultivars to cellular disruption suggests the presence
of quantitative resistance of root tissues to S. sclerotiorum. Similarly, the differences in
accumulations of pre-formed polyacetylene falcarindiol in the root periderm of several
carrot cultivars was associated with varying levels of resistance to S. sclerotiorum
(Olsson & Svensson, 1996). The concentration of falcarindiol was negatively correlated
with the susceptibility of cultivars to S. sclerotiorum, suggesting that it may contribute
to disease suppression in storage. Falcarindiol has antifungal properties and is
intensively accumulated in young, active roots, but concentration declines with age
(Lewis & Garrod, 1983). Recent studies have demonstrated that insertion of specific
genes in the carrot genome that express antifungal proteins (e.g., thaumatin-like
proteins) may also enhance resistance to S. sclerotiorum (Punja, Chen & Yip, 2003).
Disease avoidance has been identified as an effective alternative strategy to improve
the control of Sclerotinia diseases in crops that lack qualitative resistance to S.
sclerotiorum. Direct associations have been observed between cultivars with an open
canopy architecture and lower apothecial production and reduced incidence of white
mold in bean (Schwartz & Steadman, 1978; Schwartz, Steadman & Coyne, 1978) and
reduced Sclerotinia stem rot in soybean (Boland & Hall, 1987b). Improved management
of white mold has been achieved by breeding bean cultivars for a combination of
physiological resistance and architecturally-based disease avoidance mechanisms, such
as an upright growth habit and lodging-resistance (Park, 1993; Saindon et al., 1993;
Huang, Mundel & Erickson, 2003).
Recently, several quantitative genetic traits (e.g., QTL) were identified in
association with resistance to S. sclerotiorum in various crops, including partial
physiological resistance, canopy porosity, plant height, and lodging in beans (Miklas et
al., 2001; Kolkman & Kelly, 2003; Miklas, Delorme & Riley, 2003) and soybeans (Kim
& Diers, 2000). Such genetic information for interactions between carrot and S.
sclerotiorum is not yet available. However, our studies involving the reduction of
canopy through clipping suggest that architecturally-based disease avoidance
mechanisms also represent a significant strategy for improving the management of SRC
(Kora, McDonald & Boland, 2005a). Carrot genotypes with upright and compact
growth traits offer an important source in selecting and breeding for these disease
avoidance mechanisms.
3.1.3. Biological Control

A wide range of microorganisms with antagonistic or mycoparasitic properties have the
potential to degrade sclerotia and suppress diseases caused by S. sclerotiorum (Adams
& Ayres, 1979). However, hyperparasites Coniothyrium minitans Campbell, and
Trichoderma spp. are among the few most effective organisms that have obtained
commercial application (Anonymous, 2003). Coniothyrium minitans can destroy
sclerotia in soil and suppress the development of mycelia and sclerotia in diseased crops
through mycoparasitism. Treatment of soil with solid preparations of C. minitans at
seeding completely reduced apothecial production under canopies of artificially
inoculated host and non-host crops (McLaren, Huang & Rimmer, 1996). Applications of
spore suspensions of C. minitans on Sclerotinia-infected crops also reduced apothecial
production by up to 90% during a 4-year rotation of crops susceptible to S.
sclerotiorum, including carrots (Gerlagh et al., 1999).
254 C. KORA ET AL.
Mid-season foliar treatment of carrot crops with a biofungicide containing C.

minitans (Contans®, Prophyta, Germany) reduced apothecial production by up to 100%
and the proportion of Sclerotinia-infected roots by up to 36% (Couper, 2001). The effect
of C. minitans in reducing Sclerotinia stem rot of rapeseed was comparable to the
control provided by a chemical fungicide, suggesting the possibility of using this
biological alternative to replace fungicides (Weber, 2003). The reduction of surviving
sclerotia and infection by S. sclerotiorum in sequential crops improved when C.
minitans was incorporated in soil in the fall (McQuilken et al., 1995) or when a spore
suspension was sprayed on previous crop residues (Budge et al., 1995).
Foliar treatments, applied immediately after the first symptoms of disease were
detected, resulted in the highest proportion (e.g. >90%) of new sclerotia infected by C.
minitans and the lowest number of subsequent apothecia (Gerlagh et al., 2003).
Coniothyrium minitans is a promising biocontrol for SRC because it can effectively
colonize senescent tissues (Huang, 1977), persist and spread in soil (McQuilken et al.,
1995), as well as reduce the viability of new sclerotia produced on diseased plants
(McLaren, Huang & Rimmer, 1996; Huang et al., 2000; Bennett, Leifert & Whipps, 2003).
Several Trichoderma spp. can suppress the disease caused by S. sclerotiorum by
parasitizing hyphae and sclerotia of the fungus (Inbar, Menendez & Chet, 1996) and
inducing local and systemic resistance in the host plant (Elad, 2000). In soil infested
with T. koningii, the number of viable sclerotia was reduced by 100% within 60 days
(Dos Santos & Dhingra, 1982). Feeding by soil mycophagous animals (e.g., fungus
gnats, Bradysia sp.) can damage and predispose sclerotia to infection by Trichoderma
spp. (Gracia-Garza et al., 1997).
Sporidesmium sclerotivorum Uecker, Ayers & Adams is another mycoparasite
responsible for destroying sclerotia of S. sclerotiorum in soil (Uecker, Ayers & Adams,
1978; Adams & Ayres, 1979; Zhou & Boland, 1998). Soils infested with spore
suspensions of S. sclerotivorum prior to planting had up to 60% fewer apothecia, and 51-
100% less Sclerotinia stem rot of soybean (Del Rio, Martinson & Yang, 2002). The fungus
is an aggressive colonizer of sclerotia of S. sclerotivorum and produces numerous spores
able to spread and persist in soil for at least 5 years (Martinson & Del Rio, 2001; Del Rio,
Martinson & Yang, 2002). These features provide for excellent residual effects and
demonstrate the viability of this mycoparasite as a biocontrol for Sclerotinia diseases.
However, mass production of inoculum for commercial purposes is difficult, because S.
sclerotivorum is very fastidious and difficult to grow in artificial media.
Compost alone, and soils amended with it, can also reduce the survival of sclerotia and
suppress apothecial production in soil (Couper, Litterick & Leifert, 2001; Yang,
Kharbanda & Tewari, 2001; Rousseau, Rioux & Dostaler, 2003). The suppressive effects
of composts in these studies were mainly attributed to the enrichment of the soil microbial
profile and to the increase in organic matter content. However, addition of compost to soil
increased the incidence of SRC through the indirect effect of promoting the growth of
carrot top and development of a dense and lodged canopy (Couper, 2001). Ferraz et al.
(1999) found similar effects of promoted canopy growth, increased production of
apothecia, and higher disease incidence in bean crops, following high compost input in
soil.
3.1.4. Chemical Control

Application of fungicides has been the most prevalent means of reducing the viability of
sclerotia in soil or levels of SRC in the field. Preplant application of methyl bromide,
formaldehyde, pentachloronitrobenzene or metham-sodium effectively reduced
populations, and carpogenic germination, of sclerotia in soil (Steadman, 1979; Ben-
Yephet, Bitton & Greenberger, 1986; Ben-Yephet, 1988). However, fumigation of soil
with some of these chemicals can be expensive for carrot crops and does not always
provide effective control of soilborne diseases (Cheah & Brash, 2001). Moreover, the
use of methyl bromide is in decline and is being phased out, due to its ozone depleting
properties (Gullino et al., 2003). Foliar application with fungicides of the benzimidazole
(e.g., benomyl and thiophanate-methyl) and dicarboximide (iprodione and vinclozolin)
groups have consistently reduced or prevented infections of carrots in the field and
storage (Tahvonen, 1985; Pritchard, Boese & Rimmer, 1992; Stack, Gudmestad & Lee,
1998; Hansen et al., 2001). Disease suppression in these studies improved when foliar
fungicides were applied closer to harvest (e.g., 1 to 10 days before harvest) and were
combined with rapid cooling of harvested carrots. A fungicide that combines iprodione
and thiophanate-methyl (Compass®, Rhône-Poulenc Agriculture, UK) also effectively
decreased the level of SRC in the field and increased marketable yield of carrots
(Couper, 2001).
Except for benomyl, which was phased out in 2002, several of the above fungicides
are currently used to control SRC in countries where they are available, e.g., New
Zealand and California, (Cheah, L-H. and Davis, M., personal communications). In the
UK, carrot growers use azoxystrobin and tebuconazole on a calendar spray schedule
basis, starting just before canopy closure ( McQuilken, M., personal communication). A
reduced risk fungicide of the carboxamide group (boscalid), with a unique mode of
action, is now available for the control of Sclerotinia diseases in a number of crops, but
not carrots (Barton & Chapman, 2002).
Proper timing of fungicidal sprays and adequate coverage of susceptible tissues of
the host are crucial for obtaining effective control of Sclerotinia diseases with foliar
applications (Hunter, Abawi & Crosier, 1978; Steadman, 1979). The fungicides are
most effective if applied during the period when ascospores production coincides with
the susceptible stage of the carrot crop. In general, adequate chemical coverage of
susceptible tissues is achieved in canopies with lower plant densities and upright growth
habits. Application of fungicides following the lateral clipping of the carrot canopy,
may improve the coverage of potential infection sites, such as the base of the plant and
crown. Adequate management of Alternaria [Alternaria dauci (Kühn)] and Cercospora
[Cercospora carotae (Pass.)] leaf bights of carrots is also important for minimizing the
accumulation of additional senescent and damaged foliage that may be a suitable
substrate for infection by S. sclerotiorum.
3.1.5. Disease Forecasting

Forecasting models have been proposed to predict the occurrence of inoculum and
diseases caused by S. sclerotiorum, and the need for fungicide applications in several
256 C. KORA ET AL.
crops such as bean and rapeseed. These models combined a selection of microclimate,
pathogen, and crop variables that influence disease development such as soil moisture,
rainfall, temperature, number of apothecia, petal infestation by ascospores, canopy
development, blooming patterns, and cropping history (Hunter, 1981; Hunter et al.,
1984; Turkington, Morall & Gugel, 1991; Twengström et al., 1998; Bom & Boland,
2000; Clarkson et al., 2004).
Figure 2. Diagram of an expert system prototype, based on crop and inoculum factors for
warning of outbreaks of Sclerotinia sclerotiorum rot of carrot (SRC), advising growers
about decisions for management actions (e.g., initial application time of fungicide sprays or
other control methods). The diagram shows the sequential order of critical thresholds for
risk factors of disease initiation, and actions for each outcome (Yes or No). The inoculum is
measured by direct ascospores counts (A) or prediction based on microclimate suitability
for pathogen development and field history (B). The system requires field validation prior
to making recommendation for commercial use. Output: seven-day severity index value that
best predicts the occurrence of inoculum in field tests (see Table 1) (adapted from Kora,
2003).
Similarly, in carrot crops, occurrence of apothecia and ascospores was most closely
associated with the closure of the canopy and high soil moisture (Kora, McDonald &
Boland, 2005b). Consequently, a risk algorithm was proposed to predict the presence of
inoculum using canopy closure of 95%, and daily mean soil matric potential of -0.4 bars
as crop and microclimate thresholds, respectively (Table 1).
In addition, a preliminary expert system was proposed that predicts the start of
disease and recommends the initiation of control measures when the canopy is 100%
closed, 70 to 80% of carrot plants have 1 to 2 senescing and 1 to 3 healthy leaves
lodged on the soil, and ≥10 colony forming units of S. sclerotiorum arising from
ascospores are deposited per 90 mm Sclerotinia semi-selective media plate (Fig. 2)
(Kora, 2003).
The combination of field history and microclimate suitability for pathogen
development were proposed as an alternative means to determine the risk of pathogen
presence when a direct measure of the inoculum is not feasible. Field studies to test the
predictive accuracy of proposed thresholds and validate these models are currently
being conducted by researchers at the University of Guelph, Guelph, Ontario.
Table 1. Crop and microclimate risk factors, factor sub-ranges, and corresponding
multiplier values of risk points used to calculate the risk for the occurrence of
apothecia and ascospores of Sclerotinia sclerotiorum.
Risk Factor Factor Sub-range Risk points x
Crop
Canopy growth < 95% of soil surface shaded 0
95 to 100% of soil surface shaded 1
Micro-climate
Soil matric potential y < -0.4 bars 0
-0.4 to -0.3 bars 1
-0.2 bars 2
> -0.2 bars 3

x
Risk points represent the degree of suitability of the factor sub-range for pathogen development
in a scale from 0 to 3. A seven-day severity index (SDSI) is computed by multiplying risk points
associated with host and microclimate risk factors for any given day, and then summing the daily
scores over 7 preceding days: SDSI = ∑(day 1 -7) (CGRP ⋅ SMPRP), where CGRP and SMPRP = daily risk
points of the canopy growth and soil matric potential sub-ranges, respectively (Kora, 2003).
Additional research is required to identify the SDSI value (e.g., 7 ≥ SDSI ≤ 21) that best predicts
inoculum in the field and could be used as action threshold for timing disease management decisions.
y
Daily mean values.
258 C. KORA ET AL.
3.2. Storage Practices

3.2.1. Cultural Control
General hygiene and sanitation measures are important to reduce spoilage in storage and
increase shelf-life of carrots. Removal of soil and plant debris through prestorage
washing and grading of carrots is recommended for improved storability, but had
inconsistent effect on suppression of SRC and other postharvest diseases (Lockhart &
Delbridge, 1972; Geeson, Browne & Everson, 1988). However, washing and culling
unmarketable roots enhances the efficacy of prestorage dip treatments (Geeson, Browne
& Everson, 1988) and rapid removal of latent heat from harvested roots (Pritchard,
Boese & Rimmer, 1992). Gentle washing using the spray-brush method and clean,
disinfected water (e.g., with chlorine or other sanitizing agents) is recommended to
minimize injury to roots and establishment of storage rots (Cheah & Brash, 2001).
Regular clean-up by removing debris and sanitizing the conveyors, grading belts, and
hydrocoolers is essential to avoid post-wash recontamination of roots. Washed (or
unwashed) roots should be placed in clean storage bins to minimize the risk of disease
originating from inoculum persisting on the surface of bin walls. Pressure washing for
debris removal and disinfestation (e.g., steam or chemical sanitation) of bins may be
necessary, particularly when previously used to store infected carrots (Kora, 2003).
Minimizing injuries during mechanical harvest or handling, and wound healing by
exposing carrot roots to high temperature treatments prior to storage, can decrease
predisposition of carrots to infection. Exposure of carrot roots to 3 s of steam at 90°C
suppressed disease caused by S. sclerotiorum, Alternaria alternata [(Fr.: Fr.) Keissler],
and A. radicina (Meier) by up to 92% in storage (Afek, Orenstein & Nuriel, 1999). The
effect of steam treatments was attributed to the killing of pathogens on the carrot
surface, removal of spores and debris, and stimulation of antifungal compounds in roots.
Although brief heat treatment, or curing, can effectively heal wounds on carrots and
reduce subsequent infection by S. sclerotiorum, it may encourage disease development
in roots that are already infected (Geary, 1978). Therefore, this method may not be
effective to suppress SRC in storage when high levels of disease occur in the field.
Maintaining air temperatures of 0 to1°C, relative humidity of 95 to 100%, and good
air circulation are optimal to reduce losses caused by SCR and sustain long term quality
of carrot in storage (Salunkhe & Desai, 1984; Geeson, Browne & Everson, 1988; Le
Cam et al.,1993). Although S. sclerotiorum is capable of growing and causing disease at
temperatures as low as 0°C, its activity is substantially reduced below 6°C (Finlayson,
Pritchard & Rimmer, 1989). Sclerotinia sclerotiorum produced higher levels of
extracellular pectolytic enzymes and decay on the surface of carrots incubated at 20 or
1°C, when exposed to 94 to 96%, than to 98 to 100% relative humidity (Van den Berg
& Yang, 1969).
Disease was reduced by up to 36% when carrots were stored in an ice-bank cooled
storage at 0.5 to 1°C and 97 to 98% relative humidity, compared to conventional storage
at 2 to 2.5°C and 85 to 95% relative humidity (Geeson, Browne & Everson, 1988). Ice-
bank cooling systems provide superior storage conditions because they deliver saturated
air at a temperature near 0°C through positive ventilation (Le Cam et al., 1993).
Modified atmosphere storage (e.g., various CO2/O2 ratios) also reduced losses caused by
S. sclerotiorum and improved long term storability and quality of carrots (Reeleder et
al.,1989). It is suggested that low O2 levels have an inhibitory effect on the growth of S.
sclerotiorum and can also increase resistance of carrot roots to infection. High CO2
levels delay senescence of roots.
Rapid cooling of harvested carrots prior to storage is important for further
suppression of disease because this reduces respiration rate and spread of
microorganisms. Increasing the cooling time from 6 to 72 h increased SRC level by
300% after 15 weeks in storage at 6°C (Pritchard, Boese & Rimmer, 1992). Increase of
storage temperature from 2 to 20°C led to progressive electrolyte leakage from carrot
roots as a result of rapid disruption and increased permeability of cell membranes
induced by S. sclerotiorum (Finlayson, Pritchard & Rimmer, 1989). In conventional
storage using a refrigeration coil or a Filacell cooling system, cooling of bulk carrots
from 6 to 1°C can take up to 75 days (Pritchard, Boese & Rimmer, 1992) and is not
uniform. Rapid and uniform cooling can be achieved by forced-air cooling or
hydrocooling. Forced-air cooling consists of ventilating by pulling (or blowing)
refrigerated air through the openings of stacked bins filled with carrots, preferably in a
tunnel setting, for up to 6 h (Fraser, 1998). An alternative approach is to cool carrots by
‘air-washing’ using ice-bank systems that move chilled air vertically through cascading
ice water. In these ice-bank cooling systems, carrots cool to 1°C in less than 6 h
(Geeson, Browne & Everson, 1988). Hydrocooling is the preferred method of cooling
because it achieves more rapid removal of latent heat (e.g., from 25 to 4°C in 25 min)
(Cheah & Brash, 2001). In this system, carrots are cooled by immersing or showering in
sanitized water held at 1°C by mechanical refrigeration, and preventing re-warming of
carrots after cooling is important.
3.2.2. Biological Control

The potential of using biocontrol agents to suppress the postharvest development of
SRC is restricted by cold temperatures during storage. Dipping carrot roots for 5 min in
a conidial suspension of a cold-tolerant mutant of T. harzianum (P1) prior to storage
reduced disease severity and increased marketable yield by 75% after 8.5 months at 0°C
(Tronsmo, 1989). Satisfactory suppression of subsequent infections by S. sclerotiorum
in storage three months after harvest was also obtained by inoculating carrots at seeding
with mycorrhizae (Glomus intraradices Schenck & Smith and Glomus etunicatum
Becker & Gerdemann) (Gotoechan & Desilets, 1999). It is suggested that disease
suppression in mycorrhizal-colonized roots is attributed to the ability of the mycorrhiza
to induce defense mechanisms in carrots against S. sclerotiorum. However, neither these
or other agents are commercially available for the control of postharvest SRC.
3.2.3. Alternative Methods
Induction of natural resistance in carrots and other vegetables to storage diseases is

receiving increasing attention as an alternative to chemical methods (Terry & Joyce,
260 C. KORA ET AL.
2004). Several biological and physical methods have been used to increase resistance of
carrots to infection by S. sclerotiorum. Chitosan, a naturally derived polysaccharide, has
been tested as a postharvest treatment for the control of SRC in storage (Cheah, Page &
Shepherd, 1997). Coating carrot roots with 2 or 4% solutions of chitosan significantly
decreased disease incidence and inhibited subsequent development of the fungus.
Later studies demonstrated that coating carrots with enzymatically hydrolyzed
chitosan at 0.2% provided a similar level of disease reduction and suggested induced
resistance in carrots as the mode of action (Molloy, Cheah & Koolaard, 2004). Chitosan
is believed to possess a dual mechanism of action: it interferes with fungal growth and
acts as an elicitor that activates defense mechanisms in plant tissues (El Ghaouth, 1994).
The use of chitosan as a prestorage treatment appears promising, but further research is
needed to optimize application rates and conditions for long-term protection.
Nonionizing ultraviolet (UV-C) radiation can elicit the accumulation of the
anti-fungal phytoalexin 6-methoxymellein in carrot roots, and induce systemic
resistance to subsequent infections by S. sclerotiorum (Mercier et al.,1993). In carrot
slices treated with UV-C irradiation at a dose of 2.20 ⋅ 105 erg cm -2, accumulation of
6-methoxymellein increased to maximal inhibitory levels (e.g., 60 µg ⋅ g-1) that reduced
the growth of S. sclerotiorum at 1 or 4°C. However, UV-C treatments should be
integrated with other control strategies for a prolonged protection during storage (El
Ghaouth, 1994; Terry & Joyce, 2004).
Ozone has demonstrated fungistatic effects on S. sclerotiorum and was proposed as
an alternative disinfectant for stored carrots (Liew & Prange, 1994). Treatments with
gaseous flow of ozone for 8 h daily during 28 days reduced the daily growth rate of S.
sclerotiorum on inoculated carrot roots by up to 56%. Ozone concentrations of 60 µl ⋅
l-1, however, caused significant physiological disruptions including increased respiration
rate, electrolyte leakage, and discoloration of carrots. An ozone supply of 15 µl ⋅ l-1
during 8 h daily at 2 °C was suggested for providing adequate disease control while
preserving carrot quality.
3.2.4. Chemical Control

Fungicide dip treatments of carrots prior to storage have provided effective control of
SRC and reduced crop losses during long term storage. As with foliar treatments,
fungicides of the benzimidazole and dicarboximide groups were most effective and
attained commercial application. Dipping carrots in 0.1% aqueous solution of sodium
orthophenylphenate (Hoadley, 1963), 0.05 or 0.025 % a.i. suspension of benomyl, or
0.05 % a.i. suspension of iprodione (Geeson, Browne & Everson, 1988; Cheah, Page &
Shepherd, 1997) significantly reduced decay during long term storage. Optimum storage
conditions (Geeson, Browne & Everson, 1988) and proper washing and grading of
harvested carrots (Lockhart & Delbridge, 1972) were important for enhancing the
efficacy of fungicide dip treatments. Although selected fungicide dips provided
adequate protection and suppressed disease development, current approval for registered
postharvest fungicides is limited, due to concerns over chemical residues on carrot
roots.
4. RECOMMENDATIONS ON INTEGRATED DISEASE MANAGEMENT

The individual use of the control methods described above can reduce the incidence of
Sclerotinia diseases to a certain extent, but these may not always provide effective
management in commercial productions. Integrating various specific methods in a timely
and orderly fashion can provide sustainable control solutions while obtaining maximum
benefit, and prolonging the usefulness of suitable methods. The traditional principles of
plant disease control, first established by H.H. Whetzel in 1929, have been reviewed to
reflect the modern concepts of disease epidemiology and management (Arneson, 2001).
Modern disease management intends not to eliminate disease, but to avoid the
occurrence or reduce the progress, keeping the disease development below an
acceptable level. In this view, control methods are re-grouped under three strategy
approaches according to their quantitative effects on specific aspects of pathogen and
disease development, and the goal they achieve, i.e. to reduce inoculum, reduce
infection rate, or reduce epidemic duration.
We used a similar approach to group the control methods for SRC integrating them
in a management system that combines all three strategies. In each strategy, we
emphasize methods that offer more than one beneficial effect or improve results of other
methods when in combination. However, agriculture practitioners can select any of the
methods based on the suitability for specific conditions, availability of the product or
technology, and ultimate goal.
4.1. Reduction of Inoculum

The importance of within-field sources of inoculum for the epidemiology of SRC
prompts the need for soil sanitation, in order to reduce the reservoir of viable sclerotia
prior to seeding. Several cultural, biological, and chemical methods can achieve
disinfestation of soil through eradication. Steam disinfestation in particular, is
recognized as a viable alternative to chemical fumigation and has been used for years to
manage several soilborne plant pathogens (Bennett, Leifert & Whipps, 2003).
Excellent control of sclerotia achieved in recent studies will likely encourage the
commercial adoption of this method. Currently, work is underway to develop more
effective and energy-efficient steam disinfestation methods (Bennett, Leifert & Whipps,
2003). Moist sclerotia are more sensitive to steam treatment and require lower minimum
lethal steam temperatures than dry sclerotia (Couper, 2001; Van Loenen et al., 2003).
Therefore, moistening soil prior to steaming is essential for the successful control of S.
sclerotiorum. In addition, biocontrol agents (e.g., C. minitans) may have higher
proliferation rates when applied in sterilized soil (Bennett, Leifert & Whipps, 2003),
suggesting an improved establishment and efficacy of these agents if biofungicides are
applied after soil disinfestation. A combination of these cultural and biological methods
is, therefore, recommended for reducing the number of sclerotia in soil.
Foliar-applied C. minitans can also minimize carry-over of newly produced sclerotia
in subsequent years if application coincides with the presence of sclerotia in the
diseased crop (e.g., on growing plants or plant residues after harvest) (Huang et al.,
2000; Budge et al., 1995).
262 C. KORA ET AL.
Organic matter and compost can be counter-effective for disease control although
beneficial for suppressing the development of sclerotia. Where input of organic matter
is important for crop management, and in soils containing high natural organic matter
(e.g. peat, muck), additional strategies, such as canopy clipping, reduced irrigation
and/or mulching are recommended to offset the undesired effects of these soil
amendments. Combination of crop rotation with no-tillage and chopped residue left in
the field was suggested as the most useful method to reduce apothecia in infested fields
(Gracia-Garza et al., 2002). Lower plant density as a result of wider inter- and intra-row
spacing, balanced nitrogen input, and/or canopy clipping can reduce production of
ascospores in the field by suppressing the germination of sclerotia. Sanitation of
containers, storage facilities, and handling equipment, combined with washing, grading,
and rapid cooling of the roots aim at reducing the mycelial inoculum entering storage.
4.2. Reduction of Infection Rate

Manipulation of crop canopy by using upright cultivars or lateral clipping can reduce
infection rate by minimizing the physical contact of the leaves with soil and removing the
potential infection sites. Further, well-timed and disease risk-based applications of
fungicides or biofungicides aim at protecting the crop from infection. Rotating fungicides
of different family groups and modes of action is essential to delay the development of
pathogen resistance to chemicals. An open canopy, achieved by manipulation prior to
applying (bio)fungicides, would improve the penetration of material to potential infection
sites at the plant base. Forecasting systems would help growers in making sound disease
management decisions by advising to apply fungicides or other control measures if and
when crop and environment conditions are conducive for pathogen and disease
development. Availability of an effective infrastructure for delivering these forecasting
systems is important for the viability and successful use of these tools.
Maintaining optimal temperature and relative humidity conditions in storage aims at
protecting the crop and suppressing SRC development from harvest until marketing. In
addition, a prestorage treatment can be used to induce natural disease resistance in roots
during storage. Adequate management of disease in the field can improve the efficacy
and the success of these methods in storage.
4.3. Reduction of Epidemic Duration

Selective harvesting, separate storage, and immediate selling of carrots from diseased
crops will minimize the spread of disease in the field (e.g., during in-situ overwintering)
and delay, or reduce, the introduction of inoculum in storage. Decisions about which
crops to be harvested first should include field monitoring and testing, in order to
identify the disease incidence, the disease and yield loss thresholds, if available, and the
knowledge of disease history of the field.
4.4. Proposed Integrated Disease Management Programs

In summary, we propose two integrated programs for the management of SRC as
examples to apply to various carrot production systems. What these programs have in
common is the use of a variety of approaches to suppress disease while sustaining the
efficacy of existing control tools.
1) For conventional carrot production using currently available methods we
recommend: applying best soil and crop management practices that maximize crop
health and minimize suitability for disease development such as: a minimum of three
years rotation with nonhost crops, seeding on well-drained fields and raised beds,
adequate spacing, planting relatively upright cultivars, and balancing nutrient and water
inputs; monitoring crop development and inoculum presence and spraying foliar
fungicides when senescing leaves start to collapse on soil and apothecia and (or)
ascospores are present; clipping the canopy prior to spraying to improve efficacy of the
fungicide by allowing for better coverage; managing other foliar diseases; rapidly
cooling the harvested roots and storing carrots in sanitized bins; and maintaining
optimum temperature (close to 0°C) and humidity (>95%) in storage.
2) For organic carrot production using currently available methods we recommend:
applying best soil and crop management practices that maximize crop health and
minimize suitability for disease development such as a minimum of three years rotation
with nonhost crops, seeding on well-drained fields and raised beds, adequate spacing,
planting relatively upright cultivars, and balancing nutrient and water inputs; sanitizing
soil and crop residues using biological (e.g., Contans, biofumigation), cultural (e.g.,
deep plowing or no-till), and (or) physical (e.g., steam) methods; monitoring crop
development and inoculum presence to determine crop susceptibility; clipping the
canopy when senescing leaves start to collapse on soil and apothecia and (or)
ascospores are present; spraying a foliar biofungicide (e.g., Contans) as an alternative or
a complement to canopy clipping (if needed); harvesting and selling first carrots from
diseased crops; rapidly cooling the harvested roots and storing carrots in sanitized bins;
and maintaining optimum temperature (close to 0°C) and humidity (>95%) in storage.
Eventually, new technologies may become available for inclusion in these
programs such as cultivars with upright, stocky, and lodging-resistant tops developed
through breeding, validated inoculum and disease forecasting models and delivery
systems, crop loss and action thresholds to determine the need for application of control
measures, low risk fungicides, and more biocontrol products.
5. CONCLUSIONS AND FUTURE PROSPECTS

Diseases caused by S. sclerotiorum are difficult to predict or control if there is
insufficient knowledge on the biological and environmental factors that influence their
development. The epidemiology of SRC is unique in that disease is bi-cyclic, involves
collapsed senescing leaves instead of flower petals in disease initiation, secondary
infection is important, and the susceptibility of the crop increases continuously with
aging and lodging.
Severe epidemics can be promoted by the use of high-yielding cultivars with large
tops, high plant densities, high fertilizer and water inputs, and insufficient rotations that
264 C. KORA ET AL.
are typical practices in current carrot cultivation. New information attained on disease
epidemiology and control methods summarized in this review contribute to a better
understanding and open new directions for the management of the disease.
Sclerotinia rot of carrot is difficult to manage because of several attributes of S.
sclerotiorum, such as the long term persistence of sclerotia in soil, the wide host range
and ubiquitous distribution, ability to produce infective propagules in synchrony with
the susceptible stage of carrot, and the ability to infect at temperatures as low as 0 to
1°C. The growth pattern of carrot crops is significant for the epidemiology of SRC in
that development of the canopy promotes production of inoculum, whereas senescing
leaves in the lower canopy provide a continuum of substrate available for infection.
Within-field production of ascospores is also significant for inciting important
epidemics because this mainly occurs when the crop and microclimate conditions are
favorable for disease development. Therefore, integrating methods that reduce within-
field sources of sclerotia, inhibit germination of sclerotia, and manipulate the crop
attributes that contribute to disease development, is pivotal to disease management.
Knowledge of relationships between the growth stages of S. sclerotiorum and carrot
phenology is required to integrate management practices that target the pathogen and
the crop. Monitoring and forecasting tools should be used to determine the presence of
inoculum during the susceptible stage of the crop and the suitability of environment for
disease initiation.
A carrot phenology model that incorporates physiological and architectural
attributes, such as foliar senescence and canopy lodging may be a useful tool for
predicting crop development and SRC epidemics and improving management of this
disease. More research is needed to develop and implement systems for the effective
delivery of advisory services to growers, to develop new biological control products,
and for breeding carrot cultivars with the traits of upright, compact, and lodging-
resistant canopy.
ACKNOWLEDGMENTS
We are grateful to Dr. Michael Davis, University of California, USA, Dr. Mark
McQuilken, Scottish Agricultural College, UK, and Dr. Lian-Heng Cheah, New
Zealand Crop and Food Research Institute, New Zealand for their valuable contributions
through personal communication.
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14
O. P. SHARMA AND O. M. BAMBAWALE
INTEGRATED MANAGEMENT OF KEY DISEASES OF

COTTON AND RICE
National Centre for Integrated Pest Management, IARI Campus,

New Delhi-110 012, India
Abstract. The major diseases of cotton and rice and the most suitable control measures are reviewed.
Practical issues related to the identification of a disease based on symptoms and presence of pathogens
are shown, as they have utmost importance for successful management. The main concepts for Integrated
Disease Management are discussed, together with the technologies advocating the combination of a
variety of control measures, including the conservation of existing natural defense system, crop rotation,
intercropping, and cultivation of pest-resistant varieties. Cotton diseases considered include seedling
diseases, bacterial blight, Alternaria leaf spot, grey mildew and leaf spots caused by Myrothecium,
Cercospora, Helminthosporium, Macrophomina, stem canker, late season Phoma blight, rust (Phakopsora
gossypii), leaf crumple, Cotton Leaf Curl Virus, Tobacco Streak Virus, root rot, Verticillium and Fusarium
wilts, new wilt or parawilt, boll rots and lint diseases. Rice diseases reviewed include rice blast, brown spot,
bacterial leaf blight and leaf streak, sheath blight, sheath rot, Fusarium wilt or “Bakanae”, stem rot, Tungro
Virus, false smut and post-harvest diseases.
1. INTRODUCTION
Indian agriculture is at a crossroads facing food insecurity, growing ecological
imbalance, stagnation of natural resources, decreasing diversity, spread of
unsustainable agricultural practices and threat from global market. Currently 142
million ha area is under cultivation contributing 27% to GDP, and dependence on
agriculture is not likely to come down in the near future. With the present growth
rate, population is expected to increase to 1.162 billion by 2010 requiring 247
million tones of food from 142 million ha, which is somewhat difficult to achieve.
There is a need for diversification and low cost alternatives to produce more food
and fiber from the same land surface without loss of biodiversity and reduction of
forest areas.
Pests are major biotic constraints in achieving self-sufficiency in quality food
production, while keeping the environment clean. Losses due to pests at national
level vary approximately around 18%, depending upon the genetic constituent of
crop, its health and the governing environment. Negligence in endemic areas often
results in complete crop failures. The general estimate of annual crop losses due to
pests in India amounts to Rs. 90000 crores per year (1 crore = 10 million).
271
272 O. P. SHARMA AND O. M. BAMBAWALE
Chemical based management with various groups of active compounds is

loosing effectiveness against major pests and paved the way for alternative
technologies, viz. Integrated Pest Management (IPM). During the 70’s the need to
increase food production to meet the demand from a rapidly increasing population
from limited land resources required the use of intensive farming systems, based on
high inputs like fertilizers, pesticides, irrigation, and multiple cropping with narrow
genetic bases. No doubt the production of major cereals tripled and met national
food requirement, but narrowing of genetic bases, changes in cropping patterns and
introduction of non local crops favoured the insurgence of diseases, which were of
mycological novelties. Diseases of lesser importance became a potential threat and
in some cases they assumed an epidemic status.
In recent years, the cultivation of transgenic cotton in larger scale has resulted in
the increase in certain hitherto unknown diseases, like parawilt, grey mildew, and
tobacco streak mosaic virus. Intensive agriculture, especially after the introduction
of new high-yielding genotypes, has become susceptible to pathogens and their
prevalent races. The changing cropping patterns, including cultivation in non-
traditional areas, have resulted in a spurt of diseases in various cropping systems,
remarkably changing the scenario of biotic stresses.
Cotton and rice are two highly intensive crops, consuming 54 and 22% of total
pesticides being consumed in the country and, on the contrary, account for only 5
and 3% of the total cultivated area (Singh, 2005; Dubey & Sharma, 2005). Since
pesticide use is maximum on these two crops, they have been selected for the
discussion in this chapter.
2. IDENTIFICATION OF DISEASES
Quick identification of a disease based on symptoms and presence of pathogens is of
utmost importance for successful management. The first step in diagnosis is to look
at affected plants in the field, and at distribution and prevailing weather factors.
Distribution patterns will help to understand the nature of pathogen viz., air-borne,
seed-borne, water-borne or soil-borne. In the case of the rice ecosystem, air-borne
pathogens are blast, brown spot, sheath blight and sheath rot (Rao, 1996). Bacterial
leaf blight of cotton as well as paddy are seed-borne and water-borne, spreading to
adjoining plants with irrigation. Sheath blight, stem rot and damping-off of paddy
and wilt, bacterial blight and Alternaria of cotton are important soil-borne pathogens
(Rao, 1995).
If diseased plants randomly occur regularly over large field, this distribution
may suggest the involvement of an air-borne pathogen. If diseased plants are found
in small circular patches, the causal agent may be a soil-borne pathogen. If a disease
affects a broad geographic area, air-borne pathogens or vector-borne pathogens e.g.,
tungro virus and grassy stunt virus in rice and grey mildew in cotton, may be
suspected. The symptoms and their location on the plant give further clues about the
nature of the disease. Quite often the lower leaves are attacked first and slowly dry-
up after contributing nutrition to the plant, and in such cases one should not worry
COTTON AND RICE DISEASE MANAGEMENT 273
for their curative management. If only the top parts of plants are affected, air-borne
pathogens can be suspected. Sheath blight, stem rot and seedling blight in paddy and
sore chin and stem canker in cotton can be diagnosed by looking at the collar or base
of plants for fruiting (sclerotial) bodies. The signs and symptoms that are observed
must be carefully compared with documented information on the respective disease,
for confirming the diagnosis. Once the causal agent and stage of a disease has been
correctly identified, it will be possible to develop curative or protective strategies by
spot application to manage the disease before it assumes an epidemic status.
3. THE CONCEPT OF INTEGRATED DISEASE MANAGEMENT

Concern over loosing effectiveness of conventional fungicides, residues and
development of resistance owing to various reasons viz., indiscriminate use of
chemical pesticides, non-observance of prescribed waiting periods, sub-standard
pesticides, incorrect advice and promotion of specific pesticides by dealers, wrong
disposal of pesticide containers and cleaning of plant protection equipments, has
prompted the development of IPM.
IPM technology advocates the combination of a variety of control measures,
including the conservation of existing natural defense system, crop rotation,
intercropping, and cultivation of pest-resistant varieties. Pesticides may still be used,
but selectively and in greatly reduced quantities (Dubey & Sharma, 2001). IPM is
more complex for the producer to implement than spraying by the calendar, which is
not only easy but also off shelf available, on a credit basis. IPM technology requires
education, skill in pest monitoring and understanding of its dynamics, and it often
involves cooperation among producers en mass, for effective implementation. At the
time IPM began to be promoted as a pest control strategy in the 1960’s, there was
very little proven IPM technology available to be transferred to farmers.
By the 1970’s, sufficient research had been conducted to provide the knowledge
to implement IPM programs successfully in important crops, such as rice, cotton,
sugarcane and vegetables. However, exaggerated expectations about the possibility
that dramatic reductions in pesticide use could be achieved without affecting crop
yields by means of IPM adoption, could not been realized. IPM is an in-built
component of crop improvement research and its various disciplines are
incorporated with the aim at evolving environmentally sound pest management.
However, substantial reduction of chemical pesticides use could be brought about
through promotion of appropriate IPM applications (Singh & Sharma, 2005).
Strategies for the effective management of plant diseases
1) Avoidance of pathogens by exclusion from a geographic area, by legislation,

and by host plant evasion, to prevent the pathogen from coming into contact
with its host;
2) Pathogen eradication or removal from the host, crop residue and soil, or from
other reservoirs;
3) Protection of plants by environmental changes, that are less favourable or

unfavourable for disease development;
4) Use of cultivars with built in multiple resistance, which may resist the biotic or
abiotic constraint and the eventual disease development;
5) Provide the crop with every possible advantage, using all available practices
in integrated disease management, and
6) Treatment with systemic or contact chemicals to kill associated pathogens.
Our efforts have been aimed at an holistic crop management approach, primarily
through host plant resistance, crop health monitoring, application of bio-pesticides at
first sight of disease initiation and spot application of chemical fungicides. The options
available for use of host plant resistance, development of disease epidemics and
control measures for the management of some important diseases of rice and cotton
are discussed in the following pages.
4. INTEGRATED DISEASE MANAGEMENT IN COTTON

Cotton is primarily the world’s major fiber used in almost half of all textiles, apart
from seed being used as a source of food. The cultivation of the cotton crop has
impacted on the economic development of human societies, since its cultivation for
the past 5000 to 10000 years. India is the third largest producer and the second
largest consumer of cotton in the world. Cotton has a large share in the Indian
agriculture output itself, accounting for about 2.5% of the agricultural output, valued
at more than Rs 20,000 crore. The area cultivated with cotton is estimated at 8.5
million hectares, which is about one-fourth (26.7%) of world’s acreage. Cotton
provides 7 million jobs in farms, at least a million jobs in mills and many more jobs
in trade, ginning, power looms, garments and other sectors.
Pest scenario has gradually changed over the decades due to the kind of genetic
material cultivated during those periods and the intensive cultivation practices of
later years. There has been a significant increase in the hybrid cotton area since
1970s, which has increased vulnerability to pests and diseases. Peculiarly, some of
the pests and diseases have a tendency to recur periodically by virtue of
adaptation/evolution of resistance and use of prone genetic material in newer
hybrids. Thus, in the early 50’s mainly the diploids (Gossypium arboreum and G.
herbaceum) were grown which were not prone to bollworms or sucking pests, but
had high incidence of grey mildew (GM) and Fusarium wilt.
As soon as cultivation of the tetraploid cotton (G. hirsutum and G. barbadense)
was introduced in the late sixties and early seventies in India, the pest scenario
changed. In the early eighties we faced a peculiar problem of a new type of wilt,
which is now considered to be a genetically controlled physiological disorder. After
the introduction of the tetraploids, there was a marked reduction of grey mildew,
since they were highly resistant to GM and Fusarium wilt.
Resistance to Fusarium wilt still continues but the hirsutum has become
susceptible to GM. A devastating epidemic of GM on some pvt hybrids in southern
and central India was noticed in 2001-02. Then, in the early nineties, there was
introduction of the infamous leaf curl virus, probably from across the border of
Pakistan in the North Zone. Soon a good number of hirsutum hybrids and varieties
resistant to the disease were developed. The disease remains contained for the
present, but hangs heavily as a potential threat. More recently, many fields of the Bt
transgenic cotton were found to have experienced heavy incidence of New wilt
(Parawilt), tropical rust and GM (Singh et al., 2004; Sharma et al., 2007).
Cotton is highly prone to diseases in the rainfed areas where opportunities for
growing alternative crops are limited. Thus, diseases are an important determinant of
the prosperity of the rainfed farmers (Puri et al., 1998; Singh et al., 2002). The pest
problem though cannot be eliminated altogether but it can be minimized through
application of appropriate IPM technologies (Puri et al., 2000).
The chemical based pest management has been losing its efficiency mainly due
to its adverse effect on nature and rising problem of resistance. The highly intensive
and remunerative irrigated north of India has witnessed deceleration in the
productivity, due to acute biotic stresses.
All parts of cotton, from root to tips, thick stem to tender shoots, vegetative to
reproductive parts are affected by one or another disease (Bambawale et al., 1998).
Cotton diseases of national and regional importance are as follows:
National
- Cotton Leaf Curl Virus (CLCV)
- Blackarm/Angular leaf spot (Xanthomonas campestris p.v. malvacearum)
- Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum)
- Root rots (Rhizoctonia spp.)
- Grey mildew (Ramularia areola)
Diseases of regional significance

- Verticillium wilt (Verticillium dahliae)
- Altemaria leaf spot (Alternaria macrospora)
- Rust (Phakopsora gossypii)
4.1. Seedling Diseases

Wet conditions predispose seed decay that often result in slow or no germination.
Fungi most often associated with seed deterioration in cotton are as follows:
Alternaria sp., Aspergillus sp., Colletotrichum gossypii, Chaetomium sp., Fusarium
sp., Pythium sp., and Rhizopus sp. The seed decay as well as seedling mortality due
to Fusarium spp. and Sclerotium rolfsii are occasional and occur in traces, which
can be managed by field sanitation and acid-delimitation. In case of non-conducive
microclimate, the pathogens remain latent and cause foliar diseases at vegetative
stage later (Alternaria alternata, A. macrospora, Cercospora gossypina,
Colletotrichum sp. Leveillula taurica, Myrothecium roridum, Phoma exigua and
Ramularia areola). The crop in fact is affected by a number of foliar diseases viz.,
Alternaria macrospora, Myrothecium roridum, Cercospora gossypina and
Colletotrichum gossypii, but the disease development remains restricted in early

stages, due to unfavourable microclimate provided by wide spacing between rows
and plants.
4.2. Bacterial Blight

The bacterial blight causal agent is a bacterium originally named as Pseudomonas
malvacearum by Smith in 1901, who later referred it as Bacterium malvacearum.
Dowson (1939) later classified the bacterium as Xanthomonas malvacearum (Smith)
Dow. However, the accepted name is at present Xanthomonas campestris pv.
malvacearum (Smith) Dye. The cotton plant is affected by the bacterial blight at all
stages of the crop development, starting from the seedling. The pathogen is seed-
borne and the disease is transmitted from the cotyledons to leaves, followed by the
main stem and bolls.
Symptoms at each stage has been given different descriptive nature, based on
the plant organ or the growth stage affected, viz. seedling blight, angular leaf spot,
vein blight, blackarm and boll lesions. Foliar symptoms are known as angular leaf
spot (ALS). Initially, the spots are water-soaked and more obvious on the dorsal
surface of the leaf. Another common leaf symptom occurs when lesions extend
along the sides of the main veins. This may be seen together with or in the absence
of ALS and is referred to as “vein blight”. In susceptible cultivars, infection spreads
from the leaf lamina down the petiole to the stem. The resulting sooty black lesions
give rise to the term “black-arm” by which the disease is commonly called. The
lesion may completely girdle the stem, causing it to break in high windy conditions
or under the weight of developing bolls. In India, where the crop is grown under
irrigation, losses of 5-20% are often experienced. Fourteen distinct races viz., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 15 and 18 have been found infecting cotton in different
areas of the country. Race 18 is the most virulent, overcoming five major blight
resistant genes viz. B2, B7, BIN, BN and B4 (Meshram, Raj & Dake, 1987).
Epidemiological studies indicate that the number of rainy days is positively
correlated with initial appearance of the disease. The number of rainy days is an
important factor governing other weather variables (Meshram & Raj, 1992). RH
above 85% at atmospheric temperatures of 30-35°C, favours blight maximum
disease development (Meshram & Raj, 1987). Evaluation of a number of
Phylloplane bacteria (Plb) isolated and tested in vitro against X. campestris pv.
malvacearus yielded six strains with antagonistic activity. Some of tested Plb were
able to reduce infection at 47% of untreated plants (Meshram et al., 2001). The
collection and quick destruction (burning) of infected crop residues help in reducing
the chances of disease initiation and of its build-up. Also, dense cropping should be
avoided. In order to contain disease, acid delinted seeds treated with carbendazim at
2 g ⋅ kg-1 should be preferred. Seeds may be soaked in 0.1% streptomycin solution
before sowing, to eradicate internally seed-borne pathogens. On the first appearance
of field symptoms, the crop should be sprayed with a mixture of copper oxychloride
(0.3%) + streptomycin sulphate (0.01%) preparations (Meshram et al., 1985; Sharma
et al., 2007), repeated at 10-15 days intervals to check for secondary spreads.
4.3. Alternaria Leaf Spot

Alternaria leaf spot is caused by the fungus Alternaria macrospora Zimm. The
disease is common in all the cotton growing areas of India. It appears in a severe
form in diploid cotton (G. herbaceum) in Karnataka, especially in “Arabhavi”,
which is considered as hot bed for this disease. The disease affected “Jayadhar”
variety and in its epidemic form caused not only the leaf spot, but also twig blight,
dry boll rot, and opening of badly affected bolls. The earliest disease symptom is the
appearance of spots on the cotyledons of seedlings.
Alternaria macrospora is well known to attack the seedlings in the Indian
climatic conditions. In favourable situations the spots can enlarge to 10 mm in
diameter, and several spots may coalesce together causing cotyledons to shed. On
green leaves, a pronounced purple coloured margin is visible all around the spot. On
older leaves, the necrotic tissues/spot is often marked by a pattern of concentric
structures. In humid weather conditions, the necrotic tissues turn to a sooty black
colour, due to intense fungus sporulation.
Severe infection of upper canopy leads to premature defoliation, a condition that
is very common among G. barbadense and certain cultivars of G. herbaceum,
widely grown in India. Although Alternaria alternata (Fr.) Keissler is the main
species involved, leaf spot is, however, most often attributed to A. macrospora
Zimm.
The optimum temperature for leaf spot disease development is 20-25°C.
Resurgence due to excessive synthetic pyrethroids was also observed (Spross-
Blickle et al., 1989). The pathogen is internally seed borne and infected seeds serves
as primary inoculum (Mukewar & Raj, 1994). Mukewar, Raj & Meshram, (1995)
reported successful management of the disease through seed treatment. Also in this
case dense cropping should be avoided, in order to reduce the disease incidence. The
seeds may be dressed with seedex/difolatan/indofil M-45 at 2-3 g ⋅ kg-1 of seeds.
Spraying with 0.2% mancozeb at 20 days interval from first symptom appearance is
effective in managing the disease and increasing yields.
4.4. Grey Mildew

The conidial stage of the causal agent is known as Ramularia areola (Atk.)
[synonyms: Ramularia gossypii Speg. Ciferi, Cercosporella gossypii Speg.]. The
fungus has an ascomycete sexual stage which is known as Mycosphaerella areola
Ehrlich and Wolf. The disease has been reported from almost all cotton growing areas
of the world and is known as “false mildew”. However, in India, it is commonly
known as “grey mildew”.
The disease appears first on the lower canopy of older leaves when the plant
attains maturity, usually after first boll-set, in the form of irregular angular, pale
translucent spots 1-10 mm in diameter, with a definite or irregular margin formed by
the veins of leaves (called ‘areolae’). The leaves dorsal surface shows profuse
sporulation (giving the lesions a white mildew-like appearance) causing light green to
yellow green coloration on the ventral (upper) leaf surface. In a due course these
becomes necrotic and dark brown in color. At this stage, they can be easily mistaken
for the angular leaf spot phase of the bacterial blight.
The fungus develops into three distinct stages during its life-cycle. The conidial
stage appears on living tissues, mainly on the underside of leaves while they are still
attached to plants for a short time after abscission. The spermogonial stage occurs later
on the fallen leaves. It is followed by an ascogenous stage which develops on partially
decayed leaves which, in turn, help the pathogen to survive in soil.
The optimum temperature for conidial germination is between 25-30°C (Raj,
Meshram & Chakrabarty, 1999). In severely affected plants, the leaves often defoliate
resulting in premature boll opening with immature lint (Lavekar et al, 2001; Sharma et
al., 2004). However, its late appearance during the crop season increases aeration and
helps in maturation of bolls. Changing scenario has resulted in occurrence of disease
and a new disease cycle often starts at an early vegetative stage causing unwanted
defoliation (Chidambaram & Kannan, 1989; Srinivasan, 1994; Sharma et al., 2005).
Grey mildew disease caused by Ramularia areola do appear in severe forms at
harvesting stage, but the boll dehiscence stage warranted no fungicidal (wettable
sulphur) application.
Analysis of various weather parameters indicates that frequent rains coupled with
high humidity favour disease development and spread. Closer spacing and high
fertility are conducive to disease development and spread. In order to manage the
disease, the crop residues should be removed and the fields must be deeply ploughed
to bury and destroy the remaining plant tissues/debris. The crop should be rotated with
cereals in disease endemic areas.
Combination of Trichoderma or Pseudomonas with chemical fungicides such as
tebuconazole, benzothiodiazole and prochloraz were tried and, except for
benzothiodiazole, combinations were highly effective in managing the disease
(Sharma et al., 2007). Foliar application of ziram (0.2%) or carbendazim 50 WP/
tridemorph 80 EC (0.1%) at 10 days interval from the day of first appearance, result
effective in controlling the disease.
4.5. Myrothecium Leaf Spot

Myrothecium leaf spot is caused by Myrothecium roridum Tode ex Fr., which attacks
young and woody stem tissues, causing the developemnt of stem lesions and dieback.
Earlier, it was known to occur mainly in northen India but during the ‘70s it was
observed in almost all cotton growing area of India. At times, it appears in a severe
form causing even plants defoliation.
The disease first appears on the young plant leaves only (4 to 6 week), but later it
may cause pre-emergence and post-emergence damping-off of seedlings. The leaf
spots are initially circular with tan coloured with violet-brown margins, which
coalesce and form large patches. The diseased spots are often surrounded by
translucent areas, which are concentrically zoned bearing black pinhead sized
sporodochia which later drops leaving a hole in the centre. In severe cases, the stem
may also break.
Myrothecium leaf spot does affect the bolls and boll lesions damage the lint by
making them brittle and discoloured It is an important disease of great significance
and only five pathotypes (MR-I, MR-II, MR-III, MR-IV and MR-V) have been
reported to occur in India. The primary source of inoculum is given by the infected
seeds and crop residues. Heavy pathogens loads delay the germination process and
also cause seedling mortality (Taneja & Raj, 1990).
4.6. Cercospora Leaf Spot

This disease is also known as anthracnose, and it occurs throughout the cotton growing
areas in India. It is often found associated with other diseases, with the appearance of
red dot marks on the leaves which expand in diameter to about 2 cm. It causes
irregular brown lesions, often surrounded by marked of chlorotic tissues.
The disease causes premature defoliation leading to decreased yields and
immature fibres. The causal organism is a hyphomycetes described as Cercospora
gossypina Cooke, characterized by a wide range of hosts. The ascomycete stage of this
fungus has been identified as Mycosphaerella gossypina.
The conditions favouring infection or sporulation and predisposing the crop to
infection are similar to those of the Alternaria leaf spot (Puri et al., 1998). Green bolls
are susceptible at all the physiological stages and show small reddish brown to dark
purple spots with depression in the center.
The pathogen survives on the infected seeds and crop residues. Crop rotation and
good agronomic practices involving field sanitation and debris burial may help in the
elimination of the primary inoculum. Seed treatments with carbendazim proved to be
an effective management practice (Raj, 2002).
4.7. Helminthosporium Leaf Spot

Apart from causing defoliation in adult plants, this fungus also causes seed rotting and
pre-emergence damping off. Numerous circular, light brown spots occur usually on the
dorsal surface of the leaves and bracts (Puri et al., 1998). Size of spots varies from 0.5
to 3.0 mm in diameter, and they later turn ashy in centre, with a dark purple ring
around. In severe cases, the central ashy coloured tissue falls apart, leaving holes.
Leaves with high disease intensity defoliate.
The disease is caused by Helminthosporium gossypii Tucker and H. spiciferum
(Bain.) Nicot. The fungi often survive during off season also as saprophytes on
diseased plant debris. Avoidance of dense cropping helps in reducing disease
incidence as well as intensity.
Foliar spray of thiabendazole or copper oxychloride at 0.2% controls the disease.
However, a number of other fungicides such as zineb, ziram and captan are effective in
controlling the secondary spread of this disease.
4.8. Macrophomina Leaf Spot and Stem Canker

The disease is caused by Macrophomina phaseolina (Tassi) Goid, which was earlier
known as M. phaseoli (Maubl.) Ashby with following synonyms, e.g., Sclerotium
bataticola (Taub) and Rhizoctonia bataticola (Taub) Butl. The fungus has been
reported to cause severe leaf spot and blight diseases, which start in two fashions. In
the first case, small pink coloured circular spot appears on originating point of
petiole of cotyledonary leaves which passes on to leaves, resulting in premature leaf
fall. The lesions extend upward and downward in a patch of 2-3 cm length,
encircling the stem. Subsequently, the bark shreds off and woody portions get
exposed. At times, the stem of seedlings at the junction of cotyledonary leaves are
affected, causing stem cankers and risk of breaking, due to high wind velocity.
Sometimes, a yellowing/browning of cotton leaves start from apical ends, finally
ending in a complete blight, covering the whole infected tissues with sclerotia. In
some cases, the stem breaking at the point of infection may also result in death of
grown-up plant.
Treating the seeds as well as soil with carbendazim significantly controls the
disease. Excessive wet conditions should be avoided. Foliar application of
carbendazim (0.1%) effectively controls the disease.
4.9. Late Season Phoma Blight

This blight caused by Phoma exigua Desm. affects all the above-ground parts with
symptoms of small, round, brownish spots (1-6 mm) and distinct purple margin on
leaves, which ultimately turns into rusty brown with whitish central portion in the
older lesions, bearing pycnidia on upper surface.
Under severe incidence, the spots coalesce to form irregular patches, followed by
drying and shedding of leaves. Similar symptoms can also be seen on petioles, bracts
and bolls. On stem, the lesions are dark brown, ranging from pin-head to 1cm, circular,
oval or elongated, often coalesced in linear rows along the length to form broad
necrotic tissue. The older lesions turn greyish white at the centre, bearing pycnidia of
the fungus. Blight does not cause shot-hole symptoms and appears late in the season.
Foliar application of carbendazim (0.1%) will help in reducing the disease.
4.10. Rust
The disease is characterised by red coloured pustules scattered over the whole green
surface of leaves and is caused by Phakopsora gossypii (Lagerh.) Hirats. Initially the
spots are purple with a red/brown center on the upper side of the leaf and brown,
powdery underneath. The incidence is more on older leaves than on the younger ones.
The uredia are formed in small, purplish brown spots, which coalesce to turn into large
patches. The disease appears during the dry season between December-March and
100-120 days after sowing and is prevalent in southern parts of India (Sharma et al.,
2007). It is of little significance and importance although it causes some loss, and
adopting chemical control measures will not result economical. However, calcium
spray (0.1%) provides good control of rust.
4.11. Leaf Crumple

Viral diseases of cotton are not common in India, but a cotton leaf crumple virus
disease was reported for the first time in India in 1977. Subsequently, another virus
disease, i.e., cotton leaf curl virus was reported in northern part of India in 1994. Both
diseases are transmitted by whiteflies (Bemisia tabaci).
The affected plants can be distinguished from healthy plants by abnormality in
leaves, mostly in the form of upward or downward curling of leaf margins. In some
cases, the affected plants remain stunted and give poor yields if the infection occurs in
early stages of growth. It has been reported from central India on Gossypium hirsutum
cv Acala glandless and on hybrid cotton H-4, as new record. However, the disease has
not caused thus far much damage to this crop.
The characteristic symptoms of the disease are hypertrophy of internal tissue,
shortening of veins in severely affected leaves, elevation in interveinal tissues,
downward curling of leaf margins. However, vein thickening or foliar out growths and
hypertrophy of petals are not reported. The disease is transmitted also through graft
transmission.
4.12. Cotton Leaf Curl Virus (CLCV)

This disease is caused by as Gemini Virus which has two components in its genome.
In nature, the disease is spread by whiteflies (Bemisia tabaci). The disease has been
reported affecting most of the G. hirsutum varieties grown in northern part of India.
The affected plants remain stunted and their leaves show distinct upward or downward
curling. The curling occurs due to the increase in veinal tissues on the abaxial side of
leaves. At a later stage, all the diseased leaves develop enations which become
prominent with time. Susceptible cultivars, presence of alternate/collateral hosts and
insect vectors are three prime factors that govern the outbreak and spread of leaf curl
disease. The disease progresses more at high temperature and intensity of infection
remains less on cotton under the shade. Disease development is maximum during
August and September (Singh et al., 1994).
4.13. Tobacco Streak Virus

A serious disease with putative virus etiology was observed to occur in the
transgenic cotton growing region of Southern Maharashtra. Symptoms comprised of
chlorotic and necrotic spots and leaf distortions. A generalised leaf and stem
necrosis extending to mid-veins, and petioles resulting in stunting, was observed in
early stage of crop development (Sharma et al., 2007).
The virus was identified as Tobacco Streak Virus (Ilar Virus) based on the
reaction to virus specific antibody in ELISA. This virus has also been recorded to
infect other hosts like sunflower, groundnut and soyabean and was found to be
transmitted by thrips. The virus is also reported to be transmitted by other arthropods
and insects of the order Thysanoptera.
4.14. Root Rot

The disease is caused by Rhizoctonia solani Kuhn. and R. bataticola (Taub) Butler
(pycnidial stage: Macrophomina phaseolina). The disease occurs as circular patches
affecting the plant at the seedling stage or after wood formation. A yellow patch
appears on the lower part which later blackens leading to drying of seedlings. Affected
plants can easily be pulled out of the ground due to the rotting of secondary roots. Tips
of roots are mostly discoloured, yellow and become sticky (Puri et al., 1998).
In severe cases black dot like sclerotia may be seen on the wood beneath the bark
and between the shredded bands of bark. The most common symptom is dry or wet
dark rot of the lower stem. On split opening, the affected plant can be easily
distinguished by discolored stele of main root and pith of stem. In severe cases, there is
dissolution of stem and root tissues. Tissue strands have been often found full of
minute sclerotia. The pathogen is morphologically characterized by brown pigmented
vegetative hyphae, branching at right angle to the hyphal cell and close to the distal
septum. The perfect state is a basidiomycetous, Thanatephorus cucumeris.
Variation in cultural, physiological or pathogenic characteristics among isolates
conforming to the morphological description of R. solani have been observed, and are
being used as a basis for isolates grouping. Anastomosis occurs between isolates of the
same group which, therefore, appear to be genetically related. Some pathogenic
specialization is also supposed to be associated with these groups. Anastomosis groups
(AGs) 1 and 4 have a wide host range but AG-2 has some specialization towards
crucifers, and isolates AG-3 come predominantly from potato. Monga and Raj (2000)
reported maximum disease incidence when the soil moisture ranged between 3.8 to
4% and soil temperature varied from 29.7 to 36.9 oC.
For effective management fields having long history of disease should be avoided
for planting cotton. The field should be deeply ploughed and left for solarization. After
harvesting, plant debris should either be completely buried or removed. Early sowing
and harvesting is recommended to avoid extreme temperatures. Sowing in April or
June instead of May helps in reducing disease incidence. Crop rotation is helpful in
minimizing disease incidence, through reduction of inoculum.
Soil amendment with FYM and de-oiled oil cakes helps in reduction of disease.
Soil application of ZnSO4 at 24 kg ⋅ ha-1 helps in disease management. Soil application
of consortium of Trichoderma viride, T. harzianum and Gliocladium virens has proved
to reduce disease incidence (Monga & Raj, 1996; Sudhakar, Reddy & Rao, 1997).
Seed dressing with bio-pesticide preparations of T. viride or G. virens or with
fungicides brassicol + captan and carbendazim slurry, may effectively reduce the
disease incidence. Green manuring with Sesbania acubeata + planting during second
week of July, and application of ammonium sulphate and intercropping with moth
(Vigna acontifolia) considerably reduces the disease incidence. Intercropping alone
with V. acontifolia also reduces the incidence quite significantly. Under severe
incidence, drenching of carbendazim (0.2%) is effective. Seed treatment with T. viride
or T. harzianum coupled with soil application of ZnSO4 at 24 kg ⋅ ha-1 reduces the
disease incidence.
4.15. Verticillium Wilt

This disease is caused by Verticillium spp., a genus of hyphomycetes of which
Verticillium dahliae Kleb. is a very important member. In India, the disease occurs
mainly in Tamil Nadu and produces wilt symptoms, surviving in soil (DeVay &
Pullman, 1984). Symptoms of the wilt first appear on relatively young plants before
the maximum temperature reaches 20 to 240C and then disappear in summer, to re-
appear when the temperature declines. Affected plants show yellowing and drooping
of young shoots, and ultimately defoliation. Plants affected during the fruiting stage
develop characteristically mosaic pattern on the affected leaves, which usually begin at
the base of plant and progresses towards the top. Leaf symptoms first appear as
yellowing of tissues along the margins and between the major veins (Puri et al., 1998).
When the intensity of infestation increases, these areas become more intensely yellow
and occasionally red, before turning white and necrotic, giving the appearance of tiger
stripes.
The disease occurs mostly in winter and may be seen affecting as circular patches
sporadically, and initially in low lying poorly drained areas. Over the years, the disease
spreads to the entire area either through irrigation water or through ploughing
operations (Kannan, 2002). Kannan and Srinivasan (1984) could isolate a most
virulent strain causing rapid defoliation, which characteristically produced abundant
microsclerotia with sparse conidia in cultures.
Fields with long history of disease occurrence should be avoided and should be
provided with good drainage systems. In absence of other options, soil treatment with
sodium dimethyl-dithio-carbamate or 1-2-dibromo-3-chloropropane at 10-15 lt ⋅ ha-1
may be followed. Seeds should be acid-delinted. Resistant varieties should be
preferred over others, and crops should be rotated with resistant varieties in order to
reduce soil borne inoculum. Rotation with puddled paddy, chrysanthemum and lucerne
(Medicago sativa) is recommended since it decreases the inoculum considerably.
Rotation with sesame, safflower, groundnut, cowpea, tomato, okra, beet, brinjal, chilli,
castor and sweet potato should be avoided. In case of potassium deficiency, its
replenishment decreases the extent of disease intensity. Seed should be treated with
bio-pesticide preparation of T. viride or T. harzianum or G. virens at 3 g ⋅ kg-1 of seeds.
Crop should not be over-irrigated. In case of severe wilt benzimidazole or benlate at
10-20 kg ⋅ ha-1 or sodium dimethyl dithio carbamate or 1-2-dibromo-3-chloropropane
10-15 lt ⋅ ha-1 should be applied in trenches.
4.16. Fusarium Wilt

Symptoms of this wilt, due to Fusarium oxysporum Schlecht f. sp. vasinfectum (Atk.)
Snyder and Hansen, may appear at any stage of crop development, depending on
inoculum density, temperature and host susceptibility. At high inoculum density or
during the very beginning of infection, plants may be killed even at the seedling stage.
Usually the first symptoms become apparent in the field between 30-60 days after
planting, quite often on the onset of flowering. The pathogen colonises plant roots and
penetrates into the vascular tissues in which it proliferates within the xylem vessels,
eventually spreading throughout the plant. In more advanced stages of infection the
fungus grows out of the vascular tissues and after the host death it sporulates on crop
residues. Fusarium oxysporum f. sp. vasinfectum has the ability to survive in soil for
long periods by producing sclerotized, thick walled resting bodies, which can resist
desiccation and lysis.
The disease can be recognised at the seedling stage by symptoms first appearing
on the cotyledons as the darkening of veins, followed by peripheral chlorosis. The
cotyledons become progressively more chlorotic and then necrotic before they shed. In
older plants the first external evidence of infection is yellowing at the margin of one or
more of the lower leaves. As the disease progresses within the plant, more leaves
develop chlorosis, which characteristically appears in patches between the main veins,
the rest of the leaf remaining green (Puri et al., 1998). Under the optimal conditions for
disease development, all leaves of affected plants succumb and shed before the stem
dries out.
The species F. oxysporum is variable, containing a large number of saprophytic
and pathogenic forms which have certain morphological features in common. Optimal
temperature for spore germination and growth through soil is 25°C, but maximum
sporulation occurs at 30°C. Spore production and germination are maximum at 100%
relative humidity (RH). No germination has been observed below 80% RH. Mycelial
growth in soil is maximum at 40% moisture holding capacity and pH 5.6 - 7.2.
Favourable soil temperature is between 22-30°C, the optimum being 24-28°C. Hot
and dry periods of long duration, followed by rains favour maximum disease
development (Sheo Raj et al., 1999).
Fields having long history of the disease should be avoided. Fields should be
deeply ploughed and left for solarization. Use of nitrogenous fertilizers, particularly
ammonium nitrate, should be discouraged and calcium ammonium nitrate should be
used instead, in place of urea or ammonium sulphate. Use of potassium fertilizers
should also be encouraged. Seed treatment with carbendazim at 2 g ⋅ kg-1 seeds may be
considered as preventive measures. Resistant varieties should also be cultivated.
4.17. New Wilt or Parawilt

Affected plants show drooping of leaves starting from the crown downwards
especially at flowering and boll development stages. The wilt, of unknown causal
agent, generally appears at flowering and boll development stages and plants show
drooping of leaves, which start from the crown downwards. The principal
morphological changes are widening of angle between the petiole and stem, epinasty,
defoliation and wilting of whole plant. Xylem vessels in the wilt affected plants were
of larger dimensions.
Scanning electron microscopy of tissue reveals formation of emboli in xylem
vessels. On the basis of symptomatology and behaviour, wilt could be categorised into
slow and quick type. In “slow-wilt”, the disease generally appears in 60-80 days old
plants. There is partial epinasty, drooping of lamina, reddening of leaf surface, petiole,
stem and branches without chlorosis or necrosis and without loss of turgor. Leaves
shed and some plants show partial recovery, whereas others may die. There is neither
vascular discoloration nor rotting of roots.
In ‘quick wilt’, there is sudden drooping of leaves and tender shoots about 45 days
after germination, and the plants wilt. There is no development of red pigments or
shedding of dried leaves and the wilted plants rarely die. New shoots start develop
from the lower nodes but remain unproductive. The causal agent is not yet fully
ascertained, however, the formation of embolii in the xylem vessels has been reported
which creates hindrance in translocation of nutrients and water (Mayee & Mukewar,
2001; Mayee, Rao & Yadav, 2001). Prolonged drought conditions followed by
downpour rain may favour the disease (Raj et al., 1991).
Drought like conditions should be avoided, and under such conditions,
irrigation may help in reducing the disease incidence. Spraying of 1% KNO3 is
effective. Soil drenching with 0.5% nitrogen + 0.5% phosphorous within 12 hrs of
initiation of the wilt, results in quick recovery of the affected plants.
4.18. Boll Rots and Lint Diseases

The disease occurs in areas of high humidity or places having dense crop or with
high vegetative growth rates. The insects responsible for infection are mainly
Dysdercus nigrofasciatus Stål, 1855 and Pectinophora gossypiella Saunders, 1844.
Microbial decay of boll takes place before dehiscence and the infection contaminates
the lint. Numerous micro-organisms (over one hundred) have been isolated from rotted
bolls. Most of these are wound pathogens causing boll rot after physical damage or
premature rupture of boll suture, while others are secondary invaders/contaminants.
Majority of fungi cause boll rot following wound inoculation. However pathogens
such as Diplodia gossypina, Glomerella gossypii, Myrothecium roridum,
Xanthomonas campestris f. sp. malvacearum and Bacillus subtilis cause decay after
contact inoculation (Puri et al., 1998).
Fungal pathogens primarily responsible for boll rot are:
Alternaria alternata Helminthosporium sp.

A. macrospora Fusarium moniliforme
Ascochyta gossypii Fusarium compactum
Colletotrichum capsici Nematospora nagpuri
C. gloeosporioides Phytophthora boehmeriae
C. gossypii Rhizoctonia solani
Curvularia lunata R. bataticola
Diplodia gossypina Myrothecium roridum
Prolonged periods of high humidity sets on the boll rot epidemics. There are
four general conditions which govern boll rot in fields;
(1) long periods with free moisture on plants

(2) long periods when RH exceeds 75%
(3) low light intensity
(4) high temperature.
Boll infection/rot can be kept under check by mechanical collection and
destruction of infested bolls, burning them or by spraying antifungal/bacterial chemicals.
5. INTEGRATED DISEASE MANAGEMENT IN RICE
In India, rice is cultivated in 42 million hectares under four major ecosystems viz.,
irrigated, rainfed lowland and upland, and flood prone ecosystems. In the history of
agriculture, the brown spot disease devastated rice crop during 1942-43 and resulted in
famine also known as “Bengal Famine” (Padmanabhan, 1973). Traditionally, farmers
practiced essentially subsistence agriculture till 1942. However, due to the population
growth and the eventual need for additional food, farmers started using petro-based
chemicals, fertilizers and pesticides, to protect and increase production. Decades later,
the green revolution ushered in an era of increased production and self-sufficiency in
cereals. The fertilizers and pesticides were used indiscriminately (Singh et al., 2005).
Soon, disease problems became severe as a result of a more complex agriculture,
involving changes in production practices and introduction of pathogens into new
areas. Many diseases caused by biotic agents like fungi, bacteria, viruses and
nematodes appeared in rice farms and reduced yields considerably. A few of them
cause concern as epidemic (blast and bacterial leaf blight) and occasional outbreaks
(sheath blight, tungro virus or brown planthopper), or appear rarely (false smut and
rots).
Modern agriculture can overcome many of these problems, but its increased
complexity and intensity demand unprecedented precision in the management of crop
diseases (Nagarajan & Muralidharan, 1995; Muralidharan et al., 1997). The abiotic
agents like drought or flood induced by the vagaries of monsoons, and the nutritional
deficiency or toxicity also resulted in the emergence of additional disease problems.
The development of pathogen resistant to chemicals and crop losses has raised
questions about the efficacy of pest management practices. Effectively limiting losses
from plant diseases requires that these should be managed in processes that are
sustainable and eco-friendly.
Diseases of normal and scented rice cultivars are similar, but their relative
importance varies in different agroecological zones (Singh et al., 2003). Many
external and internal factors influence rice crop health and growth. These include both
biotic and abiotic factors that are widely prevalent in rice ecosystems. Pathogens -
fungal, bacterial and viral - are the main biotic factors affects productivity. The main
abiotic factors that affect rice include nutrient deficiency from major and minor
elements, ambient temperature regimes (high or low), and rainfall - insufficiency
leading to drought, or excess leading to flood. Nutrition-induced physiological
disorders have been reported as “Khaira” a zinc-deficiency disease which is dominant

and widely prevalent (Nene & Gairola, 1965).
Economic threshold level (Muralidharan, Shind & Siddiq, 1990) has been made
only on theoretical presumptions and difficult to adopt. The climatic conditions,
cultural practices and the host resistance or susceptibility can totally alter the estimated
threshold values. For the poly-cyclic pathogens, generally the onset of primary
infection, the time taken to complete one generation, duration to crop maturity and
reproductive ability are considered. With monocyclic pathogens, information on the
degree of primary infection and the rapidity of spread within and between plants are
considered in addition to the crop maturity duration.
5.1. Blast
The disease, caused by Pyricularia grisea Sacc. (telomorph Magnaporthe grisea
(Hebert) Brarr) occurs at all stages of crop growth in rainfed, irrigated and hill rice,
and severe incidence results in heavy or total loss in yield. The sexual phase of the
blast pathogen has not been detected in nature. Infection by P. grisea leads to
formation of spindle-shaped lesions with brownish margins and grayish center. The
spots usually begin as small water-soaked, whitish, greyish or bluish dots. Fully
developed lesions in 2-3 days reach 1.5 cm in length, and 0.5 cm in breadth (Rao,
1994).
Adjacent lesions often coalesce under favorable conditions turning a major part or
the entire leaf to produce a burnt appearance. Brown or black patches may appear
around nodes and the nodes so infected often break apart. At a later stage, the fungus
attacks the base of panicle at neck region and this infection is known as panicle blast
or neck blast or spikelet blast. Neck infection causes the panicles to break and fall
over, resulting in the loss of grains (Muralidharan &Venkatarao, 1993). Small brown
to black spots formed by pathogen can also be seen on the glumes.
The factors that influence blast epidemics are the susceptible variety, availability
of inoculum to initiate the disease, excessive application of nitrogen fertilizer, low
night temperature (24oC), high humidity and drizzle weather. Cloudy weather
encourages blast spread; leaf wetness has a direct effect, and the longer the wet
period, the greater is the infection.
Heavy doses of nitrogenous fertilizers and soils deficient in silica content and
zinc deficiency influence the blast incidence. When conditions are conducive, the
pathogen multiplies rapidly to produce abundant conidia from lesions. The disease
moves quickly from field to field by producing myriad number of spores that are
disseminated by wind in all directions. These spores upon falling on rice plant, initiate
further disease to progress rapidly through the entire field. The repeated cycles of
spore production and infection continues throughout the crop growth. Under
favourable conditions, the green lush crop growth is turned into burn up appearance
(Muralidharan & Venkatarao, 1987).
Rao (1971) described the occurrence of conidial shapes of various isolates
available in India agroecological regions. Rice blast outbreak rules have been
developed (Muralidharan & Venkatarao 1980a; 1987) with which early and accurate
warnings can be issued to farmers.
For leaf blast outbreaks:
1) moderate to high incidence of leaf blast must be observed in trap nurseries

sown at weekly intervals with any locally susceptible cultivars under high
plant population density and fertilizer, and
2) continued prevalence of low night temperatures (< 20°C) coupled with
high relative humidity (> 90%) for 7 to 10 days, or 3) cloudiness, dewfall
or light drizzle, should be present.
For neck blast outbreaks:
1) at least a low to moderate leaf blast incidence during tillering stage, or in

susceptible trap plants, must be observed and
2) low night temperatures (< 20°C) during panicle initiation to flowering
stage, or
3) cloudiness, dewfall or light drizzle during the reproductive phase should be
present.
If one rule is not fully satisfied, more conducive changes in any other rules may
compensate and lead to outbreaks of leaf or neck blast. Example, intense dewfall,
extended dew period, or frequent drizzle may compensate for high temperature
(>20°C) regimes up to 26°C or more (Pasalu et al., 2006).
Cultural practices exert a deep influence on blast development. Generally, using
over aged seedlings and delaying in planting of rice seedlings will increase the severity
of the disease (Venkatarao & Muralidharan, 1982b). Dense planting is commonly
advocated for obtaining maximum yields. Leaf and neck blast infections were found to
increase significantly with the increase in the density of plants (Venkatarao &
Muralidharan, 1982b). Over wintering conidia found on grass hosts, namely
Panicum repens, Brachiaria mutica, Digitaria sanquinals and Leersia hexandra
(Veeraraghavan & Padmanabhan, 1965) were observed as source of primary
inoculum. Nitrogen fertilizers have a remarkable effect on the susceptibility of rice
(Amin & Venkatarao, 1979).
Nitrogen supply induces a heavy incidence of blast regardless of application of
other fertilizers (Amin & Venkatarao, 1979). Chakrabarti (1992) reported that the
disease becomes serious by poor fertility of soil caused by low nitrogen, phosphorous
and potassium levels. Due to lodging problems in scented rice, usually a low dose of
nitrogen is applied without actual information of soil nitrates, which often results in
high incidence. Pyricularia grisea can be managed by seed treatment, followed by
foliar spray of mancozeb, carboxin, bitertanol etc. Application of Si also reduces
disease intensity (Datnoff et al., 1989). Microbial biocontrol agents such as Bacillus
subtilis in the form of seed treatments or soil application and foliar spray have been
considered effective (Nanda & Gangopadhyay, 1983).
Blast continues to be a threat limiting yield potentials of cultivars in all rice

ecosystems, especially in scented rice growing areas of northern India. Studies
indicated that resistance to blast disease in rice cultivars was governed by monogenic
dominant or recessive, or by digenic/polygenic genes (Padmanabhan, 1975). Thirteen
dominant resistance genes were identified at eight loci. By analyzing 132 isolates,
Padmanabhan et al. (1970) identified 31 races. Tadukan plants carrying resistance
gene Pi-ta showed small lesions infecting < 2% of the leaf area, indicating a very high
level of durable resistance to blast disease. They clearly demonstrated the expression
of a high degree of resistance in A57 carrying three resistance genes (Pi-1, Pi-2 and Pi-
4). The performance of BL 245 with two resistance genes (Pi-2 and Pi-4) and
C101LAC (Pi-1) was comparable to A57.
At present, there are over 250 rice cultivars resistant to P. grisea. Nitrogen
management with need-based application of fungicides may be adopted to control
blast on a susceptible variety. It is advisable not to top dress with nitrogen fertilizer
until fungicide is applied, if conditions are conducive for disease development.
Neem coated urea (at 60 and 90 kg N ⋅ ha-1) was effective in reducing blast
disease and increasing yields. Fungicides like ediphenphos 50 EC (1 ml ⋅ l-1) or
tricyclazole 75 WP (0.6 g ⋅ l-1) or carbendazim 50 WP (1 g ⋅ l-1), may be used to check
further development of the disease.
5.2. Brown spot

In thw mid ‘40s, serious damage from brown spot, caused by Drechslera oryzae
Subramanian and Jain (syn. Helminthosporium oryzae Bred De Haan), was considered
as nutrition deficiency. However, with the introduction of dwarf high-yielding rice
cultivars, fertilizers and irrigation, it became a minor disease. The characteristic
symptom occurs on the leaf as circular to oval spots, that are dark brown to purplish
brown. The spots may also be distinct, isolated and scattered throughout the leaf
surface. They frequently coalesce together, causing withering and drying of entire leaf.
In wet weather, they may also appear on the coleoptile, leaf sheaths, panicle branches
and glumes. Black or dark-brown spots appear on the glumes, and the entire surface of
glumes may be covered with spots.
Weeds like Panicum repens (L.), Echinochloa spp., and Brachiaria mutica
(Stapf.), acts as collateral hosts. Brown spot disease is often associated with soils
deficient in nutritional elements such as silica, potassium, manganese or magnesium.
Brown spot infection is prominent when nitrogen deficiency is induced at late
growth stage. Cloudiness, humid weather and poor growth of plants further favour
the disease development. The disease is seed-borne. Fungicide seed treatment (with
copper oxychloride) is useful in reducing the disease on seedlings. If brown spot
symptoms are observed in the later growth stages of the crop, iprodione 50 WP (2 g ⋅
l-1) or mancozeb 75 WP (2.5 g ⋅ l-1) can be used to control the disease. Other methods
suggested and practiced include field sanitation, crop rotation, proper fertilization,
good water management and use of soil amendments. Stubbles help to carry the
pathogen and enable its survival during the absence of rice crop.
Adoption of green mulching (Sesbania-Black gram) helps in addition of organic

carbon and nitrogen in soil. Enriching farm yard manure (FYM) may be obtained
with Trichoderma at 3-4 kg per 200 kg and leaving for 48 hrs before spraying in the
field (Sharma et al., 2006). Proper management of plant nutrition by using silicon
fertilizers (e.g., calcium silicate slag) in poor soil conditions considerably reduces
disease intensity. A number of cultivar resistant to brown spot disease are also
available.
5.3. Bacterial Leaf Blight

Symptoms of bacterial leaf blight disease appear at the leaves tip or edges as yellow
water soaked and undulating lesions, parallel to the veins, that later turn to straw or
yellow colour (“Kresek”). The disease, due to the bacterium Xanthomonas oryzae pv.
oryzae (Ishiyama) Swigs et al., initially starts from either one or both sides of leaf
margin. As the disease progresses, the drying spreads downwards and inwards of leaf
blade, causing leaf drying and death. Often amber coloured bead-like bacterial
exudates are present on lesions. In systemic infection, seedlings wilt and die. Grains
are either partially filled or become chaffy. The rod-shaped bacterium is Gram-
negative, not sporigenous, motile with a polar flagellum, and measures 1.75 × 0.60 μm
in size.
Bacterial cells are surrounded by mucus capsules and are joined to form
aggregated and stable masses. After the introduction of dwarf, high-yielding TN1 rice,
which was known for susceptibility to bacterial blight, the disease assumed greater
importance in India. Its incidence and severity are very much influenced by rainfall,
winds, rainy days, susceptibility of the cultivar and nitrogen fertilizer application. The
yield losses have been reported to vary from 2-74% in such epidemics (Rao &
Kauffman, 1977; Muralidharan & Venkatarao, 1979). Severe epidemics of bacterial
leaf blight in two consecutive wet seasons (1979 and 1980) in northwestern India,
reduced grain yields drastically. Every year, bacterial leaf blight causes damage to rice
crop, in several districts in India.
The disease occurs in two phases. The wilt phase usually occurs during the active
tillering stage and results in the death of tillers. Seedlings are killed by Kresek if
inoculum pressure is very high (Srivastava, 1972; Rangareddy, 1987). The leaf blight
phase is common in all the rice growing areas and it appears from maximum tillering
to heading stages. Sometimes, farmers mistakenly consider the disease as a crop
drying due to unfavorable conditions, and they expect the crop to recover. But under
favorable conditions, the disease spreads quickly to devastate the entire crop. In
diseased plants, panicle emergence is poor and spikelets are partially filled and
discolored. Bacterial leaf blight causes more damage in rice fields where high doses of
nitrogen fertilizers are used.
Epidemics of bacterial leaf blight occur frequently all along the Indian coast
exposed to cyclonic storms and intense monsoon rains. Mechanical injuries to rice
leaves caused by rain storms, strong winds, sap sucking insects and intercultural
operations offer entry points for the pathogen. Once inside, the bacteria multiply
quickly to millions of cells and cause blight. Affected leaf lesions ooze out exudates
containing numerous bacterial cells (Datta et al., 1970).
The studies on pathogenic variation from India deal with the leaf blight phase
only and Gupta, Sharma and Saini (1986) reported the presence of 11 virulence
genotypes in X. campestris pv. oryzae indian populations. DNA finger printing of 67
isolates of X. oryzae pv. oryzae collected during 1994 and 1995 from 18 locations in
India belonged to a single lineage representing pathoype Ib. The resistance to bacterial
leaf blight disease in some cultivars was considered to be due to a combination of two
or more genes or to new genes that were often described as dominant, recessive,
inhibitory, complementary or polygenic.
Heavy rains coupled with high temperature, presence of deep irrigation water
and severe winds favour disease, but severe summer and drought suppress the
disease. Soil moisture at and above saturation favours development of the kresek
phase of the disease, and symptoms appears within 10 days at about 30ºC.
High rate of nitrogenous fertilizer increases disease development. Rain splashes
and wind aid in the bacterium dissemination. Field to field irrigation also aids in the
pathogen spread. Field sanitation aiming at removing weed hosts, rice straws,
ratoons, and volunteer seedlings helps in the reduction of the field inoculum. Seed
treatments with “bleaching powder (100 µg ⋅ ml-1)” and “zinc sulfate (2%)” reduce
disease incidence. Dipping the seedlings with suspensions of Pseudomonas
fluorescens based products at 2% before transplanting and proper plant spacings
help in management of the disease. Field crop needs to be constantly monitored for
initial symptoms and sprayed with P. fluorescens.
There are no effective chemicals to protect the crop from bacterial leaf blight
disease. Streptocycline and cow dung applications have been reported in production
oriented surveys to contain the disease spread. Extensive studies, however, have
shown their ineffectiveness in controlling bacterial leaf blight. The options for control
of bacterial blight include use of resistant cultivars and judicious nitrogen
management. If favorable weather persists and disease is already incident on a crop, it
is advisable to withdraw application of nitrogen fertilizer. A number of resistant
varieties are also available commercially .
5.4. Bacterial Leaf Streak

Symptoms of the disease, caused by the bacterium Xanthomonas oryzae pv. oryzicola
Fang et al. (Dye.) appear on leaf as linear water soaked, translucent streaks of 1 to 4.5
cm in length. The streaks are dark green in colour at first and later turn yellowish-
orange brown. The progress of the leaf streak is longitudinal. Several such steaks
however, may coalesce causing total blight of leaves. Yellowish droplets of bacterial
exudates are often visible on the surface of leaf streaks. Generally, symptoms are more
pronounced in indica cultivars. Infection of spikelets results in death of stamen and
browning of glumes. The exudates (with bacterial cells) on infected leaves serve as
inoculum for rapid secondary spreads of the pathogen.
The rod-shaped bacterium is a Gram-negative, motile by a polar flagellum, not

sporigenous and measures 1.2 × 0.3-0.5 μm in size. Usually the bacterium is
capsulated (Devadath & Dath, 1970). The pathogen gains entry either through injured
epidermal cells or directly through the stomata, colonizing the sub-stomatal cavity and
ebentually invading intercellular spaces of the mesophill. The bacterial invasion is
confined to the interveinal parenchyma, the bundle sheath parenchyma the and
sclerenchyma extensions, connecting the vascular bundles to lower and upper
epidermis. Hence discrete streaks limited by veins are formed.
The disease is transmitted through seed (Srivastava, 1969). The pathogen is
carried from field to field by irrigation. It is also known to survive in infected straw.
Humid conditions, intermittent rainfall and leaf or plant injuries from storms or
cyclones favor disease incidence. However, the bacterial leaf streak occurs
sporadically and it is often restricted to only a few fields. Occasionally, damage from
bacterial leaf streak may be encountered on seedlings in nurseries. Streptocycline
spray application may check the disease from any further damage to seedlings. Nayak
et al. (1975) found three pairs of independent resistant genes governing resistance.
5.5. Sheath Blight

Sheath blight caused by the fungus Thanetophorus cucumeris Frank Donk. (anamorph
Rhizoctonia solani Kuhn) is widely prevalent, particularly in irrigated rice ecosystems,
but it seldom assumes epidemic proportions. The asexual phase of the pathogen
actually incites the disease in plants, just above the water line in fields, to cause
damage to yields. Although sheath blight occurs, its impact on rice yields is
demonstrated to be relatively less in India, when confronted to blast. Losses from
sheath blight in irrigated ecosystems may be around one tonne per hectare
(Muralidharan et al., 2003).
Rhizoctonia solani as a pathogen causes lesions and necrosis in plant tissues. The
profuse saprophytic external growth visually appears to be more threatening due to
thick mycelia and numerous sclerotia. The pathogen affects all plant parts, viz.
sheaths, internodes, upper leaves and panicles. On sheaths, infection leads to
appearance of spots that are grayish in colour and ellipsoid or ovoid in shape. The leaf
of the affected sheath dries up. In humid weather, white threads of fungal body can be
seen all over the surface of leaf sheaths. The infected leaves and internodes turn grey
to straw colour with lateral brown bands. Initially white mycelial knots appear on the
infected portions which later turn to brown and dark brown sclerotia loosely adhering
to plants. Detached sclerotia are carried by water streams, and upon coming in contact
with plants, initiate the disease (Rao, 1995).
The majority of isolates belongs to Ag-1 IA group. AG-1 IA and AG-1 IB are the
only isolates capable of producing typical sheath blight symptoms on rice. The
infection then ascends to the upper part of the foliage and to flag leaves. Plants are
more susceptible at booting and flowering stage than at tillering and panicle stage
(Roy, 1982). Kannaiyan and Prasad (1978) has established a correlation between the
development and yield, paving the way for developing a mathematical model for
yield loss assessment. Aging plants are more susceptible, due to loosening of leaf
sheath from the culm. Early planting and dense plant populations encourage disease
development.
Application of nitrogenous fertilizers at high doses increases the severity of sheath
blight disease. The progress of the disease is governed by ambient humidity and
temperature. Sheath blight epidemics occur in highly humid conditions with an
average daily temperature of 30°C. Under favorable conditions, the disease spreads to
top portions of the plant. Rhizoctonia solani also invades spikelets, causing sterility or
improper grain filling (Rao et al., 2000).
The pathogen is ubiquitous and has a wide host range affecting all grasses and
broad-leaved weeds that grow on rice bunds. Since sclerotia are lighter in weight, they
float on water with water current and accumulate at the fields periphery. Similar
symptoms and sclerotia are produced on all hosts. Even if leaves of rice plants come in
contact with infected weeds on bunds, they pick-up infection and spread the disease.
Hence, keeping bunds clean of weeds will help in checking the disease spread from
primary sources. Disease spreads very slowly and environmental changes from humid
to dry weather will stop its progress.
Removal of infected stubbles or crop residues from the field is recommended to
reduce the amount of inoculum for the succeeding cropping season. Seed treatment
with Pseudomonas aureofaciens reduced the disease incidence and increased yields.
Spraying of crops with useful bacteria or antagonists was successfully demonstrated
by a number of workers (Vasanthadevi et al., 1989; Vidyasekaran et al., 1995).
Seeding rate or plant spacing should be optimized to avoid closer plant spacing or
dense crop growth which favors the build up and spread of the disease. Higher N
content of soil seems to increase disease severity (Kozaka, 1965; Roy, 1978),
wherease high K+ level disfavours the disease development (Basu & Sengupta,
1992).
Sclerotia have very poor competitive saprophytic activity in presence of local
soil fungi (Roy, 1985). Mycoparasitism of the fungus by Trichoderma spp. has been
reported as early as 1980s (Roy & Sayre, 1984). Dath (1982) reported inhibitory
effect of green manuring on viability of sclerotia. Sclerotial population was
drastically reduced in the soil amended with green manures viz. Dhaincha (Sesbania
spp.) and sunhemp (Crotolaria juncea). Enriching FYM with T. harzianum at 3-4 kg
per 200 kg and leaving for 48 hrs before spraying in the field was helpful (Sharma et
al., 2006). Farmers should look for initial symptoms on weeds growing on bunds
and spray to contain them. Spraying with suspensions of Pseudomonas fluorescens
cells at the initial stage of the disease also may be applied. If the humid weather is
likely to persist for a prolonged time and disease is noticed all along the peripheral
areas in the field, a foliar spray application of either validamycin 3L (2.5 ml ⋅ l -1) or
hexaconazole 5 EC (2ml ⋅ l -1) will prevent its spread.
In addition to keeping bunds clean, soil solarization helps to reduce sheath blight
incidence. Use of antagonistic bacteria was also suggested for the control on sheath
blight disease (Krishnamurthy & Gnanamanickam, 1997). Three selected bacteria viz.,
fluoroscent Pseudomonas sp. (PF-9), Bacillus sp. (B-44) and chitinolytic bacterium
(CH-1) were tested rigorously (Laha & Venkataraman, 2001) and were found effective
in reducing disease severity, either alone or in combination with one foliar application
of carbendazim. Transgenic IR 64 rice plants over-expressing rice chitinase, a

pathogen-related protein, have also been generated (Datta et al., 2002; Datta, 2004).
5.6. Sheath Rot

Sheath rot caused by Sarocladium oryzae (Sawada) Gams and Hawksworth, was first
reported to occur in India in 1978 (Agnihothrudu, 1973). Wounds and entry points
created by leaf sap sucking pests facilitate early infection. Lesions begin to grow as an
oblong or irregular brown spots with margins. The spots enlarge to coalesce affecting
entire sheath. Severe outbreaks may cause considerable yield losses (Muralidharan &
Venkatarao, 1980b; Raina & Singh, 1980).
The pathogen produces dark-brown to chocolate-brown spots on upper sheaths,
resulting in either partial or incomplete emergence of the panicle along with seed
sterility. The disease usually appears during heading to maturity stages. A whitish to
light-pink powdery growth of the fungus may be found on the panicle, inside the
sheath, and young panicles may rot completely. Grains inside the choked panicles and
on the partially emerged panicles may be chaffy, light to dark-brown and covered by a
white to light-pink mat of mycelium and spore mass. If the pathogen attacks after the
panicle emergence, the grains may be partially or completely filled and a glume
discoloration is caused (Rao et al., 2000).
Availability of moisture in the form of rain or dew at flowering encourages an
outbreak of sheath rot disease. Singh and Raju (1981) reported that the causal agent
survives up to 4 months in seeds and 7 months in leaf sheaths, stored at room
temperature. Deka and Pookan (1992) carried out pathogenicity tests and found the
fungus is pathogenic to several weeds (Echinochloa colona, Monochoria vaginalis,
Hymenachne assamica, Leersia hexandra, Panicum walense, Oryza rufipogen and
Eleusine indica).
Narasimhan et al. (1994) reported use of gypsum at 500 kg ⋅ ha-1 as a top
dressing as capable of reducing the disease quite significantly. In the endemic areas
application of carbendazim (0.1%), twice at fortnightly intervals from boot leaf stage,
is effective in preventing damage. Thiophanate-methyl, ediphenphos (0.1%),
hexaconazole, propiconazole and mancozeb are also useful in controlling the sheath
rot infection. Many cultivars possessing resistance or tolerance to sheath rot are
available.
5.7. Fusarium Wilt or “Bakanae”

Foot rot or “bakanae” disease occurs in almost all the rice growing areas in India and
is caused by Fusarium moniliforme Sheld (teleomorph Gibberalla fujikuroi (Sawada)
Wr.). At places, yield losses may vary from 3 to 95%, due to foot rot disease.
Diseased seedlings in nursery are lanky, pale yellow and taller than the healthy
and uninfected plants. Such seedlings die either before or after transplanting. Diseased
plants in fields show brown discoloration of tissues at lower nodes that facilitate an
easy separation of the root system at collar region, resulting in foot rot symptoms.
Infected plants have fewer tillers and their leaves dry, one after the other. They wilt
and die within a few weeks. Presence of white or pink mycelial growth, adventitious
roots from the lower internodes, and wider leaf angles with stem are diagnostic
features of the disease. The fungus also causes necrotic lesions on the leaves, stem,
panicle or kernel leading to withering of growing shoots.
The disease is both seed-borne and soil-borne. The panicle infection is caused by
secondary air-borne conidia and ascospores discharged from diseased plants, from
heading til harvest. The fungus grows intercellularly in stigma and anthers and finally
reaches to cover the ovary. The fungus remains viable for 16-28 months in seeds and
infected plants (Sunder & Satyavir, 1997). Diseased debris also serve as a primary
source of inoculum as the fungus can survive in plant debris for 10-28 months. Lower
temperature (5-10°C) and humidity conditions (RH 30-35%) favor the survival of the
pathogen in infected grains and stubbles. Cultural practices that aid in disease
management include selection of healthy seed, late sowing of early maturing cultivars
and crop rotation. Foliar application of benzimidazoles like carbendazim 50 WP or
benomyl 50 WP (1 g ⋅ l-1) can control foot rot disease in fields. Seed treatment with
carbendazim or benomyl (2 g ⋅ kg-1 seed), checks the seed-borne infection.
5.8. Stem Rot

This disease is common in Haryana (Ahuja & Shrivastava, 1990) and a few other
states growing scented rice. It is caused by Sclerotium oryzae Catt. [anamorph
Helminthosporium sigmoideum Cav. and telomorph Magnaporthe salvinii (Catt.)
Krause and Webster]. Stem rot infection starts with sclerotia in the soil or plant debris.
Severe disease incidence causes a premature lodging in crop. The sclerotia are
disseminated by irrigation. The first symptoms appear as small, black, irregular lesions
on the outer leaf sheath near the water line.
The fungus penetrates mostly through wounds into the inner leaf sheath and
finally rots the basal portion of the stem. Numerous black, round and shining bodies
(sclerotia) are formed externally and internally on the affected sheaths and stems. The
plants lodge and put-forth a number of green secondary tillers but the earheads
produced are chaffy. Enriching FYM with Trichoderma helps in reduction of
disease. Microbial aided decomposition of stubble/crop residue (after harvest) with
bio-compost consortium of anaerobic fungi helps in reducing the inoculum load.
Draining excess water from the field can also reduce the density of sclerotia in the
field. A balanced use of potash and lime may locally increase soil pH, reducing
infections to increase yields (Sharma et al., 2006).
5.9. Tungro Virus

Tungro virus disease occurs if a susceptible crop, a viral inoculum and green
leafhoppers (Nephotettix virescens Distant.) acting as virus vectors, are available in a
rice field. The disease attracted the public attention for the first time following an
epidemic outbreak in the Eastern India (Raychaudhuri, Mishra & Ghosh, 1967).
Severe tungro virus disease outbreaks threatened rice productions in Southern India.
Currently, the disease is under check at the national level. Stunting of infected plants
and turning leaf colour from green to yellow and then orange-red, characterize tungro
disease incidence.
Newly emerging leaves of infected plants are often pale with chlorotic intervenial
areas. The leaf lamina is often twisted following the virus attack. While orange-yellow
colouration of the foliage is characteristic, variations often exist ranging from green to
pale, or intensely orange, red and sometimes with brown spots. If the plants are
infected in early growth stages, there is no flowering. If plants are infected late, there is
a delayed and uneven flowering. Tungro virus reduces the number of spikelets in
panicles and hence yield. It also decreases filling, weight and starch content in grains
(Chowdhury & Mukhopadhyay, 1975). Two viral particles namely spherical (RTSV –
an RNA virus) and bacilliform (RTBV- a DNA para retrovirus) were considered to be
associated with the tungro disease (Saito et al., 1976; Hibino, Roechan & Sudarsman,
1978). Recent studies, however, raised the question about the involvement of the
bacilliform virus as a pathogen in tungro disease.
Viruliferous green leafhoppers Nephotettix virescens Distant, N. nigropictus Stahl.
and Recilia dorsalis Motsch., introduce the virus into rice leaves when they probe to
suck nutrients. Thus, a tungro-infected plant suffers from damage caused by both the
virus and its insect vector. The disease can occur at any stage from nursery onwards.
The initial disease on the rice crop is seen along the weedy border of rice fields and
later spreads into the main field. Tungro is found only in irrigated and rainfed lowland
rice ecosystems. Applying any chemical cannot directly control tungro virus disease.
However, the spread of tungro disease can be checked indirectly by controlling the
vector with a pesticide application. A low-dose application of imidachloprid 200 SL
(100 ml ⋅ ha-1) in nurseries after a reported outbreak in the earlier crop can effectively
control tungro from affecting new crops. Practicing a fallow or introducing a pulse or
oilseed crop can also break the continuous availability of virus inoculum in fields.
5.10. False Smut

False smut of rice, caused by Ustilaginoidea virens (Cooke) Takahashi [telomorph
Claviceps oryzae-sativae (Hashioka)] is a well-known minor disease first described
and named by Cooke in 1878 from Tamil Nadu. In the past, a few spikelets were
infected by the pathogen, and big (1-2 cm) and dark green olivaceous smutted balls
used to appear. Farmers always considered the appearance of the false smut incidence
in their fields as a sign for bumper harvest (Rao, 1964). The conditions which
generally favour crop growth also favour smut development (Singh, 1974). During the
‘80s, damage to grain yields was estimated to be up to 70% (Muralidharan et al.,
1990).
The effects of the pathogen on the host plant are visible only after flowering,
when the fungal infection of the ovary in individual kernels transforms them into
velvety-green smut balls, initially confined between the glumes. They gradually
enlarge and reach 1cm or more in diameter, engulfing majority of floral parts. The
young spore ball is flattened, smooth and light yellow in colour, and then changes to
orange, yellowish-green, green, olive-green and finally to greenish-black.
Pseudosclerotia are covered by a membrane which bursts, releasing the spore mass. In
high altitude paddy true sclerotia can be found loosely attached to pseudosclerotia
(Singh, 1980). The true sclerotia contain asci in perithecia, embedded in a stroma.
Pseudosclerotia retain their viability up to 7-months at room temperature (25-35oC).
Primary infection originates from pseudosclerotia hibernating in soil, and from
chlamydospores. Conidia play an important role in the secondary host infection.
Chlamylospores are air-borne. The fungus has been recorded also on maize, Digitaria
marginata Link. and Panicum spp. High humidity (>92%) and rainfall accompanied
by cloudy days during flowering favour the disease incidence. Use of sclerotia–free
seeds and crop rotation are important to check disease incidence. At the time of
harvest, the diseased plants should be removed and destroyed to prevent sclerotia from
falling on to the ground. Low and high temperature favour infection and appearance
of symptoms, respectively, while high RH favours the disease development. The
disease is also favoured by application of excess nitrogen fertilizers. Split application
of nitrogen (each at 10 kg ⋅ ha-1) should be recommended in disease prone areas. The
fungus is primarily soil-borne and the sclerotia in soil remain viable for a long period
of time. Secondary infections occur with the help of air-borne sporidia. Spray
application of mancozeb 75 WP (2.5 g ⋅ l-1) or chlorothalonil 75 WP (2 ml ⋅ l-1) around
flowering by targeting sprays only on emerging panicles, can help to control the
disease incidence.
5.11. Post-Harvest Diseases

At ambient temperature and humidity certain fungal diseases may cause damage to
stored grains. The stocks are kept in sheds, old buildings, unused space under cover of
tarpaulin, black polythene or plastic sheets. As a consequence of seed-borne infection,
grains are discolored, with eventual quality losses. The discoloration may be present
externally on glumes or internally on kernels or on both organs. Drechslera,
Curvularia, Fusarium, Alternaria padwickii (Ganguly) Ellis. and Aspergillus have
been found to infect harvested rice grains. Discoloration normally occurs when crop is
left in the field itself. A pre-harvest spray of copper oxychloride, particularly in humid
areas, controls grain discoloration (Govindarajan & Kannaian, 1982).
6. CONCLUSIONS
There are some constraints in implementation of IPM as “most of the plant
protection techniques used are not very attractive to the pesticide industry. They
view IPM promotion as a threat but not a lucrative proposal for business. Chemical
control is still seen as a ‘progressive’ approach by the farmers and easy to apply on
large scale. The chemical companies, who push their products much more
aggressively, provide further impetus such as credit facilities”.
Integrated approaches to disease management involving host resistance,
fungicides and cultural practices are much more common and give effective results
(Singh et al., 2000). Successful disease management often leads to profitable crop
production with higher C:B ratios. For this reason, farmers need to be active on a
community basis, and practice crop health monitoring on a regular basis. The area
should be contained by spot application, to avoid becoming source of secondary

inocula. General management practices listed below will help in curtailing diseases to
a greater extent.
- Destroy crop residues if found infected or infested, otherwise plough the field
and recycle organic matter as much as possible.
- Prefer resistant cultivars. If these are not available select high-quality seed,
treated with seed dressing pesticides. Use proper row spacing and seeding
rates as history of soil sickness.
- Rotate crops with soil resident diseases and give preference to growing
legumes.
- Assay soil for nutrient status, fungal propagules like sclerotia, spores or other
resting bodies or nematodes, and practice soil solarization.
- Determine planting date based on weather forecast to prevent seed rot and
seedling diseases.
- Apply biological control agents with the pest crossing epidemic threshold
level.
- Apply eco-friendly pesticides, in view of prevailing pest-predator index.
Monocrops often suffer significant yield losses as the result of damages due to
diseases. Often the climatic conditions during the crop growth stage encourage rapid
multiplication and spread of pathogens, resulting in either restricted or widespread
damage. Imbalance or excessive application of fertilizers, particularly nitrogen makes
the host susceptible and leads to the development and expression of diseases.
Therefore, the primary goal of IDM/IPM should be to educate and motivate farmers to
use the correct dose of appropriate fertilizers at the right time, when plants need them,
supporting plants’ health, with a lower susceptibility to diseases.
Timely intervention to control diseases substantially reduces losses by containing
diseases in limited areas and eventual spreads. Regular and periodic information on the
intensity of occurrence of diseases and insect pests, at different locations, are gathered
at national level through surveyes and surveillance programs and the same may be
used for initiating protective and curative control measures. National Centre for
Integrated Pest Management, Delhi (India) has initiated an online pest reporting
system (http://www.ncipm.org.in/ipmnetwork/Main.asp), which can be accessed by
any individual or organisation from any part of the country. By this way the real time
pest and disease data can be used for decision making processes. This information
helps in advancing planning for production and deployment of such items as bio-
pesticides, microbials and eco-friendly chemical fungicides. Safe, efficient, and
effective management practices must be carried out in a compatible manner. The
farmer needs to communicate, through farmers field schools, about the role and action
of each management practice, and about how and under which circumstances their
potentials can be exploited at best. Experiences in rainfed cotton and scented rice
indicate that disease intensity can be significantly contained in the fields by adopting
strategies explained in text and discussed under different disease headers.
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15
SALVATORE MORICCA AND ALESSANDRO RAGAZZI
BIOLOGICAL AND INTEGRATED MEANS TO

CONTROL RUST DISEASES
Dipartimento di Biotecnologie agrarie,

Sezione di Patologia vegetale, Università di Firenze, Italy
Abstract. This chapter reviews strategies in rust control, with a special emphasis on biological control, in
the light of evidence produced in recent years showing that plant disease control is most effective when
an integrated management approach is followed. A survey of the fungal antagonists (hyperparasites) most
effective against rust pathogens is given. The mode of action of these antagonists is described, and the
main problems concerning biological control are discussed, on the basis of the optimal characteristics of
an antagonist or biocontrol agent. The value and limitations of other control measures besides biological
control (eradication, definition of hazard areas, quarantine, cultural practices, chemical treatments, and
plant breeding for disease resistance) are also outlined. A consideration of all control measures suggests
that crop protection requires a holistic approach integrating a broad range of control techniques.
1. INTRODUCTION
Modern agriculture is currently confronted by a dilemma: on the one hand it has to
feed a growing world population, so that food production must be constantly
increased (De Waal, 1991). On the other hand the public is becoming ever more
concerned about food safety and clamoring for better and safer foods (Caswell &
Mojduszka, 1996; Gilg & Battershill, 1998). These two demands are obviously
contradictory, since even today maximum and optimum harvests can only be
guaranteed by a massive input of pesticides to protect crops from parasites.
However, pesticides not only lower the safety of the foodstuffs produced, but also
pollute the ground water, infiltrate the food chain, harm numerous wildlife forms,
and by an unintended process of selection they cause insurgence of plant pathogens
resistant to the very pesticides designed to kill them (Moricca, Ragazzi & Assante,
2005).
Because of the pervasive and negative effects that pesticides have on man,
animals and the environment, plant health workers, while not neglecting the benefits
that synthetic pesticides can offer in crop protection, have long since begun to direct
their attention to other means of control that will be less harmful, and equally or
more durably effective (Cook, 1993; Waard et al., 1993).
303
304 S. MORICCA AND A. RAGAZZI
The rust fungi (Basidiomycota, Urediniomycetes) are among the most serious
and widespread plant parasites, infecting a wide and heterogeneous range of hosts,
from primitive ferns to vegetables, grain crops, ornamental plants and forest trees.
These pathogens have a biotrophic lifestyle, i.e. they live in nature only on living
plant tissue. Some members of this group are capable of alternating their life cycle
between two unrelated hosts (heteroecious rusts) whereas others re-infect repeatedly
the same host in a reduced life cycle (autoecious rusts).
Although growth and reproduction occur primarily on foliage, their attacks
debilitate and kill whole plants, besides reducing foliage and root development,
impairing photosynthesis, increasing transpiration rate, decreasing translocation of
photosynthetic products, etc. (Agrios, 2005). Because of their destructiveness, rust
fungi were historically the cause of food shortage, hunger and famine for many
human populations.
Rust diseases are controlled by various means, depending primarily on the
particular context (environment) in which the control action is to be carried out.
Clearly, different measures are required to control the rust agent of an economically
important crop, such as coffee, cereals, or a forest tree. In particular, the disease is of
highest concern when the rust agent is spread transnationally or even
intercontinentally by airborne spores, rather than when the causal agent only
colonises one or a few ornamental plants, in a circumscribed space such as a private
garden or a nursery.
The context in which the rust manifests itself is important, and a thorough
knowledge of the causal agent itself is also needed to choose the type of control to
be carried out. The decision making process requires that the characteristics of the
rust agent should be known, including its biology, ecology and epidemiology, as
well as the host characteristics and those of the pathosystem. These include also the
pathogen virulence, its life-cycle (including the reproduction rate, and hence its
biomass), the modes of production and dispersal of the inoculum, the host plant age
and planting density, the type of culture (pure or mixed stands), the range of the
main host and its spatial diversity (manifesting itself in a mixing of genotypes,
especially of those bearing different resistance genes), the occurrence of alternative
or intermediate hosts, and the range of the latter. All these factors have a bearing on
the likelihood of rust infection and eventual epidemic outbreak, and therefore should
be taken into account.
In this chapter, the control of rust agents will be discussed under the following
heads:
1. biological control
2. eradication
3. defininition of hazard areas
4. quarantine
5. cultural practices
6. chemical control
7. plant breeding (for resistance)
CONTROL OF RUST DISEASES 305
2. BIOLOGICAL CONTROL
Biological control consists in using an organism to suppress or limit the growth of
another, harmful, organism, whether naturally or by manipulating the environment,
so that the harmful organism no longer represents an economic threat. The natural
antagonists of plant pathogens are exploited to eliminate the pathogen and protect
the crop. Biological control was until recently considered as a marginal strategy, but
in the last twenty years it has undergone very considerable development and has
now become one of the most promising fields in applied biological research.
Admittedly, the concept of biological control is not a new one. In the first decades of
the last century, Smith (1919) described it as a possible alternative way to limit
populations of harmful exotic insects (it was initially envisaged and developed as a
way to combat these parasites). Biological control has been understood over time in
various ways, and a final agreement on all the aspects attributed to these
technologies of control has not yet been reached. Noteworthy, for the clarity and
completeness of its terms, is the definition given by Baker and Cook (1974):
biological control is ‘a way to reduce the inoculum density or the pathogenic activity
of a pathogen or a parasite, whether active or dormant, by exploiting one or more
antagonistic organisms, whether naturally, or by manipulating the environment, the
host, or the antagonist, or by introducing massive amounts of one or more
antagonists’.
Biological control exploits the natural phenomenon of parasitism. Parasitism is
an antagonistic symbiosis between two organisms. When parasitism occurs between
two fungi it is called ‘mycoparasitism’ (Schroth & Hancock, 1981). Mycoparasitism
is widespread in nature, occurring in many groups of fungi, ranging from simpler
Chytridiomycetes up to Basidiomycetes. A narrower meaning is given to the term
‘hyperparasitism’, which is used when beneficial organisms parasitise fungal
parasites or other damaging agents: beneficial organisms are mostly other fungi, but
they can also be bacteria or viruses (Kranz, 1981; Brasier, 1990; Yuen et al., 2001).
In plant pathology, diseases are generally viewed as the result from an
interaction involving two partners: the pathogen and the host plant. Such a view
appears far too simple to portray what really occurs in nature, where these two
organisms are themselves part of complex, multitrophic interactions. Harmful
parasites are often attacked and destroyed by hyperparasitic or antagonistic fungi,
whose density in turn increases to form a further trophic interaction over the
traditional host-parasite relationship (Kiss, 2001; Duffy, Keel & Défago, 2004).
Processes of this sort are common in complex niches, like most types of undisturbed
habitats.
The importance of microbial interactions in the epidemiology of plant diseases
has long been understood, and the natural enemies of harmful agents (which for
plant pathogens are the indigenous antagonistic microflora) have been exploited to
control the pathogens and to protect crops. This is the principle of biological control
which has been refined and became more complex over time, so that it now
represents a fully fledged branch of plant pathology.
One of the reasons for the growing interest in biological control is that it seems
to be the only possible alternative to the use of pesticides (Butt et al., 2001).
Chemicals very effectively reduced, for several decades, many diseases and
controlled weeds, especially in intensive agricultural systems. But this control
technology is becoming ever more questionable, since it is too burdensome,
inadvisable on large, homogeneous cultivations (monocultures), inapplicable in
some natural areas such as forests, and in any case disrespectful of the environment.
Biological control, on the other hand, since it does not introduce foreign or toxic
molecules into an ecosystem (inasmuch as the agents used are already an integral
part of it), is considered as an ecologically compatible approach, safeguarding both
the integrity of the environment and human health. The concept of environmental
safety is becoming ever more rooted in the culture of more industrialized societies,
which define themselves by a concern for a higher quality of life, and as a result the
need for environmental safety has become an ecological imperative. For this reason,
biological control is gaining increasing support among the public, which is
becoming ever more alarmed by daily reports about the hazards of chemical
pesticides.
2.1. Suppression of Rust Agents

Hyperparasites of rust fungi are characterised by distinctive properties related to the
specialised lifestyle of the biotrophic agents, and that distinguish them from
hyperparasites of necrotrophic fungi.
Necrotrophic and biotrophic fungi differ in both the time and place in which
they can be parasitised. Necrotrophic fungi kill the tissue of the host before they
colonise them, so that the hyperparasites attack occurs mainly when they are present
on dead plant material, or later, when they grow into the soil in their saprophytic
phase. As a result, there is no direct contact between the host plant and the
hyperparasite of the necrotrophic fungus, since the host-parasite interaction and the
parasite-hyperparasite interaction occur at different places and times.
Biotrophic rust fungi, on the other hand, do not survive in a saprophytic phase
because they can only thrive on living plants, and therefore hyperparasites must
attack them in or on the host living tissue, where they are found. As a result, given
the parasitism biology of rusts, a further structural interaction, the interface host
plant–hyperparasite, occurs in rust hyperparasitism.
For economic, technical and environmental reasons, synthetic fungicides often
cannot be used to control the Urediniomycetes, while the use of biological agents is
a practical approach that has been tested on various pathosystems. Kranz (1981)
stated that about 84 species of fungi from more than 50 genera were reported as
hyperparasites of rusts, although he added that this number might be overestimated
due to synonymies. Furthermore, many organisms reported as hyperparasites were
probably only common saprophytes, growing on ageing fructifications.
2.1.1. Tuberculina spp.

Among the first fungi reported to be associated with the sori of rust agents were
some species of hyphomycetes in the genus Tuberculina, stated to be hyperparasites
of the spermogonial, aecial and uredinial stages. One of the first reports was by
Rostrup (1890) who detected Tuberculina maxima Rostr. on stems of Pinus

monticola infected with the pine rust Cronartium ribicola J. C. Fisher.
Cavara and Saccardo (1899) found Tuberculina sbrozzii Cavara & Sacc. on
leaves of Vinca major, and somewhat later Biraghi (1940) reported that there was a
close relationship between T. sbrozzii and Puccinia vincae Berk, on this host.
However, it was not demonstrated at the time that the relationship between the rust
and the antagonist was in fact a nutritional one.
Grasso (1954) reported a case of Tuberculina persicina (Ditm.) Sacc. acting as
an antagonist against a species of Gymnosporangium on leaves of apple with a
severe rust infection. The hyperparasite wrapped its hyphae around those of the rust,
covering and surmounting them, and penetrated into the aecia, separating the aecial
mass from the hymenium below.
In a recent study, Bauer, Lutz, and Oberwinkler (2004) examining the
ultrastructure of the interfaces between T. persinica and Puccinia sylvatica J. Schröt
or Tranzschelia pruni-spinosae (Pers.) Dietel, found that the antagonist was a
contact parasite. While not forming appressoria or causing other hyphal alterations,
it dissolved the host cell-wall at the contact point with its own hyphal apices,
suggesting that an enzymatic action was involved. These authors also postulated that
there was a horizontal transfer of genes between the two microorganisms. Horizontal
gene transfer also takes place between other fungi, and we may speculate that this
phenomenon might have some possible evolutionary consequences.
Sundaram (1962) reported that T. costaricana Syd. parasitised the uredinia of
Puccinia penniseti Zimm. and other species of Puccinia and Uromyces in India.
Puccinia arachidis Speg. was severely parasitised, in its uredinial phase, by T.
costaricana following artificial inoculation (Sharma, Vyas & Jain, 1977).
The Tuberculina species most thoroughly studied and widespread, and whose
parasitic action on rust has been amply demonstrated, is certainly T. maxima. This
fungus is commonly found on various pine species infected with some agents of
blister rust (Powell, 1971a). A number of researchers (Goodding, 1932; Hubert,
1932; Mielke, 1933) reported this hyperparasite as a strong suppressor, especially of
rust aecia. Its hyphae penetrate the cell walls, and the cytoplasm and the nucleus of
the spores are also disintegrated. Histological studies showed that the antagonist
destroys the nutritional basis of the biotrophic rust agent, preventing the formation
of spermogonia and aecia, and thereby reducing the amount of inoculum produced
(Wicker & Woo, 1973). Such action is assumed to delay the rate at which the host
plant becomes damaged, giving it the opportunity to heal over the wound and expel
the rust stroma (Tubeuf, 1930).
2.1.2. Verticillium spp.

The genus Verticillium comprises parasites of various damaging agents, such as the
arthropods and the plant pathogens. Several members in this genus are antagonists
of obligate parasites, such as the powdery mildews and the rusts.
Among the first cases there were Verticillium coccorum (Petch) Westerd., V.
album-minimum (Sartori et Meyer) Westerdijk, V. compactiusculum Sacc. and
V. malthousei Ware, reported on rusts on greenhouse grown cereals in Germany

(Hassebrauk, 1936).
Various cereal rusts were also reported recently to be attacked by V. psalliotae
Treschew (Leinhos & Buchenauer, 1992). This fungus seems to have a rather wide
range of hosts, since it also parasitises the urediniospores of the destructive coffee
rust Hemileia vastatrix Berk. et Broome in Malaysia (Lim & Nik, 1983) and the
soybean rust Phakopsora pachyrizi Syd. (Saksirirat & Hoppe, 1990).
In the coffee leaves lesions produced by H. vastatrix, another hyperparasite, V.
hemileiae Bouriquet, is also found with a certain regularity. This fungus, first
reported from Madagascar, causes a moderate lowering of the viability of the rust
urediniospores but has only a slight impact on the overall disease progress
(Bouriquet, 1942).
Verticillium coccorum was also reported on Puccinia chrysanthemi Roze
(Kotthoff, 1937) and, at a later date, on Cronartium asclepiadeum (Willd.) Fr. [syn.
Cronartium flaccidum (Alb. et Schwein.) G. Winter], the bark blister rust agent of
two-needled pines in Italy (Castellani & Graniti, 1949). Verticillium coccorum
produced a thick mycelial mat on the aeciospores of C. flaccidum, which then
caused a conspicuous whitish felt to form on the aecia. Rust aeciospores colonised
by V. coccorum lost their typical orange colour. Microscopic inspections revealed
that the relationship of this hyperparasite with the rust was a trophic one, with the
hyperparasite penetrating the rust propagules and emptying them of their contents.
A further hyperparasite that is noteworthy for its aggressiveness, ubiquitous
distribution and strong capacity to devitalise the propagules of its host is
Lecanicillium lecanii (Zimm., Viégas) Zare & Gams (syn. V. lecanii). It colonises
many rust fungi, including some important ones: Uromyces appendiculatus (Pers.)
Link (Allen, 1982); Uromyces dianthi (Pers.) Niessl. (Spencer, 1980); Puccinia
chrysanthemi Roze (Kotthoff, 1937); Puccinia horiana Henn. (Srivastava, Defago &
Kern, 1985; Whipps, 1993); Puccinia recondita Dietel et Holw. (Spencer & Atkey,
1981) and H. vastatrix (Locci, Ferrante & Rodriguez, 1971). Ultrastructural and
cytochemical examinations showed that L. lecanii displays its antagonistic activity
in various ways, including antibiosis, production of hydrolytic enzymes which
dissolve the host cell wall. This fungus also damages the host cell plasma
membrane, which seems to withdraw in presence of the hyperparasite structures,
which then degrades the cytoplasm (Benhamou & Brodeur, 2000).
2.1.3. Cladosporium spp.

The heterogeneous anamorph genus Cladosporium includes more than 500 species
with diverse lifestyles. Some of them are common epiphytes of herbaceous plants
and trees. Several members are endophytes, being frequently isolated from the inner
tissues of various higher plants. Others are true plant pathogens or are found on
plant surfaces only as saprophytes. Many species perform a hyperparasitic action
against a number of phytopathogens (Elllis, 1976; Petrini, 1991; Moricca, Ragazzi &
Mitchelson, 1999; Dugan & Lupien, 2002; Larran et al., 2002; Abdel-Baky &
Abdel-Salam, 2003). The hyperparasites in Cladosporium include several taxa
known to be effective in colonising and killing rust agents at different sporigenous

stages.
Cladosporium uredinicola Speg. is a widespread necrotrophic hyperparasite that
may coagulate and disintegrate the cell cytoplasm of a certain number of hosts:
Puccinia cestri Dietel et P. Henn. (Spegazzini, 1912); P. recondita (Ellis, 1976);
Cronartium quercuum f. sp. fusiforme (Berk.) Miyabe ex Shirai (Morgan-Jones &
McKemy, 1990); Puccinia violae (Schum.) DC (Traquair et al., 1984), and P.
horiana (Srivastava, Defago & Boller, 1985; Srivastava, Defag & Kern, 1985).
Several historical and geographic reports testify of the importance of this group
of antagonists. Spegazzini (1922) reported that Cladosporium uredinophilum Speg.
colonised and killed propagules of Uredo cyclotrauma Speg. in Paraguay. Steyaert
(1930) stated that Cladosporium hemileiae Steyaert was a strong hyperparasite of H.
vastatrix in the Democratic Republic of Congo. Powell (1971b) also detected
Cladosporium gallicola B. Sutton in the aecial pustules of Cronartium comandrae
Pk. on Pinus contorta var. latifolia and concluded that the antagonist parasitised the
aeciospores, and significantly reduced their production.
Sutton (1973) reported a close association between C. gallicola and
Endocronartium harknessii (J. P. Moore) Hiratsuka on Pinus banksiana and found
that the hyphae of the antagonist penetrated the rust aeciospores. Tsuneda and
Hiratsuka (1979) found that C. gallicola parasitised E. harknessii spores either by
growing on them, disintegrating their cell walls, and by penetrating the spores when,
with or without forming an appressorium, it caused the cytoplasm to coagulate and
disappear. These authors also found that during parasitism by contact, the
hyperparasite secreted enzymes that disintegrated the host cell walls. Cladosporium
aecidiicola Thüm., a common hyperparasite in Europe and the Mediterranean basin
(Hulea, 1939; Rayss, 1943) acts in much the same way against E. harknessii on
Pinus contorta, Pinus muricata and Pinus radiata in California (Byler, Cobb &
Parmenter, 1972). This hyperparasite also heavily colonises the aecia of Puccinia
conspicua (Arth.) Mains. in Arizona (Keener, 1954), and conserved urediniospores
of Melampsora medusae Thüm.
Srivastava, Defago and Kern (1985) found that C. sphaerospermum Penz.
regularly parasitised Puccinia horiana. Sharma and Heather (1981a; 1881b) stated
that C. tenuissimum Cooke, thought to be a common inhabitant of the phylloplane of
tree species, also actively colonised the urediniospores of Melampsora larici-
populina Kleb. on Populus × euroamericana. Cladosporium tenuissimum was also
identified in collections of aeciospores of the heteroecious rust Cronartium
flaccidum and its corresponding autoecious form Peridermium pini (Moricca &
Ragazzi, 1998; Moricca, Ragazzi & Mitchelson, 1999). Cladosporium tenuissimum
was identified as an antagonist by some in vitro antagonism assays, where it
inhibited a wide range of host fungi, among which the rusts Uromyces
appendiculatus, Melampsora pinitorqua Rostr., C. flaccidum, Puccinia recondita, P.
sorghi Schwein., and various other plant pathogens in important genera such as
Alternaria, Cercospora, Fusarium, Phytophthora, Rhizoctonia, etc. (Assante et al.,
2004; Moricca, Ragazzi & Assante, 2005).
Investigations into the host-parasite interface at the cytological, histological and

ultrastructural level revealed the many different ways in which C. tenuissimum
suppresses the pathogen. These include the formation of appressoria; the production
of an amorphous fibrillar material ensuring the adhesion of the antagonist to the host
cell wall; the disintegration of the hyphae; the physical destruction of the inoculum,
with degradation of its contents. Penetration of the rust propagules occurs either by
mechanical pressure or by secretion of lytic enzymes (Moricca et al., 2001).
Stereochemical analysis of the metabolites produced also revealed that the
antagonist secretes a family of antimicrobial compounds (cladosporols A-E) with a
strong antifungal activity, that are considered important virulence factors (Nasini
et al., 2004).
2.1.4. Sphaerellopsis philum

Sphaerellopsis philum (Biv.) B. Sutton [teleomorph: Eudarluca caricis (Biv.) O. E.
Erikss. = Darluca philum (Biv.) Castagne] is a mitosporic fungus that has long been
of interest to researchers because of its pronounced tendency to parasitise various
Urediniomycetes. It is a polyphagous fungus and Kranz and Brandenburger (1981)
listed 369 rust agents that it attacks. It has an almost world-wide distribution, being
found everywhere except in Antarctica. The teleomorph is more common in the
tropical zone, the anamorph in more temperate latitudes. Most important hosts in the
temperate zone are: Puccinia graminis Pers.; P. coronata Corda; P. recondita; P.
sorghi; P. striiformis Westend.; Melampsora epitea Thüm.; M. capraearum Thüm.;
C. fusiforme; Cronartium strobilinum Hedgc. et G. Hahn. and Peridermium peckii
Thüm.
The infection biology of this antagonist and the mechanism with which it
suppresses other fungi have not been yet completely elucidated. Hulea (1939)
suggested that it colonised the rust sori through the lacerations caused on leaves by
the sori when they erupted onto the leaf surface. Whatever its mode of entry, it
draws nutrients from the rust sori (Pei & Yuan, 2005). According to Carling, Brown
and & Millikan. (1976), S. philum is a destructive parasite that disintegrates the cell
walls of the host by a combination of physical and enzymatic processes.
Recently, this antagonist was employed with success in northern Europe to
control some species of Melampsora that are causing serious problems to stands of
poplar and short-rotation willow coppices cultivated for biomass production. In
particularly favourable years these rusts can cause heavy leaf fall, with losses in
yield of up to 50% (Whelan et al., 1997). The hyperparasite reduces the parasite
spore production by up to 98% (Pei & Hunter, 2000).
2.1.5. Scytalidium uredinicola

Scytalidium uredinicola Kuhlman, J. W. Carmich. et T. Mill was first reported by
Kuhlman, Carmichael & Miller. (1976), who described it as a new parasite of
Cronartium quercuum f. sp. fusiforme on Pinus taeda and P. elliottii var. elliottii.
The epidemiology of this hyperparasite has been studied especially in relation to its
interaction with E. harknessii. It was shown that the fungus does not penetrate the
spores of this host, but it releases enzymes and other metabolites ahead of its line of
advance, whereby disintegrating the host cell wall and depleting the cells of their
contents (Tsuneda, Hiratsuka & Maruyama, 1980).
In Canada, the beetle Epuraea obliquus Hatch (Coleoptera: Nitulidae) was
identified as the real vector of S. uredinicola (Currie, 1995). It is not yet clear how it
persists in the forest from one year to the next, or in which time of the year it is most
active. A recent study of perennial infections showed that the hyperparasite does not
occur on or inside plant tissues, that have been infected with S. uredinicola for 3-5
years. However, it does occur on plant tissues that have been infected for 6-9 years,
but only on the plant surface, while with plants that have been infected for 10 years
or more it occurs both on and inside the tissues. The fungus overwinters under the
host periderm and colonises the aecial pustules in April, before the peridium breaks
(Moltzan, Blenis & Hiratsuka, 2001). It is clear from this that the fungus requires a
long time to become established and persistent in the ecosystem, and this should be
taken into account when its use as a biological control agent (BCA) is envisaged.
2.1.6. Aphanocladium album

Aphanocladium album (Preuss) W. Gams inhibits many foliar pathogens. It
penetrates the aeciospores, urediniospores and teleutospores of various rust agents
(Koc & Défago, 1983). The process of penetration is accompanied by the secretion
of degrading enzymes, such as chitinases (Srivastava, Defago & Boller, 1985).
Studies on Puccinia graminis f. sp. tritici Erikss. et Henning showed that, in
infected spores, the cytoplasm disintegrates and then disappears. When conidial
suspensions of the antagonist were sprayed on leaves infected with the rust agent, a
characteristic cottony whitish felt mat consisting of mycelium of the hyperparasite
developed in a few days, and massively parasitised the host spores (Koc, Forrer &
Défago, 1983).
3. DISEASES SUPPRESSION MECHANISMS

The purpose of the use of antagonistic microorganisms is finalised to restrict the
target pathogen density. A microorganism (bacterium or fungus) acts as a BCA
when it inhibits a target pathogen, by disrupting its life cycle or by living in close
association with it. The pathogen is subsequently suppressed by a reduction in its
inoculum, or by protecting the plant surfaces from its attack (Manocha, 1985).
Antagonists suppressive actions occur through several mechanisms, some of which
are related to each other, and/or evolved independently.
3.1. Competition for Nutrients and Space

Competition for nutrients occurs when one organism attempts to obtain a limited
food resource for itself at the expense of other organisms competing for the same
resource. Competitive success may result from a faster growth or a greater capacity
to metabolise organic molecules (Boosalis, 1964). Microbes generally compete with
each other for carbohydrates, growth factors, nitrogen, iron and other micronutrients.
Competition for space takes place on the surface of the host, starting from the areas
around the infection site. It includes oxygen as a space competition-related form.
3.2. Direct Parasitism

This effect consists in the physical destruction of a pathogen inoculum (Barnett &
Binder, 1973). It can occur by a simple interaction of the hyphae, with the antagonist
hyphae entangling themselves around those of the pathogen, disrupting its mycelium
and restricting or blocking its growth. Parasitism can occur by contact, either direct
or mediated by specialised structures of the antagonist, such as appressoria, but
always causing the cell walls of the host hyphae or spores to be penetrated and their
contents to be degraded. Penetration can be by physical pressure or by chemical
action, with the release of degrading enzymes such as the chitinases and glucanases.
An effective antagonist reproduces itself and sporulates abundantly on the fruiting
bodies of the target parasite.
3.3. Antibiosis
This phenomenon consists in the production of antibiotics (e.g. toxins) or other
compounds that are toxic for the pathogens or that cause fungistasis, lysis, or
necrotic effects inhibiting the pathogen growth (Howell & Stipanovic, 1995). All
these molecules help the BCA to secure and maintain an ecological niche on the
plant, overcoming any competitive action of the resident mycoflora (inhibiting
saprophyte growth) and displacing the pathogen from the host surface.
3.4. Induction of Plant Resistance

Induced resistance is the ability of an antagonist to cause, in the host plant, a pre-
infectional resistance to a given infective agent (Van Loon, 1997). It is an aspect of
antagonism that has been studied in a number of host-pathogen systems using
various species of Trichoderma (Elad, 1996; Bigirimana et al., 1997). It seems to be
fairly common in nature and to cause a systemic (Harman et al., 2004) or more
rarely a localised (Howell, 2003) resistance in the host plant.
3.5. Improvement of Host Fitness

Pathogenic infections lower the fitness of a plant, since they impair its structure and
functions. Plants treated with hyperparasites, on the other hand, generally have a
greater photosynthetic efficiency, better growth and produce a higher and/or better-
quality yield (Kiss, 2001). Abo-Foul et al. (1996) found that cucumber plants
infected with powdery mildew had a much greater photosynthetic activity and a
higher levels of chlorophyll when they were treated with an antagonist
(Ampelomyces sp.). Plants treated with a BCA and freed from the damage caused by
a pathogen become more vigorous and more resistant to parasites.
All these mechanisms will control a disease if the antagonist succeeds in

reducing the growth, survival, rate of reproduction and hence the biomass of the
pathogen. Advances in technology over the last few years offer interesting new
prospects for biological control.
The knowledge on the mechanisms affecting the way microorganisms interact
with each other (exchange of molecular signals) is moving rapidly. Today it is
possible to enhance the effectiveness of some BCAs by genetic manipulation (Lim,
Kim & Kim, 1991; Glick & Bashan, 1997). Appropriate administering and
formulation of the antagonist are also very important (Sutton & Peng, 1993).
Inoculations with antagonists or hypovirulent strains of a pathogen make it possible
to suppress important diseases effectively. Innovative techniques such as coating
seeds with a biofilm based on propagules of an antagonist, lower the rate of
pathogen infection and thus may favour eventual plant growth (Bais, Fall &
Vivanco, 2004).
4. MAIN PROBLEMS WITH BIOLOGICAL CONTROL

The many pathosystems studied, and tests on the biological control of rusts, have
shown that hyperparasites can significantly reduce rust diseases. However,
biological control should not be seen as a panacea. In protecting crop plants, all
phytosanitary measures have their limits, and biological control is no exception to
this rule. Obstacles arise in devising BCAs that fully meet the justified expectations
of agricultural and forestry workers, in terms of effectiveness, formulation and
manner of application, as well as in the extent to which plant biopesticides (products
whose active principles are living microorganisms) can be combined with other
products and other control methods, as part of an integrated disease management
programme (Mathre, Cook & Callan, 1999; Wraight, Jackson & Kock, 2001; Fravel,
2005).
Field workers faced with a need to control pathogenic fungi have indeed not
been excessively attracted by biological control so far, because BCAs have too often
been:
1. difficult to apply;
2. variable in their effects, which in many instances are not immediately
noticeable;
3. less spectacular as concerns the results produced, in comparison with those
obtained with other approaches (e.g. pesticides);
4. biologically too complex to be easily understood and applied;
5. at times, impossible to combine with other approaches (for example,
chemical pesticides sometimes render BCAs less effective);
6. in some pathosystems, quite uneconomical.
Other negative aspects must also be considered, relating to the biology of the
three interacting partners (pathogen, antagonist, host plant), each of which can cause
the failure of a biological control strategy. The pathogen must not be too variable at
either the inter or the intraspecific level, as this may make it less susceptible to a
BCA (Deacon, 1994). Host plants, even individual plants, may also vary in their
response to a BCA, and this factor should not be neglected. However, undoubtedly,
it is the characteristics of the BCA itself that are most often to blame for failure. A
BCA may:
1. not establish itself with perfect uniformity in all fields and environments;
2. not always express the traits necessary to produce antagonistic activity in
response to signals from the host plant, the target organism, or the
environmental parameters;
3. not have a range coinciding with that of the pathogen, leading to an erratic
effectiveness of the antagonist;
4. not produce sufficient inoculum to suppress the propagules of the pathogen
effectively;
5. have an insufficient infective capacity;
6. have a too low virulence;
7. not possess enough suppressive mechanisms to reduce the disease
significantly.
Because of these drawbacks, which often derive from the complexity and
variability of the environment, research on biological control has mostly
concentrated on eliminating diseases in a controlled environment, focussing on
greenhouse crops, or crops in post-harvest storage (Paulitz, 2001). Alternatively,
attention was given to managing the incompatibility between the host and the
pathogen by cross-breeding different varieties, or by inoculating the host plant with
low virulent or avirulent pathogen strains. At the same time attempts were made to
develop methods of cultivation that favour biological control (Whipps, 2001).
Frequent initial failures have induced researchers to carry out a number of in
vitro assays on antagonistic organisms before proceeding to field tests. In in vitro
tests the environmental parameters can be kept constant and favourable for the
establishment and optimum activity of the antagonist organisms, which can colonise
the plant surface without having to face the intense competition of other native
organisms, that often occur in a natural environment.
Nevertheless, numerous experiments that were initially successful in vitro have
yielded very poor results when they were transferred to the field afterwards (Cook,
1993; Harman, 2000). The transfer of a laboratory or greenhouse test to a large-scale
field trial is always a complex issue, since the antagonist should face, in the field, a
wide range of environmental conditions. Any disease is the consequence of a
dynamic interaction between a pathogen, a plant and a particular environment in
which they exist. The environmental component of this triad can be critical in
ensuring the success of a BCA, whose insufficient ecological stability may represent
its weakest point.
The antagonist must indeed actively suppress the parasite, over a period that
could vary from a few weeks up to several months, depending on the pathogen
reproductive life-cycle, the crop and its phenological stage. In these circumstances it
must also withstand any changes that may occur in the physical environment and
resist competition from indigenous microflora. The lack of a sufficient ecological
fitness can hence impair the effectiveness of a BCA, while a lack of survival
capacity may induce a low persistence rate, so that its suppressive effect cannot last
for a time period long enough (Whipps & Lumsden, 2001).
It is therefore clear that devising an effective biological control method is by no
means a simple task. For implementing an effective and durable biocontrol strategy,
a deep knowledge about the reproductive biology of both the pathogen and the
antagonist is needed, as well as about the impacts that environmental factors exert
on pathogen aggressiveness, antagonist persistence, and host plant susceptibility
(Whipps, 1997). Other important aspects that must be carefully considered concern
the specific mechanisms that the antagonist uses to result suppressive, the changes
of its survival rate, its activity in blocking the disease process in the host plant, and
the mechanisms of plant resistance induction.
For all these reasons, it must be admitted that despite a vast amount of
experiments that have been carried out and despite the many potential
microorganisms tested, the actual use of fungal antagonists to control rusts and other
fungal diseases in the field is still rather limited.
There are, however, some beneficial species that, after rigorous testing, have
joined the ranks of plant biopesticides. One of these is Phlebiopsis gigantea (Fr.)
Jul., a fungus employed in the biological control of Heterobasidion annosum (Fr.)
Bref., a rot agent of tree stumps. This antagonist has been developed and approved
as a product marketed under the name of PG suspension® in the UK, and as
Rotstop® in Finland (Roy et al., 2003). However, even though much knowledge has
been acquired, initial results with BCAs did not lived up to expectations due to a
number of environmental, biological and anthropogenic factors in agricultural and
forest ecosystems.
Only in recent years it was understood that biological systems rely on a fragile
balance and that biological control must be rooted on a thorough knowledge of the
pathogen, antagonist and host plante ecology. These three interacting components
interact with agronomic (crop microclimate; existence of alternative means of
control) or silvicultural (type of stand, stand management and practices) factors.
The dynamics of epidemics can hence be understood, in its complexity, only if
the resident microbial flora is taken into account, and if it we realise that all the
factors involved in this interaction are in turn influenced by the physical
environment.
The experimental findings on hyperparasites of rust agents shed an important
light on how the variables mentioned above affect the biological control of a disease.
Studies on biocontrol of Urediniomycetes have made it clear that if BCAs are to
protect agricultural crops, the agricultural worker must have a knowledge of cultural
specialisation (selection of resistant varieties) that is kept constantly up to date. This
is due to the crop diversity (arising from the spatial diversification of the host plant
genotypes, achieved through the use of multiline cultivars and/or of cultivar
mixtures endowed with different resistance genes), which has a direct bearing on the
evolution of the pathogen (the physical barrier represented by resistant plant
genotypes reduces the probability of infection of migrating spores) and that of its
antagonist, and hence also on the disease epidemic progression (Mundt, 2005).
It is known, for example, that short-rotation (3-5 years) coppice stands of tree
species (poplar, willow) grown for biomass production for energy are more suitable
for biological control because of a carry-over effect, the inoculum of the antagonist
surviving, and accumulating, from each growing season to the next one (Pei &
Hunter, 2000).
A completely different situation, with the pathogen having a complete
advantage, is seen with long-rotation stands (rows of roadside or urban trees, high
forests, etc.). Here the long life-cycle of individual trees cannot keep up with the
continuing genetic adaptation and variation of phytopathogens that have a much
more rapidly evolving life-cycle. In cases such as these, it is advisable to prefer
antagonists with certain characteristics.
The most important traits for an effective BCA are as follows. A BCA should:
- be genetically stable;
- be effective even at low concentrations;
- not endanger human health;
- come as a product that is easy to apply;
- be resistant to pesticides;
- be combinable with other means of control (physical or chemical);
- not be harmful to the host plant;
- not be too specialised, so that it will kill as many parasites as possible, and
all the pathotypes that occur of each pathogen, if possible in a variety of
systems;
- occur naturally in the area where it is applied; cultural practices that
encourage its reproduction should be favoured;
- be able to colonise various matrices (other organisms, dead organic
material) so that it can survive in large numbers even when the target
organism is absent, i.e. it is not eliminated at harvest, but will persist to be
available at levels protecting future crops;
- have various mechanisms of aggression;
- spread its propagules in an effective way;
- be able to overwinter in perennial infections, so that at the start of a new
growing season, which is generally a propitious time for pathogen
reproduction, it is i) already present and well distributed at all the infection
centres; and ii) ready to sporulate, profusely and precociously;
- have a propagation cycle synchronous with the reproductive phases of the
pathogen;
- result easy to grow in culture on an economical medium, and sporulate
profusely so that it can be applied in the requisite quantities;
- display adequate ecological amplitude, so that it can survive its application
in the field, establish itself in the ecosystem, and remain active until it
encounters the target organism that it has to suppress. It should therefore

also be able to withstand unfavourable weather conditions and compete
effectively with the resident microflora, to result persistent and perennial.
5. ERADICATION
Eradication is a preventive measure, like the delimitation of hazard areas and the
imposition of quarantine, to be discussed below. Its purpose is to prevent the
outbreak of epidemics. For it has always been understood that with plant diseases,
just as with diseases affecting men and animals, prevention is the best policy. Only
when prevention fails, other methods should be considered. Effective ways to
prevent, limit, or control rust agents have been developed and validated in the field,
and are sometimes enforced by law.
A long time before the advent of synthetic pesticides, it was in fact realised that
the best weapon against plant parasites was to interrupt their life-cycle by removing
their food source, that is their host (Davis, 2001). Since rusts are biotrophic
organisms, this would necessarily result in their complete elimination.
Eradication is usually carried out to remove recently established rusts, but in
some instances it was also attempted to remove native rusts (Moriondo, 1975).
Eradication consists in the uprooting and destruction of the single host for
autoecious rusts (for which no other hosts are known), and of the intermediate
host(s), in the case of heteroecious species.
Eradication must be carried out thoroughly over very extensive areas (entire
districts or geographic regions), otherwise its effectiveness may be greatly impaired
by the air-borne dispersal capacity of rust spores over long distances, by the great
number of hosts that some rusts have, and by the widespread occurrence of the
intermediate host(s). For example, Vincetoxicum hirundinaria (white swallow wort),
the intermediate host of the agent of the two-needled pines blister rust Cronartium
flaccidum, is so ubiquitous within and on the edge of pine forests and stands, and in
the surrounding clearings, that it was immediately clear that any attempt to eradicate
the disease was doomed to failure (Moriondo, 1975).
Other attempts at eradication have been, however, more successful. In 1903
Hemileia vastatrix, the agent of coffee rust, was accidentally introduced into the
island of Puerto Rico. Since this was an island far away from any inoculum sources,
it was possible to eliminate the pathogen in one year by immediately eradicating and
destroying all infected coffee plants. In this way coffee cultivation, so important for
the island economy, was saved, and coffee rust was kept out of the Western
Hemisphere for many years, until it was accidentally introduced into Brazil in 1970
(Littlefield, 1981).
Coffee rust was also eradicated with success in Papua New Guinea in 1965.
Here, when the rust was first detected, an eradication programme was immediately
initiated to remove the first centres of infection, and this action made it possible to
stop the disease in its tracks, even before it became properly established. The
programme was facilitated by the mountainous nature of the country and the
prevalence of rain forests, which acted as barriers to the spread of the parasite
propagules towards the main coffee-growing areas (Littlefield, 1981).
In the case of heteroecious rusts, attempts to control the disease by eradicating
the intermediate hosts, on which some rusts complete their sexual cycle, are rather
more frequent. Eradicating these hosts achieves a double benefit: not only it
substantially reduces the mass of rust inoculum, thereby slowing down the spread of
the disease, but also it prevents the rust sexual recombination, reducing the
likelihood that it will evolve new races and pathotypes.
As early as 1660 an attempt was made in France to eradicate the common
barberry (Berberis vulgaris), the intermediate host of the wheat rust agent Puccinia
graminis, with the aim of controlling the disease. In the last century the eradication
of Berberis spp. was again tried to control the same rust in Denmark, USA, Ireland,
England, Switzerland and Bavaria, with conflicting results. In the first two countries
and in Germany, barberry was not completely eradicated, but a reduction in wheat
rust levels was achieved.
In England the eradication effort had no prospect of success since P. graminis
inoculum constantly arrived via air-borne spores from the Iberian peninsula. In
Switzerland and Ireland the eradication effort failed because the barberry was simply
too widespread to be eradicated (Littlefield, 1981).
A massive campaign to eradicate Ribes spp., which are the intermediate hosts of
Cronartium ribicola, the blister rust of white pines in North America, was initiated
in the thirties in the USA (Kinloch, 2003). All Ribes plants growing within a 350-m
radius of fir stands were eradicated manually or with herbicides. The campaign was
initially a success, somewhat alleviating the severity of the rust epidemic, but later
economic considerations, and the realisation that it was impossible to reduce currant
populations to levels safe for pine trees, led to the campaign being abandoned.
6. DEFINING HAZARD AREAS

Defining hazard areas is a method to elude the pathogen by avoiding planting of
susceptible hosts near centres of infection, or near areas where intermediate host
plants are growing (CMI, 1977; Gross, Ek & Patton, 1983). In the USA special
Cronartium quercuum f. sp. fusiforme ‘hazard maps’ have been created, showing
the main infection areas of this fungus, as well as those areas where its intermediate
hosts occur (Anderson et al., 1988).
Dozens of species growing in North America are the primary hosts of this rust.
They include the native species Pinus taeda, P. elliottii, P. banksiana, P. virginiana,
P. palustris, P. radiata, P. serotina, and the exotic species P. canariensis, as well as
species also found in Italy, P. halepensis, P. pinea, P. sylvestris, P. laricio, and P.
nigra.
The secondary hosts of the fungus include Quercus nigra, Q. rubra, Q.
marilandica, Q. cinerea, all native, and some species of chestnut, Castanea
mollissima, C. dentata and C. sativa. Although, in this case, the wide spectrum of
hosts makes hazard maps a hopeless procedure as a control strategy, it remains
imperative to eradicate all oaks growing near pine nurseries, and to locate such
nurseries only in areas where the above Fagaceae are absent.
As in the USA for the hosts of C. quercuum f. sp. fusiforme, in Italy for the
white swallow wort, the intermediate host of Cronartium flaccidum, hazard maps
indicating the plant distribution were drawn up for the country as a whole and for
Tuscany in particular (region in which such herb is exceptionally widespread).
These maps should be consulted before establishing pine stands, so as to avoid
placing them near areas where white swallow wort plants are growing, and thus to
reduce the risk of rust infection in pine trees (Ragazzi & Moricca, 1986).
6.1. Quarantine
Quarantine is a lawful requirement enacted in order to prevent the accidental
introduction of hazardous rust agents into areas that are currently disease-free
(Schrader & Unger, 2003). It is carried out by means of stringent controls carried out
by phytosanitary services at the nation’s main entry points (ports, airports, etc.) of
the goods to be controlled. It is enforced by a lawful prohibition to import into an
area any potentially infected plant material, proceeding from areas where a disease
occurs. It also lays down rules to prohibit local residents from frequenting or passing
through infected areas and to prevent wild or domesticated animals from going
through or grazing into quarantined zones. Accredited international organisations
such as the International Plant Protection Organisation (IPPO) and the European and
Mediterranean Plant Protection Organisation (EPPO) publish constantly updated
lists of quarantine organisms. In the A1 list of EPPO (http://www.eppo.org), listing
pathogens that are at high risk of being introduced into Europe and updated to
September 2007, the following rust agents are given: Chrysomyxa arctostaphyili,
Cronartium coleosporioides, Cronartium comandrae, Cronartium comptoniae,
Cronartium fusiforme, Cronartium himalayense, Cronartium quercuum,
Endocronartium harknessii, Gymnosporangium clavipes, Gymnosporangium
globosum, Gymnosporangium juniperi-virginianae, Gymnosporangium yamadae,
Melampsora farlowi and Puccinia pittieriana. Rust agents are the largest group of
quarantine organisms, comprising fully 37% of the total. This considerations
shows a clear indication of the danger of rust agents and the risk they represent,
for various tree species in the European Union.
6.2. Cultural Practices

One of the oldest, most common and still one of the most effective ways of
controlling a disease is to separate the host and the pathogen in time and space. This
is done by a practice called crop rotation (Cook, 1993; Schippers, Bakker & Bakker,
1997). Crop rotation can control autoecious rusts, such as the bean rust agents
Uromyces spp. and the agent of flax Melampsora lini. In temperate zones the
teleutospores, overwintering on infected leaves lying on the soil, transfer the
infection to the host plants in the following spring. Since they do not survive more
than one growing season, crop rotation in such cases is an effective control measure.
If crop rotation as such is not feasible, rust inoculum can be significantly
reduced by burning infected plant residues, or ploughing parasite propagules into the
soil (Littlefield, 1981). This is a common practice in ornamental stands and in

vegetable crops, but it would be impractical as a means to control rusts in forests.
A lowering of relative humidity curbs the growth of Urediniomycetes (Chen,
2005). When growing geraniums, carnations or other greenhouse flowers, it is
advisable to provide enough ventilation to the growing environment and to avoid
overhead watering, using instead surface or drip irrigation. These irrigation methods
do not increase the relative humidity and do not favour the spread of the
urediniospores, as is the case with overhead irrigation.
All cultural practices favouring host growth generally also increase the
susceptibility of the plants to the rust agents. Forcing plant growth by means of
fertilisers or cultural manipulation always substantially increases the rate of
infection. Plants treated in this manner exit earlier from dormancy, and present the
succulent tissue where rust infections tend to be largest, for a longer period of time.
In young pine stands, pruning branches that exhibit rust infection reduces the
likelihood that the infection will spread to the trunk (Moriondo, 1975). This measure
is obviously useless if the trunk is already affected. Nevertheless, new infections
also sometimes arise even after pruning the infected branches. In some cases, high-
value trees can be cured by excising the infected areas on the trunk, if they represent
only a small portion of the total trunk area. The infected bark, as well as part of the
healthy tissue surrounding it, is carefully removed, and the excised area is covered
with protective sealant. In this way the tree may be saved.
Pruning and removing infected tree parts, and felling and burning all trees that
are beyond cure, should in any case be recommended since these practices reduce
the amount of parasite inoculum in the area. Felling and removing infected trees also
serves to weed out the most susceptible genotypes from the host population, and, by
allowing more solar radiation to enter the forest, favour the mortality of the rust
basidiospores, which are sensitive to light.

Most experimental studies on fungicides controlling rusts deal with cereals rusts
(Buchenauer, 1982). Fungicides can be sprayed on plants or applied to the soil,
depending on the active molecules or their mode of action. When sprayed on plants,
fungicides act by inactivating the rust agents and preventing their penetration into
the host plant (protectant fungicides). Fungicides applied to soil are absorbed and
translocated systemically throughout the plant, where they have a double function,
being both protective and curative. A third category of fungicides consists of
compounds that are merely fungistatic.
The first fungicide used to control cereal rusts, in the ‘20s and ‘30s, was
inorganic sulphur. It did not, however, become very common since it was too
expensive. Other fungicides were also available on the market, i.e. pycric acid,
borax, lithium salt, maleic hydrazide and various sulphonamides, but they were all
either too toxic to the plants, too difficult to handle properly, or ineffective.
Copper oxide was particularly effective against coffee rust (Littlefield, 1981).
The organic fungicides, such as the dithiocarbamates, have more recently been
found to achieve a good control of some cereal rusts, while the triazoles seem
particularly effective in seed dressing.
For economic, but even more for obvious environmental reasons, chemical
control is altogether impracticable as a means to control rust in forest trees (Maloy,
1997). Nevertheless, some attempts have been made to control conifer rusts in
species of the genus Picea used for Christmas trees, and the fusiform rust agent of
pine Cronartium quercuum f. sp. fusiforme, using oxycarboxin. This is a systemic
fungicide that protects the plant either preventively, by penetrating the sprayed
tissues before infection, and curatively, by eliminating infections that have already
started. However, these attempts were in most cases carried out on trees outplanted
in plantations, or in ornamental plantings, or in nurseries (Littlefield, 1981; South
& Zwolinksi, 1996).
Besides oxycarboxin, systemic fungicides like myclobutanil, cytotropic
fungicides such as triforin and benodanil and dithiocarbamates like ferbam, zineb
and maneb are also widely employed in the control of rust attacking biomass energy
plantations, cereals and flowers (Dickens, 1990; Dawson, McCracken & Carlisle,
2005).
Variables critical in determining the success or failure of chemical control are:
the susceptibility of the plant species (or cultivar) to the disease in question; the
expected yield that will be obtained at the end of the growing season; its economic
value and the environmental parameters, which can favour or suppress the disease.
6.4. Plant Breeding for Resistance

Ever since the dawn of agriculture, when many plants of the same species began to
be cultivated together at the same time, it was noted that the incidence and severity
of the disease were not always the same, since some genotypes were less affected
than others. Many centuries before Gregor Mendel, farmers had discovered that
plants varied in their resistance to disease, and they begun to crossbreed them
empirically, in order to obtain superior lines.
Interesting experiments on the variability and hereditability of resistance were
carried out at the end of the 18th century by Knight, who developed some wheat
hybrids resistant to, presumably, Puccinia striiformis, and almost a century later by
Farrer, who bred, around 1880, numerous rust-resistant wheat hybrids in Australia
(Littlefield, 1981).
Nevertheless, plant breeding as a scientific discipline got under way only at the
beginning of the 20th century. It was at the start of that century that Biffen (1905),
working with the yellow rust of wheat, Puccinia glumarum, and the wheat cultivar
‘Rivet’, demonstrated that disease resistance in plants is inherited in a Mendelian
fashion. Biffen’s pioneering studies also proved that disease control can be achieved
by incorporating resistance genes into plant varieties in special breeding
programmes. As a result of this discovery, intensive genetic improvement
programmes were initiated in many parts of the world, and many plant varieties with
resistance to important pathogens were bred.
Plant breeding today is one of the most tried and tested means of disease
control, and one of the most desirable in view of its effect on the environment, since
the use of resistant plant material can allow fungicide treatment to be reduced or
even dispensed with, completely.
The first step in every plant breeding programme is always to identify the
source of resistance. This can be obtained:
- from a population of diseased plants (in cases where the plant

population has a sufficiently wide genetic basis, as it is often found in
land races);
- from the ancestors or wild relatives of the domesticated plants (often in
the centres of origin of the plant species);
- by intergeneric crosses (a difficult and tedious task, often attended by
problems of sterility and other disadvantages);
- by mutation (natural or induced with the use of mutagenic agents);
- by introducing exotic genes using recombinant DNA technology.
A major finding that became the main focus of early breeding work was the
discovery of specific resistance. Most plants have genes that confer resistance
specifically on certain rust pathogens, the particular resistance conferred differing
according to the genetic make-up of both the plant and the pathogen. Flor (1956)
studying the Melampsora lini - Linum usitatissimum system, elegantly demonstrated
that to each pathogen gene responsible for its pathogenicity there corresponded a
complementary gene in the host plant, governing its response to the pathogen (the
gene-for-gene theory).
This type of resistance, known as vertical or race-specific resistance, gave rise
to an intense activity of breeding rust-resistant varieties in cereals, which at first
yielded spectacular results. The prospect of obtaining completely resistant
germplasm by incorporating one or only a few genes (monogenic/oligogenic or race-
specific resistance) favoured the creation of new cultivars, which were grown as
monocultures, often over large areas.
However, the large-scale cultivation of varieties with race-specific resistance
exerted a strong selection pressure on the pathogens. Some rust fungi, like the cereal
rusts, produce huge masses of spores during the growing season, and therefore the
chances that mutant spores capable of infecting cultivars with specific resistance
will arise, are statistically very high. When this happens, it causes the breakdown of
the built-in resistance, and the newly successful pathogen genotypes can then spread
epidemically throughout the new host population over vast areas (a boom and burst
cycle).
Another form of resistance, which is quite common in natural populations is
that of race–nonspecific resistance. Many cultivars naturally possess a “generalized”
or “horizontal” resistance against all the different races of a particular rust agent.
Such a resistance is less spectacular than race-specific resistance since it is not
complete, and the crop remains partially susceptible to the pathogen, with some
minor yield losses that gradually occur in the cultivated area, due to the slowing
down of the rust disease (‘slow-rusting’ crops). This type of resistance is, however,
more enduring, since many genes in the host contribute towards it, so that it is called
a “polygenic resistance”. Since such resistance is generally permanent, it is also
termed “durable resistance”. Important rust epidemics, such as the southern maize
rust caused by Puccinia polysora in Africa in the middle of the last century, declined
to insignificance as the pathogen encountered many minor genes, each conferring
some partial resistance, on the maize population (Harlan, 1976).
Less elusive varietal control of rust diseases can also be achieved by combining
more specific resistance genes into a single plant variety. The discovery that several
genes each conferred specific resistance to a particular rust agent prompted a big
effort to incorporate as many of these genes as possible into a single cultivar. This
effort was however in some cases nullified by linkage effects and by allelism at
some loci (Carlile, 1988).
Nowadays, various schemes have been developed to manage host-plant
resistance in order to control rusts. All these schemes aim at diversifying the host
genotypes grown in a given area, so that the non-susceptible plants create barriers to
inoculum spread, and pathogen inoculum is diluted (Wolfe, 1985). Among the most
effective control strategies are the use of multiline cultivars (i.e. mixtures of lines
bred for the phenotypic uniformity of their agronomic traits) and cultivar mixtures
consisting of agronomically compatible, cultivated varieties that were not bred for
phenotypic uniformity (Garrett & Mundt, 1999).
Other means that have been hypothesized to be effective in reducing the
severity of pathogen attacks include induced resistance (Lannou et al., 1995;
Lannou, Hubert & Gimeno, 2005), and disruptive selection, caused by the
quantitative adaptation of the pathogen to the genetic background of the different
cultivars grown in the mixture (Wolfe, Barrett & Jenkins, 1981).
Currently, there is an increasing pressure on the world’s agricultural and forest
resources to produce an ever greater yield. The rapid development that many
countries are now undergoing, and the efforts they make to raise their economic and
social standard of living are inadvertently having a strong negative impact on the
yield of farmlands and forests, putting their sustainability at risk. The over-
exploitation of agricultural crops and forests to meet human needs is creating new
problems, as well as exacerbating older ones. Rust diseases of forest trees have long
been and still are the cause of enormous losses in wood production in many parts of
the world. In the USA the two pine rust agents C. ribicola and C. quercuum f. sp.
fusiforme have caused heavy losses in pine stands (Littlefield, 1981). In Europe too,
which has a flora impoverished by the last glaciation, pine forests are suffering from
increasing losses due to rust fungi. Countering the problem by planting new pine
species with a promising level of resistance to some rusts, is often hampered both by
the occurrence of other native or exotic rusts to which these trees are susceptible,
and by the poor adaptation of these tree species to their new habitat.
Control measures are very often expensive, and their success cannot always be
guaranteed. The genetic improvement of forest trees, which are very long-lived, is
therefore the best approach to develop rust resistance, over the long term. Indeed,
the development of genetic resistance is not only one of the most effective means to
control disease, but it will also achieve a greater and better yield, improving air
quality, protecting the soil and providing the aesthetic and recreational qualities that
an area forested with healthy trees normally provides.
7. CONCLUSIONS
It must be stressed out that single control measures taken in isolation are rarely
effective. In several cases, satisfactory results can only be obtained if a combination
of two or more control methods is applied. Plant health management should
therefore be founded on an holistic approach, in which the totality of the biological
and technical factors of disease control must be taken into account and coordinated
to minimise the impact of plant diseases. This means that an effective and affordable
control of plant diseases can only be achieved with integrated disease management.
Measures of control must both prevent and cure, and they must also be
diversified. The choice of a given control method must be based on a thorough
knowledge of the ecology of the interacting organisms and of the habitat they grow
in. Only in this way can biological control measures be harmonised with other
control methods and provide a significant contribution to the suppression of the
disease. A mounting number of studies demonstrated indeed that integrated disease
management can prevent the losses in food and wood production caused by plant
parasites, and can help to ensure a sufficiency of food and a good standard of living
to the growing world population.
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Section 3
Advances in Management Tools

16
KEITH R. MITCHELSON1, 2 AND SALVATORE MORICCA3
DNA FINGERPRINTING METHODS FOR MICROBIAL

PATHOGENS: APPLICATION TO DIAGNOSTICS,
TAXONOMY AND PLANT DISEASE MANAGEMENT
1
National Engineering Research Centre for Beijing Biochip Technology,
Beijing 102206, China
2
The Medical Systems Biology Research Center,
Tsinghua University School of Medicine, Beijing 100084, China
3
Dipartimento di Biotecnologie agrarie,
Sezione di Patologia vegetale, Università di Firenze, Italy
Abstract. Advanced molecular genetic techniques enhance our capabilities to identify and characterize
microbial pathogens, resulting in accurate testing for pathogen identification, sub-species-level DNA
fingerprinting, pathogen-load testing and disease spread monitoring. These applications are instrumental
to the study of plant disease epidemiology, so that adequate control measures can be accordingly
implemented. In this chapter, a survey of the most popular DNA profiling techniques is presented together
with some of the newer and most discriminating molecular methods. Combinations of different analytical
techniques are also proposed as a useful approach for low throughput bioassays. Advantages and
disadvantages of each single test are taken into account and key issues (sampling, validation, large-scale
testing, etc.), encountered in the practical application of these assays, are discussed. An outline of
emerging high-throughput molecular technologies expected to improve diagnostic approaches and aid
disease management is also provided.
1. INTRODUCTION
Among the main obstacles frequently encountered by plant pathologists are the
identification and taxonomic positioning of new pathogens, or the identification and
differentiation of known microrganisms whose sub-specific entities are difficult to
determine by classical approaches. Traditional methods of identification of
microbial pathogens, which are essentially based on the inspection of macro- and/or
micro-morphological characters, have inherent limitations (Ward et al., 2004). In the
Mycota, the major component of the plant parasitic microflora, somatic structures
333
334 K. R. MITCHELSON AND S. MORICCA
(hyphae and hyphal modifications) are often of little utility for identification
purposes, whereas the organs best serving as basic taxonomic criteria, such as the
reproductive cells (meiospores or mitospores) with relative sporophores, are only
obtained with difficulty in the laboratory, either from liquid or plated media or from
infected tissues. Moreover, considerable experience is necessary to induce the
formation of such distinguishing structures in vitro, and substantial expertise is
needed, as well as for their subsequent microscopic observation and recognition.
These procedures are in any case not applicable for unculturable microbes such
as biotrophic fungi, since special (selective) growth substrates may not be available
for microorganisms requiring particular nutrients. Additionally, at subspecific
taxonomic levels, differences are more and more reduced numerically and
taxonomically, becoming so inconsistent that traditional techniques may be
completely ineffective (Martin, James & Lévesque, 2000; McCartney et al., 2003).
Similarly, the use in the field of traditional techniques for scoring of symptoms for
recognition of a disease, especially for identifying early symptoms, is particularly
difficult and always retains a subjective component (Moricca et al., 1998).
During the last 20 years, with the advance of comprehensive genetic and
genomic data on many plant pathogens and other microbes, the use of natural
genetic variation present in DNA has been exploited to provide molecular genetic
markers capable of identifying, differentiating and characterizing phytopathogens
(Schaad & Frederick, 2002; Rementeria et al., 2004). This great advance in plant
pathogen diagnostics is driven by the need of accelerating identification and
differentiation techniques at the level of sub-species, variety or pathovar, specialized
forms and races, beyond traditional techniques based on morphological and
physiological differences. The identification based on cultivation and on
conventional traits is slow, and might under-represent microorganisms requiring
particular growth conditions or those occupying special niches in communities, for
example the endophytes harboured in the tissues of higher plants (Carroll, 1995;
Moricca, Hantula & Müller, 2004).
Molecular approaches can provide a detailed genetic sub-classification which is
complementary and additional to traditional techniques. Once molecular genetic
markers are comprehensively established, the need to isolate and culture a pathogen
is diminished, allowing its determination directly from host tissues or other
environmental matrices. Typically, the level of information provided by these
analyses is determined by the phytopathologist need for diagnostic specificity, and
for convenience of being undertaken within particular laboratory facilities.
Presently, the genomes of some 50 fungi have been sequenced or are being
sequenced (http://fungal.genome.duke.edu/). These are principally Ascomycetes and
their anamorphs. A number of biotechnologically important fungi and economically
important phytopathogens, including Magnaporthe grisea, Fusarium graminearum,
Fusarium verticillioides, Trichoderma reesei, Botrytis cinerea, Sclerotinia
sclerotiorum, Stagonospora nodorum, Ustilago maydis and Phanerochaete
chrysosporium are presently under study. In addition, partial genomic sequence data
DNA METHODS FOR PLANT DISEASE MANAGEMENT 335
are available for several hundred fungal species, among which numerous plant
pathogens and other agriculturally important fungi are included.
The information from these complete or partial genomes is central to the creation
of new tools for molecular diagnostic analysis of these taxa, yet we are only now
beginning to appreciate the tremendous genetic diversity among the species and
strains of this heterogeneous assemblage of microorganisms. The substantial
differences in genetic content characterizing even the smallest fungal entities today,
detectable by combination of highly discriminatory PCR-based amplification and
DNA fingerprinting or genotyping technologies, pose a formidable challenge to the
accurate design of specific molecular detection assays (Wu et al., 2006). In
addition, for in situ studies, the plant host and numerous other soil and plant-
associated organisms must not interfere with the accurate and sensitive detection of
low numbers of the target cells at early stages of infection, or in bulk assays of
combined samples to determine a pathogen load.
This chapter, after analysing the main weaknesses and drawbacks inherent with
conventional diagnostic assays, focuses on the most popular and traditional
molecular DNA markers. It then reviews some innovative DNA fingerprinting
approaches or combined analitical devices that could provide detailed molecular
identification and genotyping of microorganisms. The objective is not to provide a
comprehensive list of phytopathogens or other microorganisms that are detected
and/or characterized with these techniques. The purpose is rather to focus on the
principle of each method, in order to demonstrate how the new molecular
approaches, whether developed in the field of plant pathology, medical science,
industry, or environmental microbiology, can be successfully applied to solve
practical problems in different biological systems. We thus aim at demonstrating
how the new technologies can overcome problems inherent with conventional
identification of plant pathogens and provide, in parallel, characters useful to
support and extend more classical taxonomic relationships as well as useful
informations for disease risk assessment (Gilles et al., 2000).
2. POLYMORPHISM DETECTION METHODOLOGIES

In plant disease research, it is of crucial importance to monitor airborne inoculum as
well as to detect early pathogen infections, possibly at a pre-symptomatic stage of
the disease, in order to determine the infection reservoirs supplying such events and
be able to timely implement adequate control strategies for preventing epidemic
spread of diseases. This demands rapid, accurate and sensitive methods for fungal
typing. Methods are also required to analyse central aspects of pathogens biology,
like the mode of reproduction (sexual vs. asexual reproduction, or a combination of
both); to analyse the demographic history (gene flow, migration); to investigate the
virulence types; to explore the mating type system, or specialised recombinational
events (e.g. parasexual phenomena) possibly affecting pathogen variability; to
analyse antifungal susceptibility patterns and investigate host resistance; to study
yield-loss relationships; to implement quarantine regulations; etc. (McCartney et al.,

2003; Ward et al., 2004).
Several different genotyping methods are widely used for strain identification
of agriculturally, medically or industrially important fungi. Most of the modern
molecular typing methods are comparable to those in use already for viruses and
bacteria (Martin, James & Lévesque, 2000; Rementeria et al., 2004). However, the
presence of mitochondria and repeated nuclear genomic DNA sequences increases
the number of applicable methods. The single most important technology
incorporated into virtually all mutation-detection methods is the polymerase chain
reaction (PCR), which permits exponential amplification of defined regions of the
genome (Yu & Pauls, 1993). The majority of genotyping methods used for fungi are
based on the amplification of chromosomal or mitochondrial DNA targets by PCR
(Martin, James & Lévesque, 2000), with subsequent analysis of the DNA fragments
either by electrophoretic separation and sizing (either with or without restriction by
endonuclease enzymes) (Mitchelson, 2003), or by specific selective hybridization
analysis (Lockhart et al., 2005). The quality of the reagents used prior to, during and
after the amplification reaction is critical to the quality of data generated. Thus the
optimization of many aspects of DNA purification (Gang & Weber, 1995; Langrell,
2005; Guo et al., 2005) and subsequent PCR reaction are required to obtain the best
yields, the best specificity and the highest sensitivity (Yu & Pauls, 1993; Zinger
et al., 2007).
In principle, each physical and chemical component of the assay can be
considered as a variable factor that may be modified to effect a potential
improvement in the quality of the amplification reaction. Increasingly computerized
software is used in the comparison of DNA band profiles, even for lower throughput
analyses, because of the ease of comparison and information retrieval (Soll, 2000).
The use of advanced microarray-based analysis is also increasing because of its high
throughput and simultaneous detection of numerous genetic markers (Chen, 2006;
Palacios et al., 2007). However, the application of high throughput microarray
technology is mostly limited to research laboratories, institutes and larger hospital
laboratories (Lievens et al., 2003; Huang et al., 2006), where advanced
instrumentation needed for microarray analysis are more likely to be found.
The food industry is increasingly demanding typing methods that identify food
contaminants, especially mycotoxin-producing fungi, as well as typing for the
selection of the best fungal strains in order to maintain both the security and quality
of food. The predominant food spoilage fungi are species of Fusarium, Penicillium
and Aspergillus. Most species in these genera are difficult to determine by
conventional techniques, yet their detection, or the detection of the mycotoxins they
produce, or of the genes involved in the biosynthesis of mycotoxins, are crucial for
food safety and trade. Much development has occured in this discipline and a
number of accurate PCR-based assays have been developed (Morgan, 1995;
Edwards, O’Callaghan & Dobson, 2002). This is also the case for traditional wine
and food industries where the selection and characterization of indigenous strains
and species has become an important objective for the production of high-quality
certified products (DiMaria et al., 2002; Valero et al., 2007; Pisano et al., 2007).
Although this review is intended to examine developments in phytopathology,
new developments in technologies employed for the study of fungi and bacteria
have broad relevance to molecular mycology and several reports are included.
Bringing these analyses out of the laboratory and into the workplace (production
facility, medical clinic, remote farm or field-site) is envisioned by many applied
biologists. Portable real-time PCR for on-site (cargo, maritime, mail inspection at
ports of entry), molecular-based diagnosis of crop diseases has become an absolute
priority in crop biosecurity and phytosanitation in the USA, following the deliberate
anthrax release of October 2001 (Shaad et al., 2003). The recent reports of
integrated miniaturized transportable devices capable of PCR amplification
(Consolandi et al., 2006) or of combined PCR and DNA fragment analysis (Liu
et al., 2007b) are other welcome developments towards achieving these goals.
2.1. Genetic Fingerprinting by Fragment Sizing

One of the most frequently applied methods in phytopathological identification is
DNA fragment sizing. Electrophoretic migration methods are used to distinguish the
PCR-amplified fragments generated from a small number of polymorphic genomic
loci, many of which typically provide similar discriminatory power for the
identification of strains and pathovars (Zhong et al., 2002; Abd-Elsalam et al, 2004).
2.1.1. Ribotyping
Polymorphism analysis of the ribosomal gene complex is commonly used for
differentiation of phytopathogenic fungi (White et al., 1990), because of the ease of
amplifying the multicopy ribosomal gene regions in many different fungi by
application of ‘universal’ PCR primers to highly conserved ribosomal gene repeats
and by the definition of characteristic polymorphisms and RFLP for particular
fungal strains or species. In phytopathology, the molecular analysis tools generally
applied for ‘ribotyping’ identification tend to be low throughput techniques.
Ribotyping is a generic term, applied to RFLP analysis of the internal transcribed
spacer (ITS) regions located between the small and large subunits and encompassing
the 5S rRNA gene (Moricca et al., 1998; Jespersen et al., 2000; Kasuga &
Mitchelson, 2000; Cadez et al., 2002; Feau et al., 2005) or to the non-transcribed
intergenic spacer between the large subunit and the following small subunit
(Moricca, Ragazzi & Mitchelson, 1999; Jurado et al., 2006; Maxwell et al., 2005),
or even to RFLP of the most part or all of the ribosomal gene repeat (Pramateftaki
et al., 2000).
Ribotyping frequently involves a series of directed techniques, such as analysis
in a ‘specific’ mode in which potential polymorphism at defined loci is examined by
DNA sequencing or by PCR-RFLP analysis (Cocolin et al., 2006), or it is
undertaken in a ‘scanning mode’ looking for polymorphism at undefined loci within
the amplified fragment. Species specific ribosomal gene amplification can be

achieved by careful definition of selective primers, providing a sensitive assay for
the detection of fungi in mixed cellular environments. For example, Feau and
colleagues (2005) created specific assays for the poplar pathogenic fungi Septoria
musiva, S. populicola and S. populi based on variants of the internal transcribed
spacer (ITS), without detectable amplification of products from 12 other Septoria
species or other fungal species collected from poplar trees, thus permitting selective
detection of these fungal species on poplars.
The phylogeny of fungi may be computed from ribotyping data via a discrete
character matrix of the presence or absence of fragments, using sequence parsimony
programs such as the PHYLIP package. This often results in complete accord with
the phylogeny derived from ribosomal gene sequence analysis. Examining sufficient
numbers of different polymorphic loci across the ribosomal gene complex by “long-
range ribotyping” may provide accuracy equal to DNA sequence analysis of the
entire locus. Although molecular identification and phylogenetic studies in many
fungi still rely largely on rDNA sequence polymorphisms, some fungal species
remain indistinguishable within a given genus, even when a large region of the
ribosomal intergenic spacer or several different spacer regions between the various
rRNA genes are examined (Pramateftaki et al., 2000; Jurado et al., 2006). Thus,
parallel or complementary detection of polymorphisms in the multicopy
mitochondrial genome (Mabru et al, 2004; Gómez-Alpizar, Carbone & Ristaino,
2007) or random genome scanning methods such as AFLP genotyping and RAPD
fingerprinting, are often used for identification of species and/or to detect a more
complete picture of genetic diversity (Cadez et al., 2002; Ware et al., 2007).
Increasingly, other gene sequences are validated as polymorphic markers to discern
species. For example in the case for Fusaria, which include numerous species
involved in both plant and animal pathologies, Hatsch and colleagues (2004) used
the genes encoding cellobiohydrolase-C and topoisomerase II, in addition to
ribotyping, as targets for phylogenetic analysis and identification.
2.2. Ribosomal RNA Detection

Methods capable of detecting rare or low numbers of microorganisms within mixed
samples are of great importance for the early detection of the first infection foci, in
preventing post-harvest food losses as well as in the food industry for eliminating
contamination and spoilage. Typically, the abundant rRNA transcripts from
multicopy ribosomal genes have been used as they can be readily detected, as well as
providing high sensitivity for identifying the presence of low numbers of infection
cells (Siripong et al., 2006). Recently, Neubauer and colleagues (Leskelä et al., 2005;
Huhtamellas et al., 2007) developed sensitive RNA-based sandwich hybridisation
assays requiring only effective cell lysis procedures to release rRNA and in vitro T7
transcription for rRNA amplification and labeling. The assay could demonstrate the
detection of gram-negative Legionella cells in mixed environmental samples, as well
as gram-positive Lactobacillus and Pediococcus cells despite the brewery slurries
containing as many as 109 yeast cells ⋅ ml-1. The specificity for such direct detection
assays depends upon careful optimization of the hybridization probe sequences and
the hybridization conditions, to ensure equal sensitivity at each locus. The
sensitivity of RNA detection assays is increased by an efficient quantative T7
polymerase RNA amplification step, to increase the amount of target within the
sample.
Several variant methods have been developed for universal direct linear
amplification of total RNA (Gao et al., 2006; Moreno-Paz & Parro, 2006). Short
fusion primers (with 6-9 random nucleotides attached to the T7 promoter) were used
for the first-strand (antisense) synthesis. The shortest primer (6 random nucleotides)
provided the highest yields, as well as being the most accurate in terms of
representative amplification of RNA species, reflecting their original abundance.
The shorter random primers might be expected to hybridize less specifically, yet the
number of initiations would be significantly greater for any target RNA than longer
primers. The most representative amplification of original RNA abundances was
obtained reproducibly using higher amounts of starting template, from 50 - 100 ng
of total RNA. These techniques could be used to amplify total RNA from mixed
environmental samples for global gene expression analysis (Parro, Moreno-Paz &
Gonzalez-Toril, 2007). Environments where such approaches are beneficial are
those where small numbers of target cells are obtainable or where the cells are
normally present at low density or where the composition of the culture media or
environment (plant tissue) severely limits the RNA yield. An approach such as this
could be useful for analysis of early stages of infection of fungal pathogens in the
host plant (or in any other organism), where the number of pathogen cells is limiting
and the responses of both the host and the pathogen might be analysed (Liu &
Slininger, 2007).
2.3. Random Genetic Loci

2.3.1. RAPD Fingerprinting
Random amplified polymorphic DNA (RAPD) fingerprinting utilizes short (9-11
bp) primers which represent abundant genomic sequence motifs (Williams et al.,
1990; Chen et al., 2007). RAPD fingerprinting scans entire genomes in a non-
directed manner, principally detecting uni-dominant single nucleotide DNA
polymorphism events of low frequency that prevent the annealing of the primer at
an amplification locus, but may also by coincidence detect other co-dominant
sources of polymorphism, such as deletions, insertions and polymorphic simple
repeat loci. RAPD markers are robust genetically and have been used widely for
genetic mapping in plants (Milczarski et al., 2007; Grimmer et al., 2007) and other
organisms (Cadez et al., 2002; Abd-Elsalam et al., 2004).
Classically identified fungal isolates may still be RAPD fingerprinted without
any prior molecular genetic information for relatively low cost (Müller, Germani &
Van der Sand, 2005). However, one limitation of the technique is that all organisms,
from bacteria to mammals, are able to generate RAPD fingerprints, thus pure fungal
cultures or highly purified DNA sources are required for such analysis, as
contamination of the target genomic DNA may invalidate the RAPD fingerprinting
(Lockhart et al., 2005). This poses a strong limitation to plant disease diagnosis,
where identification of the target pathogens from infected tissues or other
environmental samples is needed.
Plant-symbiotic fungi, such as mycorrhizal species, are usually identified on
the basis of the morphological characters shown by fruit bodies, spores, vegetative
mycelia or symbiotic structures. RAPD fingerprinting may be undertaken using
genomic DNA extracted from pure cultures of symbiotic or pathogenic fungi
important in agriculture (Müller, Germani & Van der Sand, 2005; Chen et al., 2007;
Ware et al., 2007) and forestry (Bourassa, Bernier & Hamelin, 2005) where the
paucity of other well defined characteristic genetic loci, or the invariety of readily
tested ribosomal genes, was previously an impetus to the development of RAPD
fingerprint analyses. When RAPD fingerprinting is used with pure fungal isolates
the high information content may be used for detailed and precise identification of
genotypes, sufficient for large scale medical or epidemiological classification of
fungal species important to human health such as Candida sp., Aspergillus
fumigatus and other anthropophilic dermatophytes (Lockhart et al., 2005; Song et
al., 2006). RAPD fingerprinting has been used widely for the identification of
strains of fungi important to quality in food production (Cocolin et al., 2006; Chen
et al., 2007; Walczak et al., 2007) and for fungi important in food spoilage
(Lopandic et al., 2006).
Within both agricultural and medicinal settings, RAPD fingerprinting of fungi
is often applied in conjunction with classical methods of fungal classification such
as nutritional requirements, colony morphology and microscopic detail of mycelia,
spores and sporogenous structures, to provide a molecular indicator which can be
assimilated into a multi-faceted database for taxonomy and future reference (Ware
et al., 2007). More extensive application of arbitrary sequence RAPD primers can
be made for the detailed examination of genomic libraries for positional cloning
(Chen et al., 2007) and using suitable fungal populations for the generation of high
density genetic linkage maps (Kema et al., 2002; Muraguchi et al., 2003).
2.3.2. AFLPs
Amplified fragment length polymorphism (AFLP) mapping is an informative high
throughput method which assesses numerous polymorphic loci through selective
restriction digestion and linked PCR amplification (Vos et al., 1995). Because of its
reproducibility, the method has become very widely used for fungal genotyping
(Bensch & Akesson, 2005; Ware et al., 2007), for definition of phenetic similarities
between industrial yeasts (Azumi & Goto-Yamamoto, 2001) and for the generation
of detailed high density genetic maps in segregating populations (Kema et al.,
2002).
Comparison amongst three methods commonly used for molecular genotyping
(AFLP, RAPD and PCR-RFLP) by Abd-Elsalam and colleagues (2004) showed that
all three types of markers were roughly equally informative, yet the assays differed
in the number of polymorphic bands detected and AFLP fingerprinting was found to
be more differentiating than other techniques. Similar observations have been made
with typing studies in plants and other fungi (Bensch & Akesson, 2005), suggesting
that AFLPs are a preferable marker system particularly for higher density mapping.
Indeed, modifications of the protocol for AFLP mapping, such that extracted
mRNA, rather than genomic DNA, is templated, makes it possible also the direct
measurement of the variation in the expression of multiple genes (cDNA-AFLP).
Yet other modification of the AFLP protocols by employing methylated-DNA
sensitive and insensitive restriction enzyme isoschizomers, allow the distribution of
genomic DNA methylation to be assessed.
2.4. STR Fragment Fingerprinting

More than a decade ago Meyer and colleagues (1995) advocated the use of
microsatellite or STR fingerprinting in fungal identification. Here, pairs of long,
locus specific PCR primers are used to generate genomic fragments of hypervariable
size. Mixed DNA samples of host and pathogen may be used, provided that
stringent thermal cycling conditions are maintained (Weising, Atkinson & Gardner,
1995; Walczak et al., 2007). Microsatellite genotyping continues to play an
important analytical role, particularly in the differentiation of phytopathogenic fungi
(Kim et al., 2000; Mohali, Burgess & Wingfield, 2005), industrial fungal strains
(González-Techera et al., 2001; Legras et al., 2005) and medically important species
(Foulet et al., 2005). The utility of STR genotyping in other eukaryotes and
mammals has resulted in the development of equipment and methods for high
resolution sizing and automated fragment scoring (see: this volume, page 352).
Application of the general tools available for STR genotyping can benefit analysis
of fungi by this marker system.
2.4.1. DNA Shape Analyses

A number of sensitive ‘locus scanning’ methods that employ a physical alteration in
the shape and electrophoretic mobility of polymorphic DNA can distinguish
abundant single nucleotide polymorphism (SNP) variants that would not be
amenable to RFLP analysis as they occur outside any restriction site. These methods
thus eliminate the need for costly restriction steps. One method is single-strand
conformation polymorphism (SSCP) analysis, which distinguishes between shapes
assumed by fold-back ssDNA fragments. SSCP has been widely applied for analysis
of polymorphism in ribosomal gene fragments of fungi important in agriculture and
forestry (Moricca et al., 2000; Bourassa, Bernier & Hamelin, 2005) or to human
health (McIlhatton et al., 2002; Gil-Lamaignere et al., 2003). Asymmetric PCR-
SSCP (Scott et al., 1998) provides stability to the conformation assumed by the
single DNA strands by eliminating the possibility of duplex renaturation and thus
increases the ease of detecting SSCP.
Another sensitive method is the heteroduplex polymorphism assay (HPA) (Gil-
Lamaignere et al., 2003) which detects mismatches between two annealed gene
alleles by virtue of retarded mobility of the mismatched duplex molecules compared
to fully duplex homopolymers. Heteroduplex analysis on high resolution acrylamide
gels can efficiently detect sequence polymorphism varying as little as a single base
pair and also discern differences between heteroduplex and homoduplexes, a
prerequisite for detection of co-dominant markers. Such simple high resolution
techniques can be used to convert sequenced fungal genes into co-dominant PCR-
based molecular markers for genetic mapping studies and chromosomal walking
strategies, as well as for the detection of mutations in particular genes or for the
identification of pathotypes. An assay with increased sensitivity over conventional
HPA, employing denaturing gradient gel electrophoresis (DGGE) (Masoud et al.,
2004; Noll & Collins, 1987), has been frequently used to enhance the sensitivity of
detection of ribosomal gene polymorphisms to distinguish between fungi (De Souza
et al., 2004; Van Elsas et al., 2000; Yergeau et al., 2005), again avoiding the need
for polymorphisms to be restrictable as in PCR-RFLP ribotyping. SSCP, DGGE and
HPA analyses have also been applied to RAPD amplified genomic fragments for
fungal strain differentiation by the detection of additional internal nucleotide
polymorphism (Gil-Lamaignere et al., 2003; Plachý, Hamal & Raclavský, 2005).
However, the complexity of interpretation may be a limit to this approach at present.
3. COMBINED ANALYSES
Combinations of several different genotyping procedures are frequently used for
low throughput phytopathological bioassays to develop sufficient resolving power,
whilst also utilizing information accumulated in databases from earlier studies.
Typically, one, out of the several methods for genotyping at distributed genomic
loci (STR, AFLP and RAPD), is used in conjunction with analysis of defined
genetic marker loci. Defined loci include ribotyping of the rDNA complex and
polymorphism analysis of other genes (Bäumler et al., 2003; Zhong et al., 2002).
The high conservation of the nuclear ribosomal genes and their associated
intergenic regions and the mitochondrial genome has resulted in a wealth of
characteristic polymorphisms which aid comparison and identification of fungal
isolates. Phylogenic information for each of these well characterized loci is readily
available in databases. For example, Coton and colleagues (2006) employed size
analysis of SDS-PAGE fractionated proteins as well as physiological growth tests to
distinguish strains of the yeast Zymomonas mobilis. These authors then confirmed
the identification with a series of molecular genetic analyses which included
genotyping at both RAPDs and repetitive extragenic palindromic-PCR loci, as well
as sequence analysis of 16-23S intergenic repeat loci and fragments of the HSP60
and gyrB genes. Importantly, the discriminatory power of different low level
techniques can be employed in combined analyses to provide higher levels of

identification of species and pathotypes.
3.1. Genetic Mapping

The efficient genetic mapping of an organism depends on the availability of a
marker system in which the polymorphic marker loci are distributed randomly
throughout the genome at high density, and which can be assayed with high
efficiency and low cost. Whilst different classes of genetic markers provide one or
another desirable characteristic to aid mapping, none are ideal in all. The sequencing
of complete genomes provides locus details of the abundant single nucleotide
polymorphisms that occur in all eukaryotes, but most assays of SNPs are relatively
expensive as specific amplification primers are needed for each locus, including
microarray hybridization SNP assays (see: this chapter, page 346). Most fungi of
interest have yet to be genome sequenced, and few would currently justify the
development of high density SNP mapping resources, hence other less abundant but
inexpensive markers are still frequently used.
A variety of medium abundance polymorphic markers have been employed as
mapping tools to generate detailed genetic linkage maps of agriculturally and
industrially important fungi (Zhong et al., 2002). Such detailed mapping studies are
a prelude to identification of the loci of important genetic traits. For example, Kema
and colleagues (2002) used a combination of AFLP and RAPD markers to generate
a genetic linkage map in crosses of two different strains of the leaf blotch pathogen
of wheat Mycosphaerella graminicola. The 17 or 18 chromosomes of M.
graminicola were confirmed by pulsed-field gel analysis. Molecular markers
isolated during the mapping were then employed during bulked segregant analysis
(Michelmore, Paran & Kesseli, 1991) to identify additional tightly linked markers to
genes regulating mating type and avirulence traits, of which the latter is important in
determining the resistance or susceptibility in the host plant.
DNA hybridization requires the co-annealing of DNA strands of full or close
sequence complementarities, and thus can potentially identify the genomic locus of
origin of RAPD fragments (Muraguchi et al., 2003). Additional tests have also been
used for markers linked or close to phenotypic loci. These include the use of RAPD
fragments in conjunction with DNA sequencing of clones or isolated RAPD
fragments to allow the construction of species specific PCR-primers based on
diagnostic RAPD bands, a process more frequently described as ‘sequenced
characterised amplified random’ DNA or SCAR markers (Robène-Soustrade et al.,
2006), Recently, a medium density genotyping methodology based on multiplex
AFLP analysis combined with high throughput DNA sequencing called SNPWave
was reported (Van Eijk et al., 2004), which assays subsets of restrictable SNP
polymorphisms across the genome, using an automated capillary electrophoresis
(CE) sequencer for efficient signal analysis. SNPWave uses highly multiplexed
ligation followed by amplification of up to 20 ligated probes in a single PCR. The
multiplexing level of the ligation reaction can be varied and then selective
amplification is achieved using AFLP technology. The reaction products are
analysed by size on a CE sequencer with multiple fluorescence labels for the
different subsets of ligation products, and requires only short run times for analysis
of the short fragments.
Many fungi have unusual, cryptic, complex or poorly understood reproduction
cycles, and yet detailed genetic mapping can aid in the molecular genetic analysis of
the organism. The important human pathogen Candida albicans is an example of a
diploid yeast with predominantly clonal reproduction, for which the complete sexual
cycle is not known. Although mating can occur under some circumstances, it is
difficult to create segregating populations of C. albicans for the genetic analysis of
different physiological traits. It is known that genome rearrangement and
heterogeneous genetic variation in C. albicans are quite common. A high density
SNP-based genetic map was created with markers located at an average spacing of
~100 kb, to facilitate analysis of genome rearrangements (Forche et al., 2004). A
microarray format assay for 23 SNP loci residing on chromosomes 5, 6, and 7 was
used to examine different C. albicans strains that had undergone mitotic
recombination at the GAL1 locus, during infection in mice (Forche, May & Magee,
2005). These were found to have detectable loss of heterozygosity (LOH), with such
mitotic recombination events occurring independently at loci distributed across the
genome. Subsequently, a major repeat sequence (MRS) was found to effect
karyotypic variation in C. albicans (Lephart & Magee, 2006). The MRS affects
karyotypic variation by expanding and contracting the number of internal repeats
and by serving as a hotspot for chromosome translocation, potentially providing a
mechanism for generating genetic diversity that aid a commensal lifestyle in
different environments. The detailed analyses required here to investigate the
molecular genetic basis of the pathogenicity and drug restance of C. albicans may
be taken as a paradigm of the studies that may be required for other important fungi.
3.2. PFGE Karyotyping of Fungi for Pathovar Identification

Pulsed-field gel electrophoresis (PFGE) of entire chromosome length DNA
molecules is a technique that provides size separation, chromosome length
estimation and karyotype analysis (Beadle et al., 2003). Many fungi possess
significant intraspecific variation in both chromosome number and size, making it
difficult to establish a standard “reference” karyotype for many species. PFGE is
also used for chromosomal assignment of linkage group markers (Muraguchi et al.,
2003), as well as for identification of very large RFLP generated by digestion of
chromosomes by rare-cutting restriction endonucleases. PFGE-RFLP and karyotype
determination in conjunction with hybridization of gene probes is used widely for
the identification of fungal strains and pathovars important to medical science
(Alemeida et al., 2007; Lee et al., 2007) or plant pathology (e.g. the Ascomycete
Cochliobolus sativus, an important pathogen of cereals) and food science (Zhong
et al., 2002). Although PFGE analysis demands pure fungal cultures, it may also
provide a way to develop a molecular linkage map in the absence of a formal

genetic system. It is significantly more rapid than parasexual analysis for the
identification of linkage relationships among genetic markers. Repeated genes and
gene families such as ribosomal genes may be used in the physical mapping of
chromosomal karyotypes (Saracli et al., 2006; Lee et al., 2007) by PFGE
hybridization mapping of linkage markers (Zhong et al., 2002) and linkage of other
polymorphic markers (Forche, May & Magee, 2005; O’Sullivan et al., 1998), or
isolated gene fragments can provide detailed physical mapping data, complementary
to the linkage mapping described above and locating markers and genes to the
chromosomes.
In addition, PFGE can provide resources for cloning for genomics projects, as
chromosome-specific DNA can be recovered from pulsed-field gels to prepare
chromosome-specific libraries. The building of physical maps can be aided by
probing electrophoretic karyotypes with anonymous pieces of DNA from bacterial
artificial chromosome (BAC) contigs or from whole genome sequencing projects
(Beadle et al., 2003; Van Het Hoog, 2007), or with genetically mapped markers such
as RAPDs, AFLPs and STRs (Muraguchi et al., 2003). Although ultra-high
throughput sequencing technology (see: this chapter, page 349) can sequence entire
genomes of microorganisms in weeks, the cost for full-scale genome sequencing of
many plant fungal pathogens and economically important fungi will be prohibitive,
and small-scale projects involving the sequencing of genome regions and genes of
interest will continue (Lasker, 2006).
4. GENE AND GENOMIC ANALYSIS

4.1.Quantitative Real-Time PCR
Quantitative real-time PCR (qRT-PCR) is a method of quantitative assessment of
the abundance of a test genomic template relative to the abundance of a control
template based on the extrapolation of the efficiency of product amplification within
a PCR reaction (Lay & Wittwer, 1997). Because of the exponential nature of the
qRT-PCR reaction, this technique can accurately assess the relative presence of
templates differing by several orders of magnitude. This has led to its adoption for
accurate quantitation of low abundance analytes such as for estimation of the
relative levels of differentially expressed gene products within cells (Perrin et al.,
2007) and for the quantitation of the genomes of fungi and other microorganisms
present in plant host tissues (Schaad & Frederick, 2002; Leisova et al., 2006), in the
environment (Castrillo et al., 2007) and in complex mixtures such as food matrices
(Bohaychuk et al., 2007).
Waalwijk and colleagues (2004) used a sensitive real-time TaqMan® assay for
four Fusarium species and Microdochium nivale var. majus found on wheat.
Inclusion of an internal control PCR product combined with serial dilution of DNA
samples purifed from wheat tissues allowed accurate determination of the fungal
load. Similar assays have been developed for the mycorrhizal fungus Glomus
mosseae (Böhm et al., 1999), important plant pathogenic fungi such as Puccinia
recondita, Puccinia striiformis, Sclerotinia sclerotiorum and Stagonospora

nodorum (Fraaije et al., 2001; Fraaije, Lovell & Baldwin, 2002), the Oomycetes
Phytophthora infestans and Phytophthora citricola (Böhm et al., 1999), as well as
for phytopathogenic bacteria (Schaad et al., 1999), viruses (Boonham et al., 2000)
and viroids (Mumford, Walsh & Boonham, 2000). Assays such as TaqMan can
provide rapid and accurate information on the biomass and infectivity of the
pathogen, allowing disease management decisions concerning fungicidal treatments
or the use of resistant cultivars or other control strategies to be made more timely
and effectively.
The sensitivity and specificity of TaqMan real-time PCR analysis can also be
enhanced by the use of detection systems which involves competitive release of
FRET fluorescent dye upon amplification of a particular allelic product (Kuimelis et
al., 1997). Other useful systems include molecular beacons (Tyagi & Kramer, 1996)
which recognize and undergo a spontaneous fluorogenic conformational change
(reporting signal) when they hybridize to their targets, and the unimolecular
Scorpions® system (Thelwell et al., 2000) which do not involve enzymatic release of
a fluorescent probe product. Interestingly, a direct comparison between Scorpions,
TaqMan and molecular beacons suggests that Scorpions performed better,
particularly during fast cycling conditions.
4.2. Microarrays for SNP Genotyping

DNA arrays of immobilized DNA or oligonucleotides are fabricated on glass or
silicon substrates, for which labeled probes are used to determine selective
complementary binding, providing the capacity for a genome-wide assay of gene
expression or DNA polymorphisms (Käller, Lundeberg & Ahmadian, 2007).
Sample DNA is amplified by PCR and a fluorescent label is inserted before it is
hybridized to the microarray.
The growth in fungal genomic sequence data (see above) has resulted in
applications for both low density analysis of important specific genes characterizing
a phenotype (Ben-Ari et al., 2005; Sotto et al., 2007) and multiple gene
polymorphisms (Huang et al., 2006), or “genotyping array systems” that permit the
simultaneous genotyping of several thousands of polymorphic DNA loci, spotted at
high density (Lievens et al., 2005; Syvänen, 2005).
Recently Queitsch and colleagues (Salathia et al., 2007) demonstrated the use
of indel arrays, where 70-mer oligonucleotides unique to two distinct genotypes
(Col and Ler) of Arabidopsis thaliana were used to efficiently map the inheritance
of recessive mutations in genomic regions of RIL populations, as well as for
mapping mixed genetic backgrounds in lines other than the target genotypes. The
authors note the cost effectiveness of their array system and the expected ease of
transfer of the technology to non-model organisms. Such array techniques could be
beneficially applied to aid high throughput molecular genetic analysis of fungi
which differ markedly in their genotype and phenotypes.
4.2.1. Microarray Chip-Based Automated Analysers

Knowledge of short repeat-sequence genomic regions of medically important fungi
in combination with the need to speed their identification, has stimulated
development of automated analysis systems that employ locus-specific template
amplification and microarray-based detection of amplified product signals (Healy
et al., 2004; Wise et al., 2007). These signal data are then linked to a database of
characteristic patterns. The systems employ a high level of similarity of patterns to
those of co-analysed reference strains and database stored typings, and provide a
rapid identification of fungal isolates to species level, and strain level in some cases.
Studies with more than 100 clinical isolates of six Candida species (Wise et al.,
2007), previously identified by classical morphological and biochemical tests as
well as ITS sequencing, showed a final level of accuracy of 99% of the automated
system, superior to the accuracy of the classical typing methods. Application of the
system to the identification of clinical Aspergillus species and isolates resulted in a
similarly high accuracy of identification (Healy et al., 2004). The development of
other automated microarray-based genotyping analysis systems and software may
be anticipated for other important fungal and bacterial species (Zhu et al., 2007a;
2007b).
4.2.2. Microarray Analysis of Gene Expression

Gene expression analysis provides significant insight to understand regulatory
mechanisms occurring in cells, yet only recently has the acquisition and
reproduction of data quality, as well as data confirmation and verification, been
validated by proper quality controlled analysis of a common expressed mRNA set
across different microarray assay platforms (Patterson et al., 2006; Canales et al.,
2006). These studies validated and verified use of both one-colour and two-colour
detection systems, providing that adequate internal and external quality controls
signals were included, as well as providing a means to compare gene expression
data derived from different experiments.
Liu and Slininger (2007) also recently employed external quality controls for
comparison across different microarray expression platforms and demonstrated that
valid mRNA detection for yeast (S. cerevisiae) and Pseudomonas fluorescens arrays
ranged over 3 orders from 10 pg to 7000 pg, whilst qRT-PCR assay for randomly
selected yeast genes ranged over 4 orders from 100 fg to 1000 pg, providing
evidence of the higher sensitivity of qRT-PCR due to the flexibility of the
amplification steps. Within an overlapping detection range from 10 to 1000 pg the
quantitative estimations of mRNA abundance by these two methods were very
similar. Interestingly, the presence of sol-gel optical multilayers involving stacks of
low- and high-index layers deposited on the surface of glass slides can increase the
fluorescence of DNA microarrays and enhance the detection of fluorescent targets
(Fouqué et al., 2007). Using comparisons between microarrays on normal glass and
sol-gel layered arrays, the detection of weakly expressed yeast genes was found to
be more sensitive. Such advanced arrays could be considered as promising tools for
the analysis of small biological samples, or rare molecules in mixed population
samples. It could then help in the identification of the mycoflora (e.g.
mycoparasites, opportunistic fungi, etc.) associated to pathogen infections in the
field, as well as in the detection of rare microbes in complex microbial communities
(soil microorganisms, endophytic fungi and bacteria).
Gene expression studies are being undertaken for numerous fungal species to
gain understanding of the genes that are induced or repressed by environmental
factors or nutritional conditions and which affect the pathogenicity, development or
beneficial utilization of a given fungus.
Quantitative real-time PCR (RT-qPCR) analysis (Liu & Slininger, 2007) and
targeted gene expression microarrays (Rajashekar et al., 2007) have been used to
analyse the activity of limited numbers of selected genes. Differential gene
expression microarray analysis is also used extensively to examine the molecular
processes occurring during host-fungal interactions either through genes induced in
both the host plant (Keon et al., 2007) and fungal cells during symbiosis
(Rajashekar et al., 2007) or examine pathogenic interactions between the fungus and
its host (Viaud et al., 2003; Johannesson et al., 2006) or fungal developmental
programs (e.g. conidiogenesis) (Kasuga et al., 2005). Expression analysis is also
being used to identify and exploit potentially useful genes and pathways for the
catalysis of reactions that might be used industrially, for example novel genes from
ligninolytic wood-rotting fungi, which may be able to degrade hazardous chemicals
(Doddapaneni & Yadav, 2005). One major limitation in microarray analysis is the
need for detailed genome sequence information for construction of arrayed probes
and target amplification probes. This problem is acute for those areas of microbial
research where the cost of sequencing and creating expression profiling microarrays
for minor, yet important species, precludes their development.
Recently, a multi-locus method, called iGentifier, capable of assessing the
differential gene expression of entire unknown/unsequenced transcriptomes of
organisms was reported (Fischer et al., 2007). Its use was demonstrated by profiling
the powdery mildew fungus Blumeria graminis f. sp. hordei. The technique is a
combination of several different elements of fragment display (Differential Display
or RMDD) and expressed tag sequencing (SAGE, MPSS). These techniques are
amenable to high throughput analysis by using conventional array capillary
electrophoresis equipment (Reinartz et al., 2002) or on microbead arrays (Brenner et
al., 2000). Although expression analysis is not currently used for fungal taxonomy,
future use of microarrays for genome-wide analysis of gene expression may
stimulate the development of diagnostic applications, as well as the identification of
novel gene targets for the control of fungi. One example is the expression analysis
of Paracoccidioides brasiliensis (Nunes et al., 2005), the fungus responsible for
paracoccidioidomycosis in humans, during mycelium-to-yeast transition. A gene
encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD) was found highly
overexpressed during differentiation. A specific inhibitor of 4-HPPD activity was
able to inhibit in vitro growth and differentiation of the pathogenic yeast phase of
the fungus, illustrating the utility of microarray analysis to identify new gene targets
for potentially controlling compounds.
5. DNA SEQUENCE ANALYSIS

The sequencing of polymorphic genes has expanded with the availability of cost
effective electrophoretic DNA sequencing methods (Lasker, 2006) which is now
used routinely in many diagnostic laboratories for in-depth gene analysis. Low
throughput sequence directed fungal ‘ribotyping’ has been employed for several
decades using simple linear-PCR DNA sequencing (White et al., 1990; Hatsch,
Phalip & Jeltsch, 2004; Rakeman et al., 2005; Lee et al., 2007) to identify isolates.
Alternative sequencing-derived methods such as dideoxy fingerprinting (Lebech,
2002), in which both DNA sequence and SSCP components are detected, can be
used for identification of polymorphic loci. Both methods are amenable to
automation and high throughput applications.
A novel approach to determine the sequence of purified and mapped RAPD
markers for conversion into SCAR involves the use of four 10-mer sequencing
primers 3'-terminal extended by one base-pair (A, T, C or G) internal to the initial
amplification locus (Mitchelson et al., 1999). This strategy permits strand-specific
DNA sequence to be read independently from each of the RAPD fragments without
recourse to strand separation or fragment cloning, and informative RAPD fragments
may then be converted into mapped STS or SCAR loci. Another important
application of high throughput sequencing is Serial Analysis of Gene Expression
(SAGE) which quantifies expressed genes by characterizing concatomers of short
mRNA fragments (sequence tags) which are quantitatively representative of the
relative proportions of different mRNA species present in the microorganism (Irie
et al., 2003; Minami et al., 2007).
5.1. Whole Genome Sequencing
Whole genome sequencing (WGS) (Venter, Smith & Hood, 1996) is a non-targeted
sequencing strategy which uses bioinformatic alignment of random sequence reads
of genome fragments to assemble contiguous original sequence. The technique
employs high level coverage of redundant overlapping sequence reads to identify
and confirm sequence alignments, saving the need for genome library production
and curation. However, the presence of multiple repeat sequences in a genome, or
significant diversity between chromosome homologues causes the misalignment of
sequences, the misassembly of contiguous elements and prevents the overall
assembly of continous sequence. WGS has been used largely for sequencing of
haploid bacteria, and for diploid genomes for comparison to closely related
reference genomes sequenced conventionally. Recently, because of its cost saving to
genome projects, WGS has been used to provide the bulk of primary sequence and
then combined with large clone sequencing data to fill in gaps and resolve
ambiguities.
Recently, a WGS approach was used to sequence the diploid yeast Candida
albicans (Jones et al., 2004). The chromosomal homologues have significant levels
of polymorphism and rearrangement which allowed only a partial set of genomic
contigs to be assembled. Recently, Magee and colleagues (Van Het Hoog et al.,
2007) employed combined hybridization of probes to PFGE fractionated
chromosomes as well as a sequence tagged site (STS) map based on a fosmid library
of clones to identify the chromosomal position of several of the contigs from the
prior Candida assembly. This new assembly was then compared to an optical map
which identified some further missembled regions. Bioinformatic alignment to two
other partial Candida genomes were also used, achieving a final assembly of 16
super contigs aligned to the 8 chromosomes of C. albicans, illustrating the variety of
physical and molecular mapping techniques that must be also used to resolve the
ambiguities of WGS analysis of such heterogeneous diploid organisms.
5.2. Massively Parallel Sequence Analysis

With the advent of massively parallel pyrosequencing (Margulies et al., 2005) and
other solid-phase sequencing systems (Shendure et al., 2004; Kartalov and Quake,
2004) the genomes of haploid microorganisms can be sequenced to sufficient depth
to achieve high level genome coverage and assembly within several days. As details
of the genome sequences of reference organisms become available, rapid
development of other genetic analysis tools for phytopathological testing should be
anticipated as a new spectrum of molecular information become available for
genotype analysis, using a variety of technologies (Käller, Lundeberg & Ahmadian,
2007). These new pyrosequencing and solid-phase sequencing techniques have
several features advantageous for random whole genome sequencing. These
sequencing methods require only relatively short DNA fragments (200 bp – 1 kb) as
template and do not require template cloning (Margulies et al., 2005). In addition,
the products of random genome amplification procedures are more likely to
represent contiguous initial genomic regions as shorter elements are sequence
analysed. Thus, one might expect in the near future that a number of
phytopathogenic fungi and other economically important fungal species are likely to
be defined by their entire genome sequences, in addition to micromorphological and
physiological classifications. It is also likely that ultra-high throughput genome
sequencing will eventually become an element of production quality control of
important industrial fungi and strains.
5.3. Metagenomic Sequencing

Whole genome shotgun sequencing (WGS) approaches have fostered the emergent
field of study of “entire” ecosystems at the level of their microbial biota –
“metagenomics” (Venter et al., 2004), by providing the capacity to sequence mixed
biota to sufficient depth, such that entirely new species of microorganisms may be
identified in their environments, without the need for classical isolation and culture.
Isothermal in vitro genome amplification methods such as (multiple) strand

displacement amplification (SDA) and rolling-circle amplification for amplification
of whole genomes to concatomer lengths (Hutchison et al., 2005) are used to
capture the genomic content. Model studies of the metagenomic approach with
bacteria (Abulencia et al., 2006; Wu et al., 2006) have demonstrated its efficiency,
yet also showed that some amplification bias may be anticipated, particularly if
initial template representation is low within the mixture. Assessment of different
whole genome amplification methods with known and tractable microbial genomes
suggest that multiple displacement amplification (MDA) is the most efficient and
generates the least sequencing bias (Pinard et al., 2006). Overall these caveats do
not detract significantly from the possibilities of the metagenomic approach, which
can reveal previously undiscoverable information about community populations by
defining environments in terms of the microorganismal gene systems present, rather
than by the identifiable culturable organisms which grow under a (more) limited set
of defined parameters (Remington, Heidelberg & Venter, 2005). The approach,
however, does demand the careful interpretation of data and independent
confirmation of findings in any attempted genome assemblies.
Recently, pyrosequencing technology has also been used and compared to
whole genome shotgun sequencing (WGS), notably for the discovery and assembly
of mixed population of microbial community genomes from marine environments
(Goldberg et al., 2006) and from other mixed community environments (Krause
et al., 2006). Algorithms developed by Krause and colleagues allow searching for
sequence similarities in mixed environmental samples, and are capable of detecting
a high fraction of the gene content of the organisms present, albeit dependent on the
number of species composing the sample and the overall size of the sample, their
genome sizes and the depth of sequencing read data.
A current limitation to the application of these WGS approaches is the size of
the target genomes. Presently, representative coverage can be determined for a large
number of the smaller genomes of bacteria and viruses present in a mixed
population sample. For mixed environmental population analysis, the coverage of
the genomes of eukaryotes, even eukaryotic microorganisms, like algae and fungi
will be very incomplete, unless SDA is used to amplify specific classes of DNA
sequence such as ribosomal genes or other highly conserved genes (Ge et al., 2002).
Quake and colleagues (Ottesen et al., 2006) reported a microfluidic digital PCR
approach to analyse the coincidence of multiple genes within individual bacteria from
a mixed environment. Single bacterial cells, taken from the complex symbiotic
community found in the hind gut of wood eating termites, were partitioned by a
microfluidic device and distributed rapidly into parallel, individual PCR reaction
mixtures containing oligonucleotide primers for the coincident amplification of rRNA
genes, and metabolic genes of interest were selected. Subsequently, PCR products
were recovered from the individual μl-volume reactions and genes were sequenced.
High frequency coincident detection of species-specific ribosomal gene signatures
and the key metabolic genes permitted discovery of the previously unknown
rRNA-based identity of several symbionts. Such community sequencing techniques

could potentially be employed to investigate the progression of beneficial and
pathogenic organisms throughout the production cycle of important foods and crops,
to provide unique insights into the complex and often interregulated events affecting
disease progression. Such data might provide new understanding of community
interactions, of the huge, still undiscovered biodiversity which characterizes natural
microbial communities, as well as possible clues for new approaches to the control
of undesirable organisms.
5.4. Analysis by Capillary Electrophoresis (CE)

5.4.1. CE Analysis by Size Separation
Capillary electrophoresis is a rapid, high throughput analytical technique which has
automatic sample handling and data collection (Foulet et al 2005; Mitchelson,
2003). The transfer of such methodologies to the phytopathological sector
confirmed their high-resolution power. Techniques such as capillary electrophoretic
analysis of polymorphic ribosomal gene loci (Sipos et al., 2007) and heat shock
protein genes (Chang et al., 2007) after restriction and fragment length (RFLP)
analysis, demonstrated the advantages of automated CE analysis and fragment
calling.
Several recent methods of analysis of ribosomal gene polymorphisms that
avoid restriction have been developed for bacterial genotype analysis and should be
applicable to fungal analysis. These include hierarchical oligonucleotide primer
extension (HOPE) (Wu & Liu, 2007), which involves strand extension reactions
using multiple oligonucleotide primers modified with different lengths of polyA at
the 5' end that target the 16S rRNA genes of different species. Its use was
demonstrated by correctly identifying 20 different bacterial species in a mixed
sample using a 10-plex primer reaction.
Secondly, a novel 16S rRNA gene PCR-ligase detection reaction-capillary
electrophoresis assay was also employed by Barany and colleagues (Pingle et al.,
2007) in which two regions within the bacterial 16S rRNA gene are amplified using
universal PCR primers and the presence of specific single-nucleotide
polymorphisms within the amplified regions were identified by subsequent ligase-
detection reaction. The different ligation products for each species varied in color
and size and could be efficiently separated by CE.
Thirdly, chemical mismatch cleavage which identifies heteroduplex molecules
and cleaves the heteroduplex to size resolvable fragments can be analysed rapidly
using CE (Tabone et al., 2006). With access to genome sequence information for
many species, numerous polymorphic elements such as SSR (STR) repeats can be
identified and developed for rapid CE amplification product size analysis. Typically,
abundant characteristic repeated DNA polymorphisms that discriminate species and
strains that may be PCR-amplified using specific primers and size resolved by CE
are used for genotyping of fungi and other organisms. Examples include the
bacterial ERIC sequence (Godoy et al., 2004), AFLPs (Huang et al., 2007) and
appropriately sized STR loci in eukaryotes (Yeung et al., 2006a; Liu et al., 2007b).
5.4.2. CE Analysis by Fragment Shape

CE techniques have also been developed that detect polymorphisms through
alteration in the electrophoretic mobility of DNA fragments, after effecting a shape
change. These methods include SSCP (Zinger et al., 2007; Larsen, Jespersgaard &
Andersen, 2007) and heteroduplex DNA (HPA) analysis (Velasco et al., 2007).
Other sensitive methods to increase the heteroduplex shape change and better
discriminate between duplex and heteroduplex include constant denaturant capillary
electrophoresis (CDCE) (Li et al., 2005) which is a modified version of denaturant
gradient gel electrophoresis. Thermal programmed capillary electrophoresis (TP-
CE), in which a variable temperature is increased during a run using computer-
controlled thermal ramping, has also been applied for increase the detection of
polymorphisms such as in ‘ribotyping’ of ribosomal gene fragments (Gelfi et al.,
1997). These approaches are useful for the identification of low frequency mutations
and for genetic screenings of pooled samples for detection of rare DNA variants.
5.4.3. Advanced Analytical Devices

5.4.3.1. Miniaturized CE-Based Devices
Increasingly, the development of integrated miniaturized, automated DNA analysis
systems have been reported. These systems have numerous advantages compared to
conventional apparatus that obviate the initial (relatively) high cost. The automation
of processing and data reading and data collection frees professional staff from
laborious tasks, as well as providing some significant speeding of the analytical
process over conventional and classical methods. Typically these systems employ
automatic microfluidic and microelectronic control of sample processes to
sequentially transport the reactants and products from one module to another.
Capillary-array electrophoresis (CAE) provide additional advantages over
conventional CE with very rapid throughput and with up to 384 samples
simultaneously analysed in parallel capillaries or microchannels (Emrich et al.,
2002; Yeung et al., 2006a). Such advanced devices are automatic with respect to the
filling and emptying of channels with separation matrix, the loading of samples and
for all aspects of fragment separation, fluorescence detection of DNA fragments and
data acquisition.
Array CE can be used for DNA sequence determination or for length
polymorphism of PCR-STR alleles or for RFLP, SSCP and HPA analysis of PCR-
amplified DNA fragments. As the ability to use these devices for microbiological
studies increases with development of appropriate tests, the advantages of such
rapid analysis will increase the scope for plant disease management and taxonomic
studies in fungi. Mathies and colleagues have undertaken significant development of
a series of miniaturized chip-based analytical devices with integrated multichannel
PCR and high throughput capillary electrophoresis (PCR-CE) (Liu, Toriello &
Mathies, 2006), requiring only nanoliter reaction volumes and capable of parallel
genetic analyses. These devices could demonstrate complete multiplex amplification

and genetic analyses of bacterial cells in less than 30 min per analysis. Their
integrated CE devices can be readily used for a range of different specific high-
throughput analytical applications, such as STR genotyping (Liu et al., 2007b),
reverse transcriptase directed gene expression analysis (Toriello, Liu & Mathies,
2007; Liu et al., 2007a) and gene specific PCR identification-size analysis (Legally
et al., 2004; Liu, Toriello & Mathies, 2006).
5.4.3.2. Portable Microelectromechanical Systems (MEMS) for On-Site Analysis

The capability to analyse samples under field conditions rather than in a laboratory
has long been a desire for phytopathologists. The recent development of
miniaturized transportable MEMS devices capable of PCR amplification
(Consolandi et al., 2006) could facilitate in situ analysis of plant tissues for fungal
infection using sensitive qRT-PCR techniques (Schaad & Frederick, 2002) or
specific amplification of abundant ribosomal genes (Feau et al., 2005). Desirable
detection systems that avoid the need for expensive optical devices may involve
electrochemical detection. One such system illustrated the detection of bacteria
(Yeung et al., 2006b). The detection process involved the capture of silver-enhanced
gold nanoparticles via specific surface-bound hybridization probes. The whole
analysis employed a multi-step reaction process including the thermal lysis of target
bacteria, magnetic particle-based isolation of the target genomes, asymmetric PCR
to amplify specific detectable sequences and finally electrochemical sequence-
specific detection using silver-enhanced gold nanoparticles.
The Mathies group has also been instrumental in the creation of several
portable forensic and pathogen genetic analysis systems but instead use sensitive
optical equipment for detection of fluorescently labeled DNA fragments. One device
(Legally et al., 2004) was shown capable of triplex PCR amplification and
demonstrated its utility for bacterial identification with a detection limit of 2-3
bacterial cells in a targeted assay of three genes from Escherichia coli or two genes
from Staphylococcus aureus, as well as providing CE separation of the
amplification products. These instruments integrate pneumatic valves for control of
transport of samples, reactants and products, thermal PCR cycling for amplification
of specific genomic loci and CE separation of products. Portable devices could be
readily employed for similar analysis of fungal species, using appropriate fungal
specific assay kits.
The second device (Liu et al., 2007b) was more advanced and able to
undertake multiplex amplification as well as having improved separation and
resolution of short tandem repeat (STR) fragments by CE and four-color
fluorescence detection of labeled fragment signals. These systems have established
that rapid point-of-analysis DNA typing is feasible, and suggest they are applicable
for remote and field situations, where convenience of on-site analysis could enable
immediate monitoring of the exposure of a crop to pathogen inoculum, thus
informing rapid disease management decisions.
6. CONCLUSIONS
DNA profiling techniques have contributed significantly to our ability to detect and
investigate plant pathogens in the laboratory and, most recently, directly in the field.
Advanced, high-throughput technologies are expected to impact significantly on
pathogen diagnosis and taxonomy, as well as several aspects of crop production. A
great benefit would come in the monitoring of cropping systems for quantification
of pathogen load. The resolution of such issues has direct implications in the study
of the dynamics of epidemics and thus in plant disease management. More
immediate pathogen detection and more effective determination of the amount of
disease will enable control measures to be implemented more timely and accurately,
supporting the set up of crop certification programs for important phytopathogens.
Instrumentation for rapid and automated pathogen analysis will particularly assist
phytosanitary services in the inspection of plant material at ports of entry to prevent
more effectively the possible introduction of exotic pathogens into uncontaminated
areas, and ultimately will inform the development of plant quarantine policies and
regulations. Such devices will increasingly become more widespread in general
phytopathological services and analytical laboratories.
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17
SUSHEEL KUMAR 1, NUTAN KAUSHIK 1, RUANGELIE
EDRADA-EBEL2, RAINER EBEL2 * AND PETER PROKSCH2
ENDOPHYTIC FUNGI FOR PEST AND DISEASE

MANAGEMENT
1
TERI University, Habitat place, Lodi Road, New Delhi, India
2
Institut für Pharmazeutische Biologie und Biotechnologie,
Heinrich-Heine-Universität Düsseldorf, Germany 1
Abstract. Endophytes are microorganisms that inhabit the interior of a healthy plants. They offer great-
untapped potentials, which can be exploited to maintain healthy crops. Many cultivated and wild type
plants have been investigated for endophytic fungal metabolites which include guanidine and
pyrrolizidine alkaloids, indole derivatives, sesquiterpenes, isocoumarin derivatives. These metabolites
show beneficial effects to crop plants and many of them also have pesticidal and antimicrobial activity
against plant and human pests and pathogens. Full potentials and efforts needed are herein discussed.
1. INTRODUCTION
The need for new and useful compounds to provide protection and relief to crop
plants from pests and thereby sustainable food production for human consumption is
ever growing. Pesticide consumption in India increased from 434 Metric Tones
(MT) in 1954 to 70794 MT in 2002-03 (www.indiastat.com). However, a sharp
reduction to 48350 MT during 2002-03 has been witnessed due to realization of the
fact that the indiscriminate use of pesticides has created numerous problems, like the
development of resistant strains in insects and plant pathogens, resurgence of pest
species, direct toxicity to the applicator, destruction of parasites, predators, and
other beneficial organisms, accumulation of pesticide residues in several agricultural
commodities, water, air and soil (see http://www.ddsindia.com/www/npm.htm).
Animals intended for human food absorb pesticides from residues in their feed,
water or during direct/indirect exposure in the course of pest control (Aulakh et al.,
2006). Pesticide poisoning even causes more deaths than infectious diseases
(Eddleston et al., 2002). A study of pesticide poisoning in South India demonstrated
that two compounds, monocrotophos and endosulfan, accounted for majority of
* Present address: Department of Chemistry, University of Aberdeen, Meston Building, Meston Walk,
Old Aberdeen, AB24 3UE, Scotland, UK
365
366 S. KUMAR ET AL.
deaths, of which two-thirds of the patients were less than 30 years old (Srinivasrao
et al., 2005). Over 80% of women are found to be suffering from acute pesticide
poisioning in the cotton growing areas, as they assist in mixing concentrated
chemicals and refilling spraying tanks (Mancini et al., 2005). To cope with the
stated problems there is a need to develop ecologically sound, environmentally safe
and economically viable methodologies for plant disease and pest management.
Natural and biological control of pest and diseases affecting cultivated plants
has gained considerable attention in the past decades as a way of reducing the use of
chemical products in agriculture. Biological control has become an utmost important
tool for Integrated Pest Management (IPM). Use of microorganisms that antagonize
plant pathogens and insects as biological control agent results in enhancement of
resident antagonist and is risk free. Antagonistic microorganisms most frequently
are from the rhizosphere or the phyllosphere, while few are also endophytes.
Endophytic fungi offer great-untapped potential, which can be exploited for the
good crop health. The present review summarizes research work done on
endophytic fungi from terrestrial plants, which are identified for pesticidal activity.
2. ENDOPHYTIC FUNGI
All microorganisms that inhabit the interior of a plant for at least one period of their
life cycle are considered as endophytes (Arnold et al., 2003). It is noteworthy that,
of the nearly 300,000-500,000 plant species that exist on the earth, each individual
plant is host to one or more endophytes (Strobel, 2006). Endophytic fungi are
widespread in all phyla of the kingdom Fungi. Most of the endophytic species
belong to the phylum Ascomycota, and they are often closely related to fungi known
to cause diseases, either in healthy tissue or as secondary invaders of damaged
tissues (Schardl et al., 1997). This suggests that endophytes may have evolved from
pathogens or vice-versa.
Figure 1. Endophytic and pathogenic interaction of fungi in a plant

(adapted from Schulz et al., 2002)
IPM THROUGH ENDOPHYTIC FUNGI 367
The fungal endophytes possess exoenzymes necessary to colonize their hosts

and they grow well in the apoplastic washing fluid of their hosts. It has been
suggested that fungal endophyte–plant host interactions are characterized by a finely
tuned equilibrium between fungal virulence and plant defence, as depicted in Fig. 1
(Schulz et al., 2002).
Most of the plant species examined to date harbour endophytic fungi within
their asymptomatic aerial tissues, such that the endophyte represents a ubiquitous,
yet cryptic, component of terrestrial plant communities (Arnold et al., 2003).
Detection of endophytic fungi inside the host tissue has been achieved by several
techniques, including tissue print immunoassay (Hahn et al., 2003), transmission
electron microscopy (Christensen et al., 2002), direct staining and aphid assay
(Wilson, Clement & Kaiser, 1991). Antagonistic effects of endophytic fungi
including Colletotrichum sp., Fusarium sp., Nectria sp., and Xylaria sp. isolated
from cacao plants, has been reported against Phytophthora pathogens of cacao plant,
in in vitro test. These fungi grow within their plant hosts without causing any
apparent disease symptoms that involve continual metabolic interactions between
fungus and host.
3. BIOACTIVITY OF ENDOPHYTIC FUNGI

Webber (1981) was the first researcher who noticed that the endophyte Phomopsis
oblonga (Desmazieres) Traverso protected elm trees against the beetle
Physocnemum brevilineum (Say). Later in the year 1985 bioactivity of this
endophytic fungus was conclusively proven when it was shown that fungi belonging
to the Xylariaceae family synthesize secondary metabolites which were found to be
detrimental for beetle grubs (Clay, Hardy & Hammond, 1985). In another study,
weight gain and survival of the insect-pest, Spodoptera frugiperda Smith, was found
to be negatively affected by the endophytic fungus Balansia cyperi Edg., isolated
from Cyperus sp. (Hardy, Clay & Hammond, 1985). Ahmad et al. (1985) verified
similar effects of the same fungus over the grasshopper Acheta domesticus L.
In a choice assay Johnson et al. (1985), showed that aphids including
Rhopalosiphum padi, Schizaphis graminum, Oncopeltus fasciatum would feed on
endophyte-free Festuca plants rather than on infected samples. Methanol extracts of
the tall fescue infected with Acremonium coenephialum were tested for toxicity
against Oncopeltus fasciatum and found likewise to be effective. Since then a wide
range of activities of endophytic fungi has been reported either by induction of host
plant resistance or by production of secondary metabolites, which in turn protect the
plant. Endophyte-grass interactions produce more metabolites useful for crop
protection than endophyte-woody plant interactions (Saikkonen, 2004).
Fungal endophytes also impart enhanced tolerance to abiotic stresses (West,
1994; Siegel et al., 1990). Enhanced host plant resistance to insects has been
reported in Acremonium endophyte host interactions (Breen, 1994).Increase in
growth rate has been observed in tall fescue plants infected with Neotyphodium
endophytic fungi, however, beneficial effects of endopohytic fungi on plant growth
diminished with increased soil moisture and nutrients (Faeth & Fagan, 2002).
368 S. KUMAR ET AL.
Root infection with endophytic fungi produces more phenolics and elicits
greater plant defense reaction (Schulz et al., 1999). Endophytes and cell-free
washings of their culture plates were reported to reduce the density and size of
Puccinia pustules in a susceptible cultivar of wheat, when inoculated 3, 7, and 14
days prior to invasion of the pathogen. Interactions between endophytes and
Puccinia are most probably mediated by defence mechanisms induced in the host
plant (Dingle & McGee, 2003).
Birch trees with high frequencies of Melanconium sp. endophytes were less
infected with pathogenic fungi Fusicladium sp. and birch rust fungus (Elamo et al.,
1998). Induction of systemic resistance of Chinese cabbage to bacterial leaf spot and
fungal leaf spot (caused by Alternaria), by inoculation of the endophytic fungus
Heteroconium chaetospira, has been reported by Morita et al. (2003). The isolates
were inoculated in the root zone and induced systemic resistance without migrating
to foliage. In another experiment eggplant roots colonized by a sterile white
mycelial endophyte were found to be highly resistant to Verticillium wilt (Narisawa
et al., 2002). Table 1 provides a summary of endophytic fungi isolated from diverse
plant species and their associated bioactivities.
4. ENDOPHYTIC METABOLITES AS SOURCE OF NEW PESTICIDES

Several metabolites showing pesticidal activity have been isolated and characterized
from endophytic fungi. A summary is provided in Table 2. Endophytic fungi in the
tribe Balansiae produce ergot alkaloids viz. ergonovine [1], ergotamine [2],
ergocryptine [3], agroclavine [4] and elymoclavine [5] (Fig. 2) which caused
reduction in larval weight and leaf area consumption of Spodoptera frugiperda at
concentrations of 77 - 100 mg liter -1 (Clay & Cheplick, 1989).
Peramine [6] (Fig. 2), a pyrrolopyrazine alkaloid with insecticidal activity
against argentine stem weevils (Rowan & Gaynor, 1986), has been isolated and
characterized from several endophytic fungi present in the stem and leaf of tall
fescue, ryegrass (Festuca arundinacea Schreb.) and other grasses (Schardl &
Phillips, 1997). These endophytic fungi are Neotyphodium coenophialum (Morgan-
Jones & Gams) Glenn, Bacon & Hanlin, N. lolli, Epichloe festucae and E. typhina
(Fries.) Tulsane.
A tetrameric acid analog, cryptocin [7] (Fig. 2), isolated from cultures of the
endophytic fungus Cryptosporiopsis cf. quercina Petr. that is present in the inner
bark of the stem of Tripterygium wilfordii Hook. F., has been found to be effective
against Pyricularia oryzae Cav., and other phytopathogens (Li et al., 2000).
Indole derivatives like 6-isoprenylindole-3-carboxylic acid [8] (Fig. 2) have
been isolated from the endophyte Colletotrichum sp. and showed growth inhibition
properties against phytopathogenic fungi including Phytophthora capsici Leonian.,
Rhizoctonia cerealis Van der Hoeven., and Gaeumannomyces graminis (Sacc.) von
Arx & Olivier var. tritici J. Walker (Lu et al., 2000).
Lolines [9] (Fig. 2) are saturated aminopyrrolizidine alkaloids occurring in
Neotyphodium-Festucae endophyte host interactions. These compounds are broad-
spectrum insecticides showing dual activity as metabolic toxins and feeding
deterrents on specific insect species (Schardl & Phillips, 1997).
370 S. KUMAR ET AL.
372 S. KUMAR ET AL.
374 S. KUMAR ET AL.
376 S. KUMAR ET AL.
Figure 2. Structure of metabolites 1 to 9.
During interactions of grasses from the genera Lolium and Festuca with the
endophytic fungi Acremonium coenophialum and Epichloe typhina the secondary
constituents loline [9], peramine [6], ergovaline [10] (Fig. 3), were produced. Loline
and peramine were shown to have detrimental effects on aphids including
Rhopalosiphum padi and Schizaphis graminum (Siegel et al., 1990).
378 S. KUMAR ET AL.
The cytotoxic alkaloid, cytochalasin [11] was isolated from Rhinocladiella sp.,
an endophytic fungus of Tripterygium wilfordii (Wagenaar et al., 2000).
Phomapsichalasin [12] an antimicrobial agent was isolated from the endophytic
fungus Phomopsis sp. fermented on shredded wheat (Horn et al., 1995). 1-N-methyl
albonoursin [13], an antibiotic alkaloid is reported from Streptomyces sp. from
perennial ryegrass (Gurney & Mantle, 1993) (Fig. 3).
Alkaloids from endophyte and grass interactions have been shown to be
protective against several crop pests (Bush, Wilkinson & Schardl, 1997). Several
steroids such as 3β-hydroxyergosta-5-ene; 3-oxoergosta-4,6,8(14),22-tetraene;
3β,5α-dihydroxy-6β-acetoxyergosta-7,22-diene and 3β,5α-dihydroxy-6β-phenyl-
acetoxyergosta-7,22-diene have been reported as constituents of the liquid culture of
Colletotrichum sp. isolated from Artemisia annua. They have shown antifungal
activity against Phytophthora capsici Leonian., Rhizoctonia cerealis Van der Hoeven.,
Helminthosporium sativum Pamm., King and Bakke and Gaeumannomyces graminis
(Sacc.) von Arx & Olivier var. tritici J. Walker (Lu et al., 2000).
Nodulisporic acid A [14], a novel and potent natural insecticide, has been
isolated from Nodulisporium sp., an endophytic fungus of woody plants (Ondeyka
et al., 1997). Insecticidal properties have been reported against Aedes mosquito
larvae and larvae of the blowfly (Lucilia seracata). Furthermore, Hensen et al.,
(1999) elucidated the structure and relative stereochemistry by spectroscopic
methods and X-ray diffraction analysis, which gave two stereoisomers A1 [14a] and
A2 [14b], out of which A1 was more potent with regard to insecticidal activity than
A2 (Fig. 4).
Figure 4. Structure of nodulisporic acid and its stereoisomers.
The sesquiterpene chokols A-G [15-21] (Fig. 5) have been isolated from
Epichloe typhina, an endophytic fungus of Phleum pratense, and found to be
fungitoxic to the leaf spot disease pathogen Cladosporium phlei (Gregory) de Vries,
(Koshino et al., 1989a). Mellein [22] (Fig. 6), an isocoumarin derivative isolated
380 S. KUMAR ET AL.
from the endophyte Pezicula sp. has been described to be strongly fungicidal,
herbicidal and algicidal (Schulz et al., 1995).
Figure 5. Structure of sesquiterpene chokols.
Rugulosin [23] (Fig. 6), a fungal product showing insecticidal activity, has been
reported from Harmonema dematoides which is an endophytic fungus of balsam firs
(Calhoun et al., 1992). Fungicidal molecules have also been isolated from Pezicula
sp. (Schulz et al., 1995) and Epichloe typhina (host: Phleum pratense L.) (Koshino
et al., 1989b).
Colletotric acid [24] (Fig. 6), a phenolic antifungal compound, has been isolated
from liquid cultures of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. which
is an endophytic fungus of Artemisia mongolica Fisch., and was shown to be
effective against Helminthosporium sativum Pammel, King & Bakke, (Zou et al.,
2000). This compound was also found inhibitory to the bacteria Bacillus subtilis
(Ehrenb.) Cohn, Staphylococcus aureus Rosenb. and Sarcina lutea.
Muscodor albus an endophytic fungus from a rainforest plant has been proven
to be a potent fumigant, which protected fruits and vegetables during storage.
Fumigant property of endophytic fungus is due to the production of volatile organic
compounds (VOCs). Most effective class of inhibitory compound were esters
(1-butanol, 3-methyl-acetate). Other components of VOCs were alcohols, ketones,
lipids, and acids. None of these compounds was effective individually. They rather
acted due to synergistic effects. These compounds also prevented the growth of
common agricultural pests, like smut, water mold and root rot (Strobel et al., 2001;
Strobel, 2006).
Muscodor vitigenus Daisy, Strobel, Ezra, & W. M. Hess, an endophytic fungus

isolated from Paullinia paullinioides Radlk., a liana growing in the Peruvian
Amazon, has been shown to produce an insect repellent. The repellent has been
382 S. KUMAR ET AL.
identified as naphthalene [25] (Fig. 6), and was demonstrated to exhibit insecticidal
activity against the wheat stem sawfly, Cephus cinctus Norton. (Daisy et al., 2002).
Production of griseofulvin [26] and dechlorogriseofulvin [27] (Fig. 6), from Xylaria
sp., has been reported for the first time.
Xylaria sp. is an endophytic fungus of Abies holophylla. In vitro and in vivo
tests of griseofulvin have shown antifungal activity against Magnaporthe grisea,
Coryicium sasaki, Puccinia recondita, Blumeria graminis f. sp. hordei (Park et al.,
2005). Other secondary metabolites occurring in different host endophyte
interactions included quinones, peptides (cryptocandin) [28] (Fig. 6), pentaketides
and phenols (Tan & Zou, 2001).
A nematicidal fungal metabolite (culture filtrate) has been isolated from an
endophytic fungus of tomato. The endophytic fungus Fusarium oxysporum E. F.
Sm. & Swingle, showed efficacy against Meloidogyne incognita (Ko. & Wh.) Chit.,
wherein 98% of juveniles were killed within one hour of exposure to the culture
filtrate (Hallmann & Sikora, 1996).
The culture filtrate of a F. oxysporum strain also reduced significantly the
growth of Phytophthora cactorum (Lebert & Cohn) Schröt., Pythium ultimum Tro.,
and Rhizoctonia solani Kühn, in vitro. Dicanthelium lanuginosum plants inoculated
with the endophytic fungus Curvularia sp. survived at a soil temperature of 65°C
whereas plants lacking the fungi did not survive even at temperatures ≥ 40°C
(Redman et al., 2002). Increased temperature resistance is advantageous to plants as
they are able to grow concurrent with soil solarization where all soil-borne
pathogens, pests and weeds would be killed at this temperature while crop plants
will survive.
Fungal endophytes of the genera Neotyphodium and Acremonium isolated from
wild wheat species served as a source of biological control agents against pests or
abiotic stress factors in wheat (Marshall, Tunali & Nelson, 1999). Several
endophytic fungi possessing insecticidal, antifungal and herbicidal activity have
been reported from plants of diverse origin (Table 2). More recently, several
endophytic species and their metabolites have been reported as plant protectants.
Endophytic fungi producing pesticidal compounds were frequently isolated
from stargrass (Ji, Song & Tan, 2004); rice (Tian et al., 2004); Melia azedarach L.
(Gries dos Santos et al., 2003); and Theobroma gileri L. (Evans et al., 2003). Pirttila
et al. (2003) have isolated plant growth hormones from endophytic fungi of Pinus
sylvestris. Many of the endophytes have so far remained underexplored for their
metabolites. Endophytic fungi from wheat (Larren et al., 2002; 2006), rice (Fisher &
Petrini, 1992; Tian et al., 2004), maize, coffee (Sette et al., 2006) and tea (Augusta
et al., 2005), have also been described but analysis of their secondary metabolites
has not yet been performed.
5. CONCLUSIONS
Endophytic fungi offer great potential in plant protection, imparting tolerance
against several biotic and abiotic stress factors. However, endophyte-host
interactions may turn to a pathogenic interaction, if susceptibility of host and/or
virulence of the endophyte increase. However, if the metabolites responsible for the
beneficial effect can be isolated and exploited, then the risk of pathogenicity can be
avoided. Structural elucidation of secondary metabolites will help in defining modes
of action as well as in preparation of right formulations for field application.
Standard protocols for isolation of bioactive molecules will be of great importance
for production on large scale by fermentation technology. This will reduce the extra
expenditure incurred in synthesis of chemical compounds. More plant species need
to be explored for their endophytic fungi and their corresponding secondary
metabolites. Endophytes that have not been investigated for their natural products so
far should be studied for their bioactive metabolites in order to tap the rich
biodiversity of endophytes.
ACKNOWLEDGEMENTS
N. K. and P. P. thank DST/DAAD for support and collaboration.
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INDEX
Abies balsamea, 373 Alfalfa, 139, 229

Abies holophylla, 382 Aliphatic compound, 374
Acer negundo, 122 Alkaloid, 365, 376, 378
Acervuli, 38 Allegheny mountains, 87
Acetic acid, 8, 20, 235 Almond, 138, 139, 141, 142, 149
Acheta domesticus, 367 Alnus cordata, 88, 92
Acholeplasmataceae, 43 Alternaria, 4, 18, 20, 163, 179,
Acholeplasmatales, 43 181–183, 187, 275, 309, 376
Acibenzolar-S-methyl, 218, 220 Alternaria alternata, 258, 275,
Acidic soils, 228 277, 285
Acidovorax avenae subsp. citrulli, Alternaria leaf spot, 271, 277, 279
191, 193–196, 198, 200 Alternaria macrospora, 275, 277
Acidovorax, 191, 193–196, 200 Alternaria padwickii, 297
Acids, 381 Alternaria solani, 179, 181
Acremonium coenophialum, 377 Alternaria tenuis, 370
Acremonium, 121, 367, 377, 382 Amaranthus gracilis, 139
Acremonium zeae, 373, 386 Amendments, 153
Actin, 47 America, 86–88, 90, 102, 103
Actinidia chinensis, 122 American chestnut trees, 86, 87, 99,
Actinomycetes, 106, 152, 230 102–104, 109
Adaptation, 274 Amino acid transport, 45
Aecia, 37 2-Amino ethanol, 193
Aeciospores, 37 Aminopyrrolizidine, 376
Aedes, 378 Ammonium sulfate, 282, 284
Aegiceras corniculatum, 375 Ammonium, 75, 230, 232
Aerial tissues, 367 Amoebas, 106
Africa, 139, 169, 323 Amplification, 335–340, 343,
Agaricales, 371 345–352, 354, 357, 360
Agaricus melleus, 89 Analytical modeling, 162
Agrobacterium, 153 Anamorph, 137, 138
Agrobacterium tumefaciens, 4 Anastomosis, 282
Agrobacterium-mediated Andes, 166, 168, 169
transformation, 232 Anilino-pyrimidine, 10
Agroclavine, 376 Anilopyrimidine, 31
Agroecological zones, 286 Antagonist, 230, 235, 293, 303, 305,
Air temperatures, 30 307, 309–316
Airborne inoculum, 335 Antagonistic fungi, 305
Airborne spores, 304 Antagonistic microorganisms, 8, 152,
Al ethylphosphite, 77 202
Albania, 88, 96 Antheridium, 65
Alcohols, 381 Anthesis, 197
389
390 INDEX
Anthracnose, 279 Arthropods, 281

Antibacterial chemicals, 192, 199 Artificial inoculations, 88, 109
Antibiosis, 308 Artimisia annua, 372, 373
Antibiotic, 56, 107, 148, 151, 204, Asci, 142
217, 312, 378 Ascocarps, 145
Antibodies, 202 Ascochyta gossypii, 285
Anticancer, 375 Ascomycete, 277, 279
Antifungal, 378, 380 Ascomycota, 138, 366
Antifungal activity, 235, 310 Ascospore, 5, 28, 29, 32, 39, 245–248,
Antifungal compounds, 258 251, 255–257, 262, 295
Antifungal mycotoxin, 373 ASM, 220
Antifungal proteins, 253 Aspergillus clavatus strain H-037, 375
Antimicrobial activity, 154, 365 Aspergillus fumigatus, 340
Antimicrobial agent, 204 Aspergillus fumigatus CY018, 373
Antimicrobial chemicals, 202 Aspergillus niger IFB-E003, 375
Antimicrobial compounds, 310 Aspergillus, 275, 297, 336, 347,
Antimicrobial, 378 358, 361
Antimycotic, 375, 376, 384 Assays, 193, 200–202, 333, 335, 336,
Antiviral, 375 338, 341, 343, 345, 357
Aphanocladium album, 311 Aster yellows, 49
Aphids, 367, 377 Asymptomatic plants, 129
Apical dominance, 44 Atmosphere, 87
Apiosporina morbosa, 4 Aurantioideae, 61
Apocynaceae, 375 Aureobasidium,, 370
Apoplexy, 119, 126, 127, 140 Australia, 48, 49, 119, 121, 123, 126,
Apothecia, 245–248, 250–252, 254, 127, 130, 193, 195, 227, 192
256, 257, 262, 263 Australian grapevine yellows, 49
Apple diseases, 27 Austria, 88, 96
Apple management, 27 Autoecious rusts, 304, 317, 319
Apple proliferation, 46, 52, 53 Automated fragment scoring, 341
Apple scab, 27–32, 37 Avellino, 87
Apple spring diseases, 27 Avirulence genes, 215
Apple, 48, 53, 138, 139, 144, Avocado, 138, 139, 146, 148,
149, 151 149, 152
Apricot, 3, 4, 6, 9, 16, 18, 20, 48, 54 Avocado root rot, 139
Areolae, 277 Avoidance, 199, 252, 253
Argentina, 63, 64 AvrXv3, 215
Argentine stem weevil, 376 AvrXv4, 215
Armillaria mellea, 137, 138, 140 Azoxystrobin, 10, 235
Armillaria, 4
Aromatic hydrocarbon, 10 Bacillus amyloliquefaciens, 236
Artemisia annua, 378 Bacillus cereus, 235
Artemisia mongolica, 380 Bacillus subtilis, 285, 288, 380
Arthrinium, 371 Bacillus, 203, 230
INDEX 391
Bacteria, 90, 106, 110, 148, 152, 191, Beet, 250, 283
197, 200, 202, 205, 211, 217, 220, Bemisia tabaci, 281
286, 290, 293, 305, 336, 337, 340, Benodanil, 321
348, 349, 351, 354, 358, 361, 362 Benomyl, 9, 10, 20, 29, 30, 38, 151,
Bacterial antagonist, 20 234, 235, 255, 260, 295
Bacterial artificial chromosome Benzimidazole, 3, 10, 151, 234, 235,
(BAC), 345 255, 260, 283, 295
Bacterial blight, 271, 272, 276, 278, Benzothiodiazole, 278
290, 291 Betula pendula, 375
Bacterial canker, 3–5, 12–15, 21 Betula pubescens, 375
Bacterial cells, 197 Betulaceae, 375
Bacterial diseases, 211, 213, 217–219 Binomial distribution, 70
Bacterial exudates, 290, 291 Bioactive metabolites, 383
Bacterial fruit blotch, 191–194 Bioactive molecules, 383
Bacterial genotype analysis, 352 Bioassay, 15, 333, 342
Bacterial leaf blight, 271, 286, Biochemical tests, 347
290, 291 Biocontrol agents, 153, 154, 203, 204,
Bacterial species, 347, 352 211, 220
Bacterial spot control, 217 Biocontrol strategy, 315
Bacterial spot management, 217 Biocontrol, 150, 153, 172, 177, 254,
Bacterial spot, 211–214, 217 259, 261, 263
Bacterial streaming, 212 Biodiversity, 383
Bactericidal, 219 Biofumigation, 250, 263
Bacteriocin, 217 Bioinformatic alignment, 349, 350
Bacteriophage strains, 219 Biological activity, 385
Bacterium exitiosa, 214 Biological additives, 110
Bacterium malvacearum, 276 Biological control, 36, 85–87, 96,
Bacterium vesicatorium, 214 100, 106, 112, 202, 204, 249,
Bacterium, 213, 214, 216, 218, 311 303–306, 311, 313–316, 366, 382
Bakanae, 271, 294 Biological control agents, 218, 219
Balansia cyperi, 367 Biological control strategies, 85
Balansia, 367 Biomastics, 109
Balsam fir, 380 Biosynthesis, 336
Bark, 67, 69, 74–77, 79, 80, 90–95, Bipolaris, 361
109, 141, 142 Birds, 91
Barley, 229 Bitter rot, 27, 28, 31, 38, 39
Barriers, 7 Blepharospora cambivora, 90
Basidiomycetes, 120 Blight, 85–88, 92, 93, 96–100,
Basidiomycota, 304 102–104, 107
Basidiospores, 122, 128 Blight forecaster, 181
Bavaria, 318 Blight pressure, 88
Bean, 139, 245, 253, 254, 256 Blight resistant genes, 276
Beaveria bassiana, 371, 385 Blight symptoms, 88
Beech forests, 106 Blister rust, 307, 317
392 INDEX
BLITECAST, 163 Cacao, 367

Bloom stages, 7 Cacopsylla pruni, 55
Blossom blight, 3, 5, 6, 7, 8, 11, 34, Cacopsylla pyri, 55
35, 36 Cacopsylla pyricola, 55
Blossoms, 191, 196, 204 Calcarisporium, 369, 384
Blowfly, 378 Calcium cyanamide, 250
Blueberries, 20 Calcium nitrate, 252
Blumeria graminis f. sp. hordei, Calcium, 228, 230, 233, 280, 284, 290
348, 382 California, 5, 8–11, 13–15, 19, 121,
Blumeriella jaapi, 4 124, 127, 130, 139, 195, 196, 198,
Bois noir, 49–52 251, 255, 309
Boll lesions, 276, 279 Calixarenes, 154
Boll rots, 271 Callus, 93, 109
Bolls, 276–280, 285, 286 Cambial layers, 90
Bontia daphnoides, 374 Cambium, 90, 91, 93, 94, 125
Borax, 320 Camellia sinensis, 72
Bordeaux mixture, 13, 29, 36 Campania, 97, 98, 101
Boscalid, 10, 11 Canada, 3, 4, 7, 165, 195, 216
Botryosphaera, 4 Candida albicans, 344, 350,
Botrytis cinerea, 3, 4, 6, 18, 357–359, 363
334, 363 Candidatus Phytoplasma mali, 53
Brachiaria mutica, 288, 289 Candidatus Phytoplasma prunorum,
Bradysia, 254 53, 54
Branches, 124, 125 Candidatus Phytoplasma pyri, 53, 55
Brasil, 63, 64 Candidatus Phytoplasma vitis, 51
Brassica napus, 245 Canker development, 77
Brassica oleracea, 250 Cankers, 65, 67, 76, 78, 87, 90, 109
Brassicaceae, 231 Canola, 245
Brassicol, 282 Canopy clipping, 251
Brazil, 195, 206, 216 Canopy, 34, 40, 62, 63, 67, 68, 70, 74,
Breeding, 31, 33, 102, 321 80, 243, 246, 248, 251–257, 262
Brefeldin A, 375, 383 Cantaloupe, 194, 195
Brinjal, 283 Capillary electrophoresis, 343, 348,
Broccoli, 229, 231, 250 352, 356, 357, 359–361, 364
Bronx Zoological Park, 87 Capillary-array electrophoresis
Brown fruit rot, 61 (CAE), 353
Brown necrosis, 123 Captan, 10, 29, 31, 38, 40, 279, 282
Brown rot, 3, 5–9, 11, 12, 20, 61–63, Carbamate, 10
65–69, 74, 76, 79, 80 Carbendazim, 151, 234, 235, 276,
Brown spot, 271, 272, 279, 280, 286, 278–280, 282, 284, 289, 294, 295
289, 290, 294, 296 Carbohydrate, 252
Bt transgenic cotton, 275 Carboxyanilide, 10
Buckland valley grapevine Carcinogen Risk Assessment, 31
yellows, 49 Carotene, 252
INDEX 393
Carpogenic germination, 250, 255 Chestnut blight, 85

Carrot, 243–260, 262 Chestnut forests, 87
Castanea crenata, 86–88, 103 Chestnut stands, 85–87, 90, 104,
Castanea dentata, 86, 87, 104, 318 106, 110
Castanea mollissima, 86, 102, 318 Chestnut, 85–90, 92–100, 102
Castanea pumila, 88 Chile, 198
Castanea sativa, 85, 88, 96, 102, 103, Chilean gymnosperm, 386
109, 318 Chili, 283
Castilla-Leon, 97 China, 63, 64, 86, 87, 195, 198
Castor, 283 Chitinases, 311, 312
Cauliflower, 229, 231, 233 Chlamydospores, 65, 67, 144
CE separation, 354 Chlorocholinechloride, 235
CE sequencer, 344 Chlorophyll, 11
Cedar, 37, 38 Chloropicrin, 234
Cedar-apple rust, 32 Chlorosis, 33, 284
Cell wall, 43 Chlorothalonil, 10, 164, 168, 170,
Cellobiohydrolase-C, 338, 358 218, 297
Cells, 196, 197, 201–203 Chokols, 379
Cellulase, 99 Chondrostereum purpureum, 4
Central America, 216 Chromosomal assignment, 344
Cephus cinctus, 382 Chromosomal karyotypes, 345
Cercospora gossypina, 275, 279 Chromosome, 232
Cercospora, 271, 279, 309 Chromosome number, 344
Cercosporella gossypii, 277 Chromosome translocation, 344
cereals, 272, 278, 286, 304, 308, Chrysanthemum, 283
320–322 Chrysomyxa arctostaphyili, 319
Certification, 72, 130, 191, 198 Chytridiomycetes, 305
Chaetomium globosum, 371 Cirsium arvense, 52
Chaetomium, 275 Cistus albidus, 231
Chemical control, 3, 13, 16, 18, 61, Citochalasin E, 146
110, 119, 164, 249, 304, 321 Citrus rootstocks, 139
Chemical coverage, 255 Citrus Tristeza Virus, 80
Chemical management, 27, 31 Citrus, 122
Chemical prevention, 119 Citrus, 61–72, 74, 75, 77, 79, 122,
Chemical products, 366 139, 149
Chemical residues, 260 Cladosporium aecidiicola, 309
Chemical sanitation, 258 Cladosporium fulvum, 32
Chemical treatments, 225, 303 Cladosporium gallicola, 309
Chemicals, 179, 181, 182, 187, 274, Cladosporium hemileiae, 309
286, 291 Cladosporium herbanum, 328, 369
Cherry rootstocks, 13 Cladosporium phlei, 379
Cherry, 3–8, 11–15, 17, 18, 138, 139, Cladosporium sphaerospermum, 309
140, 145 Cladosporium tenuissimum, 309,
Chestnut blight pandemic, 86 310, 361
394 INDEX
Cladosporium uredinicola, 309 Conidia, 5–7, 15, 16, 29, 30, 32, 33,
Cladosporium uredinophilum, 309 38, 39, 40, 90, 100, 225, 226, 229,
Cladosporium, 308, 309, 361 236, 283, 287, 288, 295
Cladosporols, 310 Conidial concentration, 7
Classification, 334, 340, 359 Conidial morphology, 138
Clavibacter michiganensis subsp. Conidiogenesis, 348
michiganensis, 213 Conidiophore, 146, 225, 226
Claviceps oryzae-sativae, 296 Coniothyrium minitans, 253, 254
CLCV, 275, 281 Connecticut, 96
Cleistothecia, 16 Conocarpus erecta, 375
Clementine, 80 Contact fungicides, 186, 187
Climate, 233 Control measures, 271, 273, 274, 280,
Climatic conditions, 277, 287, 298 298, 303, 333
Clipping, 252, 253, 262, 263 Control practices, 217
Clonostachys, 153 Convolvolus arvensis, 52
Clover, 229 Conyza bonariensis, 139
Cochliobolus sativus, 344, 364 Cooling time, 259
Coffee, 138, 139, 304, 308, 317, Copper oxychloride, 276, 279,
318, 382 289, 297
Coffee rust, 317, 320 Copper, 29, 31, 34, 36, 74, 79, 80,
Cold storage, 244 168, 191, 204, 211, 217–220
Coleoptera, 311 Coppices, 97–99, 104, 108, 110
Collar, 94 Cordana musae, 370
Colletotric acid, 380 Coremia, 146
Colletotrichum acutatum, 38 Corn, 250
Colletotrichum capsici, 285 Cornus mas, 122
Colletotrichum coccodes, 371 Cortex, 66–68, 226
Colletotrichum crassipes, 370 Cortical parenchyma, 94
Colletotrichum gloeosporioides, Coryicium sasaki, 382
285, 380 Corylus avellana, 122
Colletotrichum gossypii, 275, Corynenum perniciosum, 90
276, 285 Coryneum kunzei var. castaneae, 90
Colletotrichum musae, 370 Costa Rica, 195, 214
Colletotrichum, 276, 367, 376, 378 Cotoneaster, 28
Colonization, 162 Cotton Leaf Curl Virus, 271, 275, 281
Colony Forming Units, 69 Cotton, 139, 228, 229, 231–235,
Combretaceae, 375, 386 271–282, 298
Comparative epidemiology, 161, 169, Cotyledons, 192, 194, 196
171, 173 Cowpea, 283
Competition, 311 Crabapples, 28
Complexity, 106, 314, 315 Craetagus, 28
Compost, 254 Crete, 230
Computer-controlled glasshouses, 213 Croatia, 49
Computing power, 161 Cronartium asclepiadeum, 308
INDEX 395
Cronartium coleosporioides, 319 Curvularia lunata, 285

Cronartium comandrae, 309, 319 Curvularia, 285, 297, 382
Cronartium comptoniae, 319 Cuticular fractures, 18
Cronartium flaccidum, 308, 309, Cutinase, 99
317, 319 Cutting, 121, 123, 128, 131
Cronartium fusiforme, 310, 319 Cylindrocarpon destructans, 135, 371
Cronartium himalayense, 319 Cylindrocarpon theobromicola, 371
Cronartium quercuum f. sp. Cynodon dactylon, 369, 373, 375, 384
fusiforme, 309, 310, 318, 321 Cyperus esculentus, 139, 151
Cronartium quercuum, 309, 310, Cyperus rotundus, 143
318, 319 Cyperus, 367
Cronartium ribicola, 307, 318, 323 Cypress, 37
Cronartium strobilinum, 310 Cyprodinil, 10, 132
Crop damage, 225 Cysts, 65, 66
Crop development, 243, 248, 263 Cytochalasin E, 378
Crop emergence, 185 Cytochalasin, 378
Crop health, 250, 263 Cytoplasm, 153, 307, 308, 309, 311
Crop protection, 303 Cytoplasmic transfers, 100
Crop records, 181 Cytospora CR 200, 375
Crop residues, 276, 278, 279, Cytosporone B, 375
293, 298
Crop rotation, 225, 228, 229, 231, Damage, 61, 63, 76, 87, 88, 92, 98,
271, 273, 279, 282, 289, 295, 103–105, 108–111
297, 319 Dark wood, 120, 124
Cropping systems, 217 Darluca philum, 310
Crotolaria juncea, 293 Databank, 179, 181
Crucifers, 229 Daucus carota, 243
Cryphonectria parasitica, 85–88, 90, Dazomet, 150, 156, 234
92, 93, 97, 99 Decay, 62, 68, 119, 125, 130
Cryphonectric acid, 99 Dechlorogriseofulvin, 382
Cryptocandin, 382 Decline, 4, 12, 44, 52, 54, 55, 62, 67,
Cryptocin, 376 74, 94, 105, 119–122, 124, 128, 140
Cryptococcus, 369 Defense proteins, 45
Cryptograms, 89 Defoliation, 62, 68
Cryptosporiopsis cf. quercina, 376 Deightoniella torulosa, 370
Cucurbit, 191–195, 197–201, 203 Delaware, 195
Cultivar, 3, 11, 13, 49, 53, 55, 191, Dematophora necatrix, 137
192, 199, 203, 205, 227, 228, 232, Demethylation, 3, 10
234, 236, 243, 249, 252, 253, 262 Democratic Republic of Congo, 309
Cultivation technologies, 213 Denaturant capillary electrophoresis
Cultural methods, 249, 252 (CDCE), 353
Cultural practices, 39, 40, 137, 144, 250 Denaturing gradient gel
Cupressaceae, 37 electrophoresis (DGGE), 342
Currant, 318 Denmark, 318
396 INDEX
Desiccation, 15, 219 Disease epidemic, 316

Detection assays, 339 Disease forecasting, 3, 18
Detection, 46, 49, 53, 335, 336, 338, Disease incidence, 248, 249, 262
342, 346, 347, 351–359, 361–364 Disease management, 3, 85, 162, 169,
DGGE, 342 211–213, 217–220, 333, 346, 353,
Diagnosis, 46, 61, 67, 272, 273 354, 360
Diagnostic applications, 348 Disease models, 179, 187
Diagnostic approaches, 333 Disease prediction, 6, 11
Diagnostic specificity, 334 Disease resistance, 27, 303
Diagnostic tools, 49 Disease risk, 251
Diaporthe CR 146, 370, 375 Disease severity, 18
Diaporthin, 99 Disease, 3, 6, 11, 18, 27, 43, 46, 47,
1-2-Dibromo-3-chloropropane, 283 49, 85, 87–91, 94, 95, 103–105,
Dicanthelium lanuginosum, 382 107–111, 119, 120, 122–124, 127,
Dicarboximide, 9, 10, 255, 260 128, 130, 132–140, 146, 147,
Dichloran, 10 149–153, 161–169, 171–177, 179,
Didymella bryoniae, 203 181, 191–193, 195–200, 205, 225,
Dieback, 61, 62, 67, 95, 98, 103 227, 228, 230, 232–235, 244,
Differential display, 348 246–254, 256–272, 274–304, 308,
Differentiation techniques, 334 313–315, 317–319, 321, 333,
Difolatan, 277 365, 366
Digitaria marginata, 297 Disease-resistance genes, 43, 56
3β, 5α-Dihydroxy-6β-acetoxyergosta-7, Disinfection, 147, 151
22-diene, 378 Disinfestation, 258, 261
3β, 5α-Dihydroxy-6β-phenyl- Distributed genomic loci, 342
acetoxyergosta-7, 22-diene, 378 Dithiocarbamates, 321
6,7-Dihydroxy-2-propyl-2,4-octadien- DMI-Piperazine, 10
4-olide, 376 DMI-Pyrimidine, 10
Diketopiperazines, 146 DMI-Triazole, 10
Dimethyl disulfide, 234 DNA, 7, 11, 43, 44, 46, 193, 201, 202,
Dinitroaniline, 235 215, 291, 296, 322, 333–341,
Diplodia castaneae var. 343–347, 349–364
radicicola, 89 DNA fingerprinting, 333, 335,
Diplodia gossypina, 285 362, 363
Diplotaxis virgata, 231 DNA fragments, 336, 353
Discoloration, 49, 67, 260, 285, DNA profiling techniques, 333
294, 297 DNA purification, 336
Disease control, 9, 10, 17, 18, 150, DNA variants, 353
163, 172, 211, 217, 218, 220, 234, Dodine, 29, 30, 38
260, 321 Dormant buds, 13, 14
Disease development, 161, 162, 164, Dothiorella strain HTF3, 375
168, 169, 276, 278, 284, 289, Double guyot, 132
293, 297 Drechslera, 289, 297
Disease diagnosis, 340 Drenching, 151
INDEX 397
Drip irrigation, 250 Enzyme-linked immunosorbent assay,

Drought, 13 46, 68
Drug restance, 344 Enzymes, 99, 226, 309–312
dsRNA, 85, 96, 100–102, 153, 156 Epichloe festucae, 376
Duplex renaturation, 342 Epichloe typhina, 376, 377, 379, 380
Epicoccum nigrum, 369
Early blight, 163, 179, 181 Epicoccum purpurescens, 369
Echinochloa, 289 Epicormic shoots, 91–93
Echinochloa colona, 294 Epidemic development, 204
Ecological fitness, 109 Epidemics, 17, 163, 165–167,
Ecology, 216 169–172, 183, 191, 196, 197, 244,
Ectomicorrhizal fungi, 107 245, 247–249, 251, 263
Ecuador, 161, 166 Epidemiological components, 173
Ediphenphos, 289, 294 Epidemiological concepts, 161, 174
Eggplant, 228, 230, 235, 236 Epidemiological data, 29
Egypt, 63, 64, 187 Epidemiological factors, 225, 228
Electrophoretic karyotypes, 345 Epidemiological studies, 11, 244
Eleusine indica, 294 Epidemiology, 146, 161, 169, 191,
Elicitins, 56 192, 195, 196, 206, 216, 243, 246,
ELISA, 46, 68, 69 247, 249, 261, 263, 304, 305, 310
Elm yellows, 47, 49, 51 Epiphytic bacteria, 36
Elm, 367 EPPO, 147, 319
Elymoclavine, 376 Epuraea obliquus, 311
Embryo, 6 Eradication, 147, 150, 192, 199, 202,
Endocronartium harknessii, 309, 319 303, 304, 317, 318
Endonuclease enzymes, 336 Eremocitrus, 61
Endophyte, 56, 334, 356, 361, Ergocryptine, 376
365–367, 376, 378, 380, 382 Ergonovine, 376
Endophytic fungi, 366, 367, 371, 373, Ergotamine, 376
376, 382, 383 Ergovaline, 377
Energy consumption, 151 ERIC sequence, 352
Enfumafungin, 385 Erosion, 106
England, 318 Erwinia amylovora, 34
Environment, 16, 43, 56, 303–306, 314, Esca, 119–121, 123–129, 130
315, 320, 322, 339, 345, 351, 359 Escherichia coli, 44, 354
Environmental changes, 274, 293 Ethyl alcohol, 205
Environmental conditions, 165, 192, Ethylene biosynthetic precursor, 232
197, 200, 314 Eudarluca caricis, 310
Environmental impact, 146 Eukaryotes, 341, 343, 351, 352
Environmental microbiology, 335 Europe, 4, 5, 7, 32, 48–53, 55,
Environmental parameters, 314, 321 86–90, 100, 102–104, 107, 123,
Environmental samples, 338, 339, 130, 138, 147, 165, 216, 309,
340, 351 310, 319, 323
Enzymatic processes, 310 European plum, 3
398 INDEX
Evolution, 274 Flyspeck, 27, 31, 39, 40

Exclusion, 3, 273 Foliar applications, 254, 255
Exoenzymes, 367 Foliar fungicides, 255
Exotic pathogens, 355 Foliar growth, 251
Exotic rusts, 323 Foliar spray, 77, 78, 79
Experiments, 162, 166, 168, 171, Foliar wetness, 33
172, 174 Fomes fomentarius, 119
Expressed tag sequencing, 348 Fomitiporella vitis, 130
Extraction protocols, 148 Fomitiporia australiensis, 130
Fomitiporia mediterranea, 120–122,
Fagaceae, 318 126, 130
False smut, 271, 286, 296 Fomitiporia polymorpha, 130
Fatty acid methyl ester, 193 Fonsecinone A, 375
FD, 46, 47, 48, 50, 51 Food chain, 303
Feeding deterrents, 372, 376 Food matrices, 345
Fenarimol, 10 Foot rot, 61, 62, 71, 72, 79, 81
Fenbuconazole, 10 Forecast, 179, 181, 185, 186, 187
Fenhexamid, 10, 20 Forecasting models, 161, 162, 173
Fermentation, 202, 203, 383 Forecasting, 161–163, 173
Fertilization, 8, 13, 35, 61, 105, 225, Forest ecosystems, 315
245, 252, 289 Forest tree, 304, 321, 323
Fertilizer, 218, 272, 284, 286–290, Formaldehyde, 255
293, 297, 298 Formulation, 313
Festuca arundinacea, 376 Forsteronia spicata, 375
Festuca pratensis, 373 Fortunella, 61
Festuca, 367, 377 Fosetyl-Al, 77, 79, 80
Festucae, 376 Fragment cloning, 349
Fibrous root rot, 61 Fragraea bodenii, 376
Field infestation, 250 France, 49–51, 54, 55, 87, 89, 96–98,
Field sanitation, 275, 279, 289 103, 111, 123, 127, 132, 139, 227
Fingerprinting, 333, 339, 340, 341, Freeze damage, 12
349, 358–360, 363 Frequency mutations, 353
Fire blight, 27, 28, 32, 34–36 Fruit clustering, 8
Fitness, 164 Fruit crops, 43, 45, 48, 56
Flavescence dorée, 46, 49 Fruit rot, 3, 5–7, 20
Florida, 63, 65, 192, 195, 211, 213, Fruit russeting, 29
214, 216, 217, 220 Fruit storability, 11
Flowers, 320, 321 Fruiting bodies, 312
Fluazinam, 151 Full-blown esca, 119
Fludioxonil, 10, 20, 132 Fumigaclavine C, 373
Fluorescence, 344, 347, 353, 354, Fumigant, 150, 234
357, 359, 362 Fumigation, 14, 150
Fluorescent probe product, 346 Fumitremorgin C, 373
Fluorescent pseudomonads, 236 Functional metabolite, 386
INDEX 399
Fungal antagonists, 303, 315 Gaeumannomyces graminis, 376, 378

Fungal extract, 369 Galls, 37, 38
Fungal genotyping, 340 Gametangium, 65
Fungal metabolites, 365 Gene, 44, 45, 53, 335, 338, 341–349,
Fungal pathogens, 339, 345 351, 352, 354, 356, 358–361,
Fungal propagules, 298 363, 364
Fungal strains, 336, 337, 341, 344 Gene expression analysis, 339,
Fungicidal treatments, 346 354, 360
Fungicidal, 380 Gene expression microarrays, 348
Fungicide, 3, 7–11, 16–18, 20, 27, Gene fragments, 341, 345, 353
29, 30, 31, 34, 38–40, 71, 72, 74, Gene mutations, 29
77, 79, 80, 131, 132, 146, 151, Gene products, 345
163, 164, 166, 168–170, 172–177, Gene sequences, 338
181, 185, 249, 254, 255, 260, 262, Gene targets, 348
263, 273, 274, 278, 279, 282, 289, Genetic basis, 322
297, 298 Genetic diversity, 335, 338, 344
Fungicide application, 7, 8 Genetic linkage maps, 340, 343
Fungicide resistance, 27, 29, 30, 39 Genetic manipulation, 313
Fungus, 5, 7, 8, 13, 15, 16, 37–39, Genetic mapping, 339, 342–344
87–92, 94, 95, 100, 101, 103, 106, Genetic marker loci, 342
109, 138, 139, 141, 143, 147–149, Genetic screenings, 353
153, 179, 181, 182, 187, 243, 245, Genetic tests, 214
246, 249, 254, 260, 279, 285, 286, Genetically engineered plants, 43, 56
293, 295, 304–311, 313, 315, 316, Genoa, 87
318, 322, 334, 336–338, 340–348, Genome, 4, 334–336, 338, 342–346,
351–353, 366, 367, 369, 376 348–352
Fusaria, 338 Genome assemblies, 351
Fusarium, 271, 274, 275, 283, 284, Genome sequencing, 345, 349,
285, 294, 297, 309, 334, 336, 345, 350, 361
367, 382 Genomic DNA, 46
Fusarium compactum, 285 Genomic DNA methylation, 341
Fusarium equiseti, 369 Genomic fragments, 341, 342
Fusarium graminearum, 334 Genomic sequence data, 334, 346
Fusarium moniliforme, 285, 294 Genotype-based classification, 46
Fusarium oxysporum f. sp. Genotypes, 304, 315, 320–323
vasinfectum, 275, 284 Genotyping, 335, 336, 338, 340–343,
Fusarium oxysporum, 284, 361, 382 346, 352
Fusarium verticillioides, 334 Gentianaceae, 376
Fusarium wilt, 197, 206, 227, 236, Georgia, 6, 9, 11, 191, 195, 196
271, 274, 275 Germ tube, 65, 66, 75
Fusiform rust, 321 Germany, 49, 88, 89, 96, 308, 318
Germination, 90, 181, 183, 233, 275,
Gaeumannomyces graminis var. 278, 279, 284, 285
tritici, 376, 378 Germplasm, 149, 252
400 INDEX
Gibberalla fujikuroi, 294 Griseofulvin, 382

Gibberellic acid, 235 Groundnut, 281, 283
Gibberellin, 35 Growing season, 244
GIS, 173 Guam, 192, 195
Gliocladium virens, 282 Guanidine, 365
Gliocladium, 106 Guignardia, 369
Gliovictin acetate, 374 Gum exudation, 67
Gliovictin, 374 Gumming, 12, 14
Gloeosporium musae, 370 Gummosis, 61–63, 65, 67, 68, 72,
Glomerella cingulata, 38 74–77, 79, 80
Glomerella gossypii, 285 Gummy canker, 74
Glomus etunicatum, 259 Gummy liquid, 124
Glomus intraradices, 259 Gummy stem blight, 203
Glomus, 153 Gymnosperm, 386
Glomus mosseae, 345, 356 Gymnosporangium, 307
Glucanase, 312 Gymnosporangium clavipes, 37, 319
Glumes, 287, 289, 291, 296, 297 Gymnosporangium globosum, 37, 319
Glycine max, 245 Gymnosporangium juniperi-
Gossypium arboreum, 274 virginianae, 37, 319
Gossypium barbadense, 274, 277 Gymnosporangium yamadae, 319
Gossypium herbaceum, 274, 277 gyrB, 342
Gossypium hirsutum, 274, 281
Grafts, 109 Hami melon, 195
Graft junction, 123, 128 Hardwood, 102, 104
Graft union, 126 Harmonema dematoides, 380
Gram-negative, 290, 292 Harvest, 6, 8, 16–21, 197–199, 203,
Gram-positive, 44 252, 316
Grape rootstocks, 139 Harvest maturity, 196, 197, 198
Grape, 149 Harvested roots, 249, 258
Grapevine yellows, 49 Harvesting, 128, 132, 244, 249,
Grapevine, 43, 45, 46, 48–52, 56, 119, 262, 263
121–123, 125, 126, 129, 130 Hawthorn, 28, 37
Grass, 229, 367, 376, 378, 383 Hazard areas, 303, 304, 317, 318
Grasshopper, 367 Hazard maps, 318, 319
Gray mildew, 271, 272, 274, 277 Hazardous chemicals, 348
Gray mold decay, 20 HcrVf2 gene, 32
Great Britain, 88 Healed canker, 91, 92
Greece, 64, 88, 96, 227 Healing canker, 91
Green manuring, 282 Health status, 148, 149
Greenhouse bioassays, 218 Helminthosporium gossypii, 279
Greenhouse conditions, 196, 199, 200, Helminthosporium oryzae, 289
203–206 Helminthosporium sativum, 378, 380
Greenhouse technologies, 213 Helminthosporium sigmoideum, 295
Greenhouse, 63, 81 Helminthosporium spiciferum, 279
INDEX 401
Helminthosporium, 271, 279, Hydrocoolers, 258

285, 378 Hydrolytic enzymes, 308
Hemileia vastatrix, 308, 309, 317 Hydroxyanilide, 10
Herbaceous plants, 226 3β-Hydroxyergosta-5-ene;
Herbicidal, 380 3-oxoergosta-4, 6, 8(14),
Herbicides, 231, 235 22-tetraene, 378
Heterobasidion annosum, 315 4-(Hydroxyethyl) phenyl, 348
Heteroconium chaetospira, 376 4-Hydroxyl-phenyl pyruvate
Heteroduplex analysis, 342, 363 dioxygenase, 348
Heteroduplex molecules, 352 3-Hydroxypropionic acid, 375,
Heteroduplex polymorphism assay 376, 386
(HPA), 342 Hymenachne assamica, 294
Heteroecious rusts, 304, 318 Hymenula cerealis, 369
Hexaconazole, 293, 294 Hyperparasite, 303, 306–313, 315
2-Hexyl 5-propyl resorcinol, 155 Hyphae, 122, 225, 229, 246, 254, 334
High throughput capillary Hyphomycetes, 106, 225, 279, 283
electrophoresis (PCR-CE), 353 Hypocotyls, 194
Honduras, 195 Hypovirulence, 85, 88, 96, 99–104,
Honeydew, 193, 195 107–111
Horizontal gene transfer, 44 Hypovirulent blight strains, 102
Hormonal balance, 44 Hypovirulent strains, 91, 100, 102,
Hormonema dematioides, 374 104, 107, 109
Hormonema, 375, 385 Hypovirus, 100, 102, 107, 109, 111
Horticultural practices, 3, 15 Hypoxia, 75
Host plant, 339, 343, 348, 376
Host range, 215 Iberian peninsula, 318
Host resistance, 27, 164, 249 Identification, 3, 333–338, 340–344,
Host susceptibility, 66, 80 347–349, 353, 354, 356, 358–361,
Host tissues, 334, 345 363, 364
Host, 137, 138, 143, 145, 146, Ilar Virus, 281
148, 149 Illinois, 195
Host-pathogen interactions, 215 Image analysis, 18
Hot water treatment, 131, 152 Immunofluorescence, 46
HPA, 342, 353 Incubation, 8, 16, 40, 63, 67, 68,
HSP60, 342 183, 184
Human health, 306, 316 India, 63, 64, 139, 198, 271, 274–281,
Human pathogen, 344 283, 286, 287, 289–292, 294, 295,
Humidity, 7, 16, 17, 20, 33, 105, 127, 298, 307
187, 212, 213, 216, 217, 278, 285, Indiana, 192
287, 293, 295, 297 Indofil, 277
Hyalesthes obsoletus, 52 Indole derivatives, 365
Hybridization conditions, 339 Indonesia, 64
Hybridization probes, 339, 354 Induced resistance, 323
Hybrids, 102, 103, 274, 275 Infected branches, 320
402 INDEX
Infected tissues, 334, 340 Interbreeding, 165

Infection, 3, 5–8, 13–15, 17–19, 62, Intercropping, 271, 273, 282
63, 66, 67, 74, 76, 79, 80, 85, Intergeneric crosses, 322
88–95, 98, 99, 102–109, 161, Intergenic regions, 342
162, 164, 165, 168, 174, 179, International Plant Protection
181, 183–187, 227, 233, 234, Organisation, 319
243, 245–250, 252, 254, 255, Iowa, 195
258, 276, 277, 279–281, 283–287, IPM, 272, 273, 275, 297, 298, 366
289, 292–297, 304, 307, 310, 312, IPM strategies, 192, 197
313, 316–321 Iprodione, 9, 10, 20, 255, 260
Infectivity, 346 Iran, 63, 64, 139
Ink disease, 85–87, 89, 90, 94, 95, Ireland, 318
104–106, 110, 111 Irrigation, 5, 13, 16, 56, 61, 63,
Inner bark, 376 65–67, 69–72, 75, 79, 144, 150,
Inoculum load, 295 169, 179, 180, 195–197, 205, 225,
Inoculum, 7, 8, 11, 13, 17, 19, 28, 233, 234, 245, 250, 262, 272, 276,
32–34, 36, 39, 40, 61, 65, 66, 283, 285, 289, 291, 292, 295
68–72, 77, 80, 99, 102, 107–109, Isocoumarin, 365, 379
111, 145, 152, 153, 164, 166, 191, Isocoumarin derivative, 365, 379
192, 195–200, 205, 206, 228–232, Isolates, 88, 99, 100–102, 107, 109, 111
234, 235, 243, 245, 247–249, 251, Isopestacin, 376
254–258, 261, 304, 305, 307, 6-Isoprenylindole-3-carboxylic
310–312, 314, 316–320, 323 acid, 376
Inocutis jamaicensis, 130 Israel, 49, 161, 166, 169, 195
Insect models, 180 Italy, 3, 43, 48, 49, 51, 54, 55, 63–65,
Insect pests, 179 85, 87–89, 95, 96, 99, 102–107,
Insect repellent, 381 110, 112, 119, 121, 123, 128, 129,
Insect vector proteins, 47 137, 139, 140, 148, 149, 225, 227,
Insect vectors, 43, 47, 49, 52, 53, 230, 231, 236, 303, 308, 318, 319
55, 56
Insecticidal activity, 376, 379, Janthinobacterium lividum, 154
380, 382 Japan, 64, 71, 86, 87, 139, 149, 151,
Insecticidal, 378 165, 195
Insecticide, 376, 378 Japanese plum, 3
Insects, 15, 17, 43, 46, 47, 91, 281, Jersey, 88
285, 290 Jesterone, 376, 385
Inspection, 191 Juniper, 37
Integrated disease management, Juniperus, 37, 38
191, 271 Juniperus communis, 375
Integrated management, 3, 27, 36, Juniperus scopulorum, 37
211, 303 Juniperus virginiana, 37
Integrated pest management, 196,
271, 272, 298, 366 Karyotypic variation, 344
Integrated strategies, 219 Kernel, 295
INDEX 403
Ketones, 381 Leucostoma canker, 3, 5, 15, 16, 21

Korea, 86, 87, 149 Leucostoma cincta, 4, 15, 21
Kresoxim-methyl, 132 Leucostoma cinctum, 15
Leucostoma persoonii, 4, 15
Laccase, 99 Liana, 381
Lactic acid bacteria, 154 Ligustrum vulgare, 122
Lactobacillus, 338 Linkage markers, 345
Lagerstroemia indica, 122 Lint diseases, 271
l-aminocyclopropane-l-carboxylic Linum usitatissimum, 322
acid, 232 Lipids, 381
Landscape, 85–87 Liquid culture, 378
Late blight, 161–177, 181, 183, 184 Lithium salt, 320
Late blight epidemics, 170 L-leucine, 193
Late blight simulators, 161 Locus specific PCR primers, 341
Late season Phoma blight, 271 Loline, 376, 377
LATEBLIGHT, 163, 167 Lombardy, 97
Latent infection, 5–7, 11 Lucerne, 283
Lateral clipping, 251, 255, 262 Lucilia seracata, 378
Laurus nobilis, 122 Lupinus luteus, 139
Lavandula stoechas, 231 Lycopersicon chilense, 232
Leaf chlorosis, 62 Lycopersicon esculentum, 215, 232
Leaf crumple, 271, 281 Lycopersicon pimpinellifolium, 227
Leaf curl virus, 275, 281 Lycopersicon pinnellii, 215
Leaf discoloration, 49 Lycopersicon, 215
Leaf distortions, 281
Leaf lamina, 276, 296 MAbs, 215
Leaf scar, 16 Macadamia, 139
Leaf spot, 271, 275–280 Macedonia, 96
Leaf streak, 271, 291, 292 Machinery, 72
Leaf symptoms, 283 Macrophomina, 271, 280
Leaf veins, 124 Macrophomina phaseolina, 280, 282
Leaf yellowing, 28 Magnaporthe grisea, 287, 334, 356,
Leaf, 376, 379 358, 382
Leafhoppers, 43, 47, 51, 56 Magnaporthe salvinii, 295
Leaves, 181, 187 Magnesium, 230, 289
Lebanon, 49 Maize, 229, 231, 382
Lecanicillium lecanii, 308 Major repeat sequence (MRS), 344
Leersia hexandra, 288, 294 Maleic hydrazide, 320
Legionella, 338, 359 Malus atrosanguinea, 32
Legumes, 229 Malus coronaria, 28
Lesion, 7, 11, 28, 37, 38, 66, 68, 76, Malus floribunda, 31
164, 166, 168, 172, 183, 194, 212, Malus iowensis, 28
213, 220, 276, 278–280, 287, Malus micromalus, 32
289–292, 295 Malus, 28, 149, 156
404 INDEX
Mammals, 340 Metabolic genes, 351

Management, 3, 8, 9, 11, 13, 15, 18, Metabolic toxin, 376
20, 27, 29, 32, 34, 36, 40, 43, 46, Metabolites, 146, 154, 156, 310, 311,
48, 49, 56, 61, 69, 71, 74–76, 80, 365, 367, 376–378
85, 87, 102, 104, 107–109, 111, Metagenomics, 350
161–164, 167, 169, 171, 179, 187, Metalaxyl, 163, 164
191–193, 198–200, 204–206, 225, Metamodel, 164
243–246, 249–253, 256, 257, 261, Meteorological stations, 186
271–275, 277, 279, 282, 286, Metham sodium, 150, 255
289–291, 295, 297 6-Methoxymellein, 260
Management practices, 27, 40 1-N-Methyl albonoursin, 378
Management strategy, 191, 200, 204 Methyilisothiocyanate, 234
Mancozeb, 29, 31, 40, 168, 170, 219, Methyl bromide, 150, 234, 255
220, 277, 288, 289, 294, 297 Methyl iodine, 151
Maneb, 10 Methylated-DNA, 341
Manganese, 233, 289 Mexico, 63, 64, 163, 165, 166, 195,
Mango, 138, 139, 149 198
Manilkara bidentata, 370, 385 Michigan, 96, 99
Mapped STS, 349 Microarray, 47, 336, 343, 344,
Marche, 97 346–349, 356–358, 360, 362, 364
Marine environments, 351 Microarray-based genotyping, 347
Medicago sativa, 283 Microbead arrays, 348, 356
Mefenoxam, 77–80 Microbes, 334, 348
Melampsora capraearum, 310 Microbial antagonists, 225, 230, 249
Melampsora epitea, 310 Microbial communities, 348, 352
Melampsora farlowi, 319 Microbial flora, 315
Melampsora larici-populina, 328 Microbial pathogens, 333
Melampsora lini, 319, 322 Microchannels, 353
Melampsora medusae, 309 Microclimate, 243, 251, 252, 256, 257
Melampsora, 309, 310 Microdochium nivale var. majus, 345
Melanconis modonia, 90 Microorganism, 45, 47, 56, 106, 251,
Melanconis perniciosa, 90 253, 259, 307, 311, 313, 315, 334,
Melanconium, 376 335, 338, 345, 350, 351, 365, 366
Melanconium betulinium, 375 Microsclerotia, 141, 144, 225–229,
Melia azedarach, 382 231, 233, 283
Melida, 70 Microscopy, 148
Mellein, 379 Middle East, 123
Meloidogyne incognita, 382 Mill’s Table, 29, 31
Melon, 192, 193, 195, 206 Missouri, 195
Membrane, 43, 44, 47 Mitochondria, 336
MEMS devices, 354 Mitochondrial DNA, 336
Mentha piperita, 228 Model, 162–166, 168, 169, 171–174,
Mesocriconema xenoplax, 13 177, 179–186, 255, 257, 263
INDEX 405
Moisture, 5, 16, 17, 33, 39, 179, 246, Mutagenic agents, 322
248, 249, 251, 252, 256, 257, 282, Mutant spores, 322
284, 286, 291, 294 Mutation, 322
Moisture sensor, 179 Mycelia, 90–93, 100, 102, 340
Molecular beacons, 346 Mycelia sterilia, 371
Molecular detection, 137, 148 Mycelial fans, 90, 91, 142
Molecular genetic analysis, 344, 346 Mycelial masses, 142
Molecular genetic techniques, 333 Mycelial strands, 144
Molecular mapping, 350 Mycelium, 65, 66, 77, 122, 141–146,
Molecular methods, 69, 333, 361, 362 149, 153, 225, 226, 245–249,
Molecular techniques, 3, 11 311, 312
Molecular technologies, 333 Mycelophagus castaneae, 89
Molecular typing, 336 Mychorrizae, 89
Mollicutes, 43, 44 Myclobutanil, 10, 29
Monilinia, 3, 5, 7, 9, 12, 19 Mycoflora, 312
Monilinia fructicola, 4–7, 9, 12, Mycology, 337
18, 20 Mycoparasite, 348
Monilinia fructigena, 5, 7 Mycoparasitic microbes, 245
Monilinia laxa, 4, 5, 7, 8, 9, 20 Mycoparasitism, 305
Monitoring, 61, 75, 80, 274, 297 Mycophagous animals, 245, 254
Monochoria vaginalis, 294 Mycoplasmas, 148
Monoclonal antibodies, 46 Mycoreovirus, 153
Mono-terpene compounds, 107 Mycorrhizal fungi, 56, 107, 153
Morocco, 64, 227 Mycosphaerella, 360
Morphological characters, 333, 340 Mycosphaerella areola, 277
Morphology, 340, 357 Mycosphaerella gossypina, 279
Mortality, 88, 96–99, 104, 108 Mycosphaerella graminicola,
Mosquito larvae, 378 343, 358
mRNA, 45, 341, 347, 349 Mycotoxins, 336, 360
Mucor rot, 19 Myosin, 47
Mugello, 110 Myrobalan, 13
Mulching, 32, 230, 250, 251, 262 Myrothecium, 271, 278, 279, 285, 371
Mullein, 374 Myrothecium roridum, 275, 278, 285
Multiline cultivars, 315, 323 Myrsinaceae, 375
Multiple cropping, 272 Myxosporium, 370
Multiple displacement amplification
(MDA), 351 Naphthalene, 382
Multiple repeat sequences, 349 Natural enemies, 271, 273
Multiplex amplification, 354 Natural stands, 88
Mummies, 8 Neck blast, 287, 288
Musa acuminata, 383 Necrophyte, 90
Muscodor albus, 381 Necrosis, 33, 44, 49, 54, 119,
Muscodor vitigenus, 381 123–127, 192, 281, 284, 292
Muskmelon, 194 Necrotic spots, 68, 281
406 INDEX
Necrotrophic soilborne fungus, 245 Nucellar clones, 72, 80

Nectarine, 3, 4, 12, 17, 18, 20 Nucleic acid sequence, 201
Nectria, 367 Nursery, 61–63, 65, 71, 72, 80, 119,
Negative binomial pattern, 70 124, 128, 130, 131, 133, 137–140,
Nemaguard, 13 147, 149, 150, 304
Nematicidal, 382 Nutrient deficiency, 56
Nematode damage, 12 Nymphs, 47, 51
Nematode, 4, 13, 14, 74, 230, 234,
286, 298 Oak disease, 173
Nematospora nagpuri, 285 Ohio, 227
Neotyphodium coenophialum, Oils, 3, 18
376, 377 Oklahoma, 195
Neotyphodium lolli, 376 Okra, 283
Neotyphodium unicinatum, 373 Olea europaea, 122
Neotyphodium, 367, 376, 382, 383 Oligonucleotide, 351, 352, 358,
Nephotettix nigropictus, 296 360–364
Nephotettix virescens, 295, 296 Olive, 65, 138, 139, 140, 149
Netherlands, 179, 182–184, 187 Oncopeltus fasciatum, 367
New York, 87, 165, 166, 172 Ontario, 7, 21
New Zealand, 9, 12, 32, 119, 123, 255 Oogonium, 65
Nicaragua, 195 Oomicota, 64
Nigeria, 63, 64 Oomycete, 89
Nightshades, 231 Oospores, 164, 165
Nigrospora sphaerica, 371 Opium, 370
Nigrospora, 371 Orchard, 3, 5, 8, 9, 13, 28, 33, 36,
Nitrate, 230, 232 38, 63–70, 72, 74, 75, 79, 80,
Nitrification, 89 85–89, 95, 104, 107–111,
Nitrogen fertilizer, 291 137–140, 143, 145–147, 149,
Nitrogen, 232, 285, 287–291, 151–153
297, 298 Oregon, 195
Nitulidae, 311 Ornamental citrus, 63
Nodulisporic acid, 378 Ornamental plants, 65
Nodulisporic acid A, 378 Orthosprin, 99
Nodulisporic acid A1, 378 Oryza rufipogen, 294
Nodulisporic acid A2, 379 Oryza sativa, 296
Nodulisporium, 378 Ostryia carpinifolia, 88
Non-hypovirulent strains, 107 Outbreaks, 183–185, 191–193, 195,
Non-transcribed intergenic 196, 198, 200, 202, 204, 206
spacer, 337 Overwintering, 262
North America, 4, 5, 7, 48, 51, 53–55, 3-Oxoergosta-4, 6, 8(14),
87, 123, 130, 139, 216, 318 22-tetraene, 378
North Carolina, 195 Oxychloride, 36
North Queensland, 193 Oxytetracycline, 217
Nostoc, 154 Ozone, 255, 260
INDEX 407
Paddy, 272, 273, 283, 297 Pathogenic interaction, 366, 382

Paecilomyces H-036, 375 Pathogenic organisms, 352
Paecilomyces W-001, 375 Pathogenicity, 133, 193, 206, 344,
Paecilomyces, 386 348, 383
Paenibacillus alvei, 236 Pathotypes, 342, 343
Painting, 78 Paullinia paullinioides, 381
Pakistan, 64, 275 PCR, 45, 46, 69, 71, 200, 201,
Pandemic dynamics, 87 335–337, 340–343, 345–349,
Panicle blast, 287 351–364
Panicle, 287–290, 292, 294, 295 PCR identification-size analysis, 354
Panicum, 297 PCR products, 351
Panicum repens, 288, 289 PCR reaction, 336, 345, 351
Panicum walense, 294 Pea, 229
Papaver somniferum, 371, 385 Peach leaf curl, 4
Paracoccidioides brasiliensis, Peach scab, 4
348, 361 Peach, 3–9, 11–16, 18, 138, 139,
Paracoccidioidomycosis, 348 141, 149
Paraguay, 309 Pear, 138, 139, 143, 144, 148, 156
Paramagnetic plastic beads, 202 Pear decline, 55
Paraphyses, 142 Pecan, 149
Paraquat, 6 Pectin, 11
Parasite, 87, 88, 90, 95, 99, 106–112, Pectolytic enzymes, 258
130, 305–307, 310, 312, 314, Pediococcus, 338
318–320 Penicillium, 236, 336, 359, 370
Parasitoids, 48, 56 Penicillium expansum, 18, 20
Parawilt, 271, 272, 275, 284 Penicillium frequentans, 8
Parenchyma, 292 Penicillium janczewskii, 374
Pasania edulis, 384 Penicillium oxalicum, 236
Passiflora edulis, 149 Peniprequinolene, 374
Pathogen, 3, 7, 13, 20, 21, 65, 77, 80, Pentachloronitrobenzene, 255
81, 90, 105, 107, 108, 111, 120, Pentaketides, 382
127, 133, 137, 139, 140–153, Pentastiridius beierii, 52
191–193, 196–199, 201–203, 205, Pepper, 228
211–213, 216, 217, 219, 244, 246, Peramine alkaloid, 376
249, 250, 256, 257, 261, 262, 304, Peramine, 376, 377
305, 310–319, 322, 323, 333–335, Perennial ryegrass, 378, 383
339, 341, 343, 344, 346, 348, 354, Periderm, 253, 311
367, 382 Peridermium peckii, 310
Pathogen eradication, 273 Peridium, 311
Pathogen groups, 216 Perithecia, 28, 29, 91, 92
Pathogen identification, 333, 364 Perithecium, 142
Pathogen load, 335 Peroxyacetic acid, 203
Pathogen variability, 335 Persimmon, 149
Pathogenesis, 191, 206 Peru, 161, 166, 168, 170
408 INDEX
Peruvian Amazon, 381 Phaeomoniella zymoides, 121

Pest, 148, 152, 271, 272, 274, 294, Phaeotracheomycosis, 124
298, 365–367, 378, 381, 382 Phage, 219, 220
Pest management, 273, 286 Phakopsora gossypii, 271, 275, 280
Pest monitoring, 273 Phakopsora pachyrizi, 308
Pestalotiopsis jesteri, 371, 376, 385 Phanerochaete chrysosporium, 334,
Pestalotiopsis microspora, 376, 384 356, 360
Pestalotiopsis, 384 Phaseolus vulgaris, 245
Pesticide, 9, 29, 39, 272–274, 282, Phellinus igniarius, 119
283, 286, 296–298, 303, 305, 306, Phenol oxidase, 99
313, 316, 317 Phenol, 376, 382
Pesticide poisoning, 365 Phenolic compounds, 149
Pesticide use, 9 Phenolics, 11
Pest-resistant varieties, 271, 273 Phenological event, 246
Petals, 246, 263 Phenological stage, 314
Petiole, 276, 280, 281, 284 Phenotype, 346
Petri decline, 119 Phenotypic uniformity, 323
Petri disease, 120, 124, 128 Phenylpyrrole, 10
Pezicula, 380 Phialophora chlamydospora, 121
PFGE, 344, 345, 350 Phialophora, 121
PFGE-RFLP, 344 Philloptosis, 62, 67
Phaeoacremonium, 119–121, 123, 127 Phlebiopsis gigantea, 315
Phaeoacremonium aleophilum, Phleum pratense, 379, 380
121, 123 Phloem, 43, 44, 47, 53
Phaeoacremonium angustius, 121, 123 Phoma exigua, 275, 280
Phaeoacremonium australiense, 123 Phoma sorghina, 369
Phaeoacremonium Phomapsichalasin, 378
chlamydosporum, 121 Phomopsis amygdali, 4
Phaeoacremonium inflatipes, Phomopsis oblonga, 367
121, 123 Phomopsis phaseoli, 376
Phaeoacremonium iranianum, 123 Phomopsis, 367, 378
Phaeoacremonium krajdenii, 123 Phosphate, 232
Phaeoacremonium mortoniae, 121, 123 Phosphorous acid, 77–81
Phaeoacremonium parasiticum, Phosphorous, 152, 285, 288
121, 123 Photosynthesis, 45, 146
Phaeoacremonium rubrigenum, 121 Photosynthetic efficiency, 312
Phaeoacremonium scolyti, 123 Photosynthetic products, 304
Phaeoacremonium subulatum, 123 Phthalamide, 10
Phaeoacremonium venezuelense, 123 Phylloplane bacteria, 276
Phaeoacremonium viticola, 121, 123 Phyllosticta, 373
Phaeomoniella, 119, 120, 121 Phylogenetic analysis, 338, 358
Phaeomoniella chlamydospora, Physalis minima, 216
119, 120 Physical barrier, 315
Phaeomoniella pinifoliorum, 121 Physiological activity, 99
INDEX 409
Physiological status, 103 Pine species, 323

Physocnemum brevilineum, 367 Pinus banksiana, 309
Phytoalexin, 77, 235, 260 Pinus contorta, 309
Phytopathogenic bacteria, 202, 346 Pinus contorta var. latifolia, 309
Phytopathogenic fungi, 350 Pinus elliottii, 310, 318
Phytopathogens, 334, 335 Pinus halepensis, 318
Phytophthora, 4, 61, 62–72, 74, 85, Pinus laricio, 318
86, 90, 94, 95, 104, 106, 107, 112 Pinus monticola, 307
Phytophthora, 61, 62, 65, 68, 75, 81, Pinus muricata, 309
179, 181, 182, 309, 367 Pinus nigra, 318
Phytophthora boehmeriae, 285 Pinus palustris, 318
Phytophthora cactorum, 112, 382 Pinus pinea, 318
Phytophthora cambivora, 85, 86, 90, Pinus radiata, 309, 318
93–95, 104–107, 111 Pinus serotina, 318
Phytophthora capsici, 376, 378 Pinus sylvestris, 382
Phytophthora cinnamomi, 64, 85, 86, Pinus taeda, 310, 318
90, 95, 104–107, 111 Pinus virginiana, 318
Phytophthora citricola, 346 Pit hardening, 6–8, 16, 18
Phytophthora citrophthora, 66–69, Plant breeding, 303, 304, 321, 322
71, 75, 77, 79, 80 Plant debris, 279, 282, 295
Phytophthora diseases, 77 Plant defense reaction, 376
Phytophthora hibernalis, 64 Plant diseases, 305, 317
Phytophthora infestans, 161, 164–167, Plant growth hormone, 382
169–171, 181–184, 346, 356, 357 Plant growth-promoting
Phytophthora megasperma, 64 rhizobacteria, 219
Phytophthora nicotianae, 61, 64–67, Plant nutrition, 290
69–72, 74–77, 79, 80 Plant pathogen, 43, 47, 334,
Phytophthora palmivora, 65 335, 366
Phytophthora parasitica, 64 Plant pathology, 161
Phytophthora rot, 75 Plant protectant, 382
Phytophthora syringae, 64 Plant spacing, 251, 293
Phytophtora root rot, 61, 62 Plant species, 367
Phytoplasma diseases, 43, 54 Plant surface, 311, 314
Phytoplasma infection, 45 Plant tissue, 245, 260, 339
Phytoplasma, 4, 43–49, 51 Plant varieties, 321
Phytosanitary services, 319 Plant vascular system, 44, 47
Phytotoxic metabolites, 99 Plantago lanceolata, 52
Phytotoxicity, 17, 36 Plantations, 88, 95
Phytotoxins, 120, 127 Planting density, 304
Picea, 321 Plasma membrane, 308
Picea glauca, 376 Plasmodesmata, 44
Piedmont, 89, 97 Plastic tunnel, 213
Pine, 307, 317–321, 323 Pleospora herbarum, 369
Pinaceae, 376 Plowing, 230
410 INDEX
Plum, 3–5, 7, 8, 12, 13, 16, 18, Prochloraz, 278

138, 139 Prokaryotes, 43, 44, 47, 49
Plum pockets, 4 Propagating materials, 137, 144,
Plum pox virus, 4 147–150
Plum pox, 4 Propagation cycle, 316
Poaceae, 373 Propagation, 121, 128, 130, 132
Podosphaera clandestina, 4, 16 Propagules, 63, 65, 69, 106, 225, 227,
Podosphaera leucotricha, 33 228, 230, 231
Polar flagellum, 290, 292 Prophylactic strategy, 187
Pollination, 196, 197, 204 Prophylactic treatment, 31
Polyacetylene falcarindiol, 253 Propiconazole, 9, 10, 26, 29, 294
Polyethylene, 229, 234 Prosopis farcta, 139
Polygalacturonase, 99 Protease, 99
Polygenic horizontal resistance, 81 Protectant fungicides, 320
Polymerase chain reaction, 45 Protoplasm, 144
Polymorphic DNA loci, 346 Prumnopytis andina, 374, 386
Polymorphic genomic loci, 337 Prunes, 3
Polymorphism, 337–340, 342, 343, Pruning, 8, 13, 15, 33, 34, 36, 40, 61,
346, 350, 352, 353, 356–364 71, 74, 108–110, 230
Polyphenol oxidases, 226 Prunus avium, 4, 55
Polyporales, 371 Prunus domestica, 3
Polystyrene trays, 205 Prunus mahaleb, 140, 143
Poncirus, 61, 71 Prunus persicae, 3
Poplar, 139, 144 Prunus salicina, 3
Portugal, 49, 50, 88, 89, 96, 104, 105, Prunus, 55
111, 139 Pseudocommis vitis, 89
Post-harvest diseases, 271 Pseudomonads, 230, 235
Postharvest fruit rots, 3, 18 Pseudomonas, 13, 153, 192, 276,
Postharvest fungicides, 260 278, 293
Potassium deficiency, 283 Pseudomonas aureofaciens, 293
Potassium phosphate, 218 Pseudomonas chlororaphis, 153
Potassium phosphonate, 77 Pseudomonas fluorescens
Potassium, 230, 233, 283, 284, 288, 289 PCL1606, 153
Potato, 161–170, 173–181, 183 Pseudomonas fluorescens, 153,
Potentilla reptans, 52 291, 293
Powdery mildew, 3, 5, 16–18, 27, Pseudomonas gardneri, 214
31–33, 34, 39 Pseudomonas malvacearum, 276
Preharvest decay, 63 Pseudomonas pseudoalcaligenes
Pre-planting infections, 119 subsp. citrulli, 192
Prevention, 72, 74, 317 Pseudomonas putida, 153
Primary inoculum, 277, 279, 288 Pseudomonas syringae pv.
Primers, 7, 12, 46, 337–340, 343, 349, morsprunorum, 4, 12, 14
351, 352, 360, 362, 363 Pseudomonas syringae pv.
Principal Component Analysis, 105 persicae, 12
INDEX 411
Pseudomonas syringae pv. Pyrus, 28

syringae, 4, 12, 14 Pyrus communis, 55
Pseudomonas syringae pv. Pythiaceae, 64
tomato, 213 Pythium, 275, 382
Pseudomonas syringae, 4, 12–14 Pythium ultimum, 382
Pseudothecia, 29, 32, 40
Psyllas, 53, 55 qRT-PCR reaction, 345
Puccinia arachidis, 307 Quantitative genetic traits, 253
Puccinia cestri, 309 Quantitative resistance, 253
Puccinia chrysanthemi, 308 Quarantine, 303, 304, 317, 319, 336
Puccinia conspicua, 309 Queensland, 227
Puccinia coronata, 310 Quercus, 88, 92, 106
Puccinia glumarum, 321 Quercus cinerea, 318
Puccinia graminis f. sp. tritici, 311 Quercus ilex, 88, 122
Puccinia graminis, 310, 318 Quercus marilandica, 318
Puccinia horiana, 308, 309 Quercus nigra, 318
Puccinia penniseti, 307 Quercus petrea, 88
Puccinia pittieriana, 319 Quercus pubescens, 88
Puccinia polysora, 323 Quercus rubra, 318
Puccinia recondita, 308–310, 346, Quercus variabilis, 386
357, 382 Quercus virginiana, 88
Puccinia sorghi, 309, 310 Quick decline, 55
Puccinia striiformis, 321, 346, 357 Quinoline, 10
Puccinia sylvatica, 307 Quinone, 382, 383
Puccinia vincae, 307 Quinoxyfen, 10
Puccinia violae, 309
Puccinia, 376 Races, 318, 322
Pulsed-field gel electrophoresis Race-specific resistance, 322
(PFGE), 344 Rain, 5, 15, 17, 19, 29, 63, 65, 67, 76,
Pumpkin, 193, 195 79, 80
Pustules, 90, 91 Rain forests, 318
Pycnidia, 40, 91, 92, 99, 101, Rain tree, 383
109, 280 Rainfall, 127, 186, 286, 290, 292, 297
Pycric acid, 320 Rainwater, 105, 106
Pyracantha, 28 Ralstonia solanacearum, 213
Pyraclostrobin, 10, 11 Ramularia areola, 275–278
Pyricularia grisea, 287–288 Ramularia gossypii, 277
Pyricularia oryzae, 376 RAPD, 338–340, 342, 343, 349, 357,
Pyricularia, 376 358, 360, 361, 363, 364
Pyrimethanil, 10, 20, 132 RAPD fingerprinting, 338, 339, 340
Pyrrocidine A, 373 RAPD fragments, 343, 349
Pyrrocidine B, 373 RAPD markers, 339, 343, 349, 358
Pyrrolizidine, 365 Rapeseed, 245, 254, 256
Pyrrolopyrazine, 376 16S rDNA, 46, 52
412 INDEX
16S rRNA, 352 Rhizoctonia solani, 282, 285, 292,

Real time analysis, 46 293, 382
Real time-PCR, 69 Rhizoctonia, 275, 309, 376
Recilia dorsalis, 296 Rhizomorpha necatrix, 138
Reddening, 140 Rhizopus, 3, 4, 18, 275
Reforestation, 95 Rhizopus stolonifer, 4, 18
Regulations, 336 Rhizosphere, 235, 236
Reinfestation, 152 Rhodotorula rubra, 369
Relative humidity, 183, 196, 197, 200, Rhodotorula, 8
284, 288 Rhopalosiphum padi, 367, 377
Remote Sensing, 187 Ribes, 318
Rennin, 99 Ribosomal gene complex, 337, 338
Reproduction, 304, 313, 316 Ribosomal gene polymorphisms, 352
Reproduction cycles, 344 Ribosomal gene regions, 337
Resistance genes, 31, 212, 289, 304, Ribosomal gene repeat, 337
315, 321, 323 Ribosomal gene signatures, 351
Resistance inducer Ribotyping, 337, 349, 353
microorganisms, 43 Rice blast, 271
Resistance induction, 315 Rice crop, 286, 290, 296
Resistance management, 11 Rice diseases, 271
Resistance, 3, 8–11, 15, 16, 18, 20, Rice ecosystems, 286, 296
27, 29, 31–35, 40, 43, 55, 56, 61, Rice, 229, 234, 271–274, 286, 382
71, 72, 79, 85, 88, 102, 103, 111, Ripening, 67
146, 149, 161, 163, 164, 166–169, Risk assessment, 179
171, 173–177, 249, 252–254, 259, Risk factors, 256, 257
260, 262, 273–275, 287, 289, 291, Risk management, 3
292, 294, 297, 335, 343 Risk model, 180
Resistant cultivars, 211, 219, 225, RNA, 338, 339, 357, 358, 360,
291, 298 361, 363
Resistant genotypes, 11 RNA detection assays, 339
Resistant germplasm, 322 RNA virus, 296
Resistant varieties, 35, 283, 291, Robinia pseudoacacia, 122
315, 322 Rocky Mountain juniper, 37
Respiration, 252, 259, 260 Roguing, 43, 49, 51, 53, 56
Restriction enzyme Root exudates, 65, 66
isoschizomers, 341 Root rot, 61–63, 66, 67, 71, 74–76,
Restriction fragment length 79, 80, 271, 381
polymorphism, 45 Rootgraft union, 35
Reverse transcriptase, 354 Roots, 244, 245, 247, 249, 252, 253,
RFLP, 45, 46, 337, 340, 341, 342, 254, 258, 259, 260, 262, 263
344, 352, 353, 360 Rootstock blight, 34
Rhinocladiella, 378 Rootstock, 13, 35, 49, 55, 61, 62, 71,
Rhizoctonia bataticola, 280 72, 77, 121, 124, 131, 149
Rhizoctonia cerealis, 376, 378 Rosaceae, 52
INDEX 413
Rose, 37 Saturated aminopyrrolizidine, 376

Rosellichalasin, 146 Scab, 28, 29, 31, 32, 34, 36, 38, 39, 40
Rosellinia arcuata, 138 Scaphoideus titanus, 47, 50, 51
Rosellinia bunodes, 138 SCAR loci, 349
Rosellinia desmazieresii, 138 Schizaphis graminum, 367, 377
Rosellinia necatrix, 137–146, 148 Schizophillum, 370
Rosellinia pepo, 138 Scilly Isles, 139
Rosellinia quercina, 138 Scion, 72, 77, 79
Rosellinia root rot, 138, 156 Sclerenchyma, 292
Rosellinia, 138 Sclerotia, 142, 245, 247, 249–251,
Rosellinic acid, 146 253–255, 261, 262, 280, 282, 292,
Rosnecatrone, 146 293, 295, 297, 298
Rotation, 205, 228, 229, 231, 234, Sclerotinia diseases, 249, 252–255
250, 263 Sclerotinia minor, 250
Row spacing, 262 Sclerotinia rot, 243, 244, 247
rRNA, 338, 351, 352, 356, 359, Sclerotinia sclerotiorum, 243–247,
361, 362 249, 250–261, 263, 334, 346
rRNA-based identity, 352 Sclerotinia stem rot, 251, 253, 254
RTSV, 296 Sclerotinia, 250, 252
Rugolosin, 99 Sclerotium bataticola, 280
Rugulosin, 380 Sclerotium oryzae, 295
Russia, 5, 88 Sclerotium rolfsii, 275
Rust, 271, 275, 280, 303, 304, Scorpions, 346
306–311, 313, 315, 317 Screening, 15, 21
Rust control, 303 Scrophulariaceae, 374
Rust diseases, 27, 37, 38 Scytalidium uredinicola, 310, 311
Rust resistance, 323 SDS-PAGE fractionated proteins, 342
Rust sori, 310 Secondary infections, 183
Rutaceae, 61 Secondary inoculum, 197
Rye, 229 Secondary metabolites, 45, 48,
Ryegrass, 376, 378 382, 383
Seed fermentation, 202
Salix gracilostyla var. Seed infection, 191, 196, 204, 206
melanostachys, 372 Seed production, 191, 195–197, 199,
Samanea saman, 383 203, 204
Sampling, 333 Seed, 191–193, 195–204, 206, 211,
Sandwich hybridization, 338, 358 213, 216, 219
Sanitary practices, 61, 71, 119, 128 Seedlings, 192–194, 196, 197,
Sanitation, 225, 230 199–201, 205, 277, 278, s280,
Sap flow, 128 282, 288–290, 291, 292, 294
SAR, 48, 219, 220 Seeding rate, 293
Sarcina lutea, 380 Seedling blight, 192
Sardinia, 97, 98 Seedling diseases, 271, 298
Sarocladium oryzae, 294 Seedlots, 196, 200, 202, 204
414 INDEX
Seeds, 191, 192, 195–204, 206, 245 Slovenija, 49

Selection pressure, 322 Smut, 381
Selection, 3, 5, 14 SNP, 341, 343, 344, 346
Selective substrates, 71 SNP mapping, 343
Senescing leaves, 246, 248, 252, 263 Sodium arsenite, 119, 131, 132
Sensitivity analysis, 161 Sodium azide, 151
Sepals, 28 Sodium dimethyl dithio
Septoria musiva, 338, 357 carbamate, 283
Septoria populi, 338, 357 Sodium hypochlorite, 202
Septoria populicola, 338, 357 Sodium orthophenylphenate, 260
Sequence polymorphism, 342 Soil, 137, 140, 141, 143–153
Sequence similarities, 351 Soil aeration, 67
Sequence tagged site (STS), 350 Soil borne inoculum, 283
Sequencing methods, 349, 350 Soil borne pathogen, 137, 140
Serbia, 49, 50 Soil condition, 105
Serial Analysis of Gene Expression Soil fumigation, 151
(SAGE), 349, 358 Soil inoculum, 225, 228
Sesbania, 293 Soil management, 61, 71, 74
Sesquiterpene, 365, 379 Soil microbial profile, 254
Setaria viridis, 52 Soil microflora, 106
Sexual recombination, 318 Soil microorganisms, 152, 348
Sexual reproduction, 182 Soil pH, 13, 145, 146
Sheath blight, 271, 272, 286, 292, 293 Soil preparation, 61, 71, 74
Sheath rot, 271, 272, 294 Soil resident diseases, 298
Shoot blight, 34, 35 Soil solarization, 152, 156, 382
Shoots, 108, 109 Soil temperature, 105, 282, 284
Sicily, 97 Soil water status, 76
Signal molecules, 45 Soilborne pathogens, 147, 150, 151, 156
Silica, 287, 289 Soil-borne pathogens, 230, 234
Silver-enhanced gold Solanum bulbucastanum, 174
nanoparticles, 354 Solanum nigrum, 52, 216
Simulated experimentation, 162 Solar radiation, 182, 229, 230
Simulation models, 161, 168, 173 Solarization, 225, 230, 234, 250, 282,
Simulation, 161, 162, 166, 168, 169, 284, 293, 298
171–177 Solarized soil, 230
Simulator, 162, 163, 167, 168, 170, Sooty blotch, 27, 28, 31, 39, 40
171, 172, 174 Sorbus, 28
Single nucleotide polymorphism, Sordariomycetes, 138
341, 352 Sori, 306, 310
Single-strand conformation South Africa, 64, 119, 121, 123, 127,
polymorphism (SSCP), 341 130, 131
16S-23S intergenic spacer region, 46 South America, 123, 130, 216
Skirin, 99 South Carolina, 7, 9, 192, 195
Slovakia, 88, 96, 97 Southern hybridization, 46
INDEX 415
Soviet Union, 3 Stem, 90, 91, 93, 94, 104, 271–273,

Soybean, 229, 245, 250, 251, 253, 254 275, 276, 278–282, 284, 295,
Spain, 49, 51, 54, 55, 63, 64, 87–89, 376, 382
96–98, 111, 139, 146, 148, 149, Stem canker, 271, 273
152, 156 Stem lesions, 278
Spatial pattern, 70 Stem rot, 271–273
Specialization, 228 Stem sawfly, 382
Specificity, 201 Stemphylium botryosum, 371
Sphaceloma, 370 Stereum hirsutum, 119, 130
Sphaerellopsis philum, 310 Steroid, 373
Sphaerotheca pannosa, 4, 16 Stomata, 127, 196, 197, 216
Spodoptera frugiperda, 367, Stone fruit crops, 3, 9, 10
376, 383 Stone fruit diseases, 3, 5, 11, 20
Sporangia, 65, 66, 68, 74, 166 Storage rots, 258
Sporangiophores, 183 Storage, 3, 19, 243–245, 247, 249,
Spore germination, 162, 168 252, 253, 255, 258–260, 262
Spore masses, 38 STR, 341, 342, 352–354
Spore production, 310 STR genotyping, 341, 354
Spore, 5, 8, 19, 29, 37, 38, 40, 162, Strains, 45, 46, 48, 51, 53, 54, 182
168, 181, 183, 307, 309, 311, 312, Strand displacement amplification
316, 317, 318, 322 (SDA), 351
Sporogenous structures, 340 Strawberry, 234
Sporulation, 11, 16, 17, 33, 77, 88, Streptomyces, 236, 378
162, 164–166, 172, 174, 277, Streptomyces canescens, 236
279, 284 Streptomyces citreofluorescens, 236
Spots, 213, 218 Streptomyces griseoviridis, 236
Spraying programs, 179 Streptomyces plicatus, 236
Sprouting, 152 Streptomyces pulcher, 236
Sprouts, 87, 88, 90, 91, 97–99, 103, Streptomycin sulfate, 36, 148, 276
104, 108 Streptomycin, 34, 36, 204, 217
Spruce budworm cell line CF-1, 376 Stress factors, 12, 382
Spruce budworm, 383 Stress, 9, 45
Squash, 192, 193 Strobilurin, 9, 10, 18, 30, 31, 34, 235
SRC, 244, 246–256, 258 Stromata, 91–93, 102, 142
5S rRNA, 337 Stumps, 88, 90, 91, 104, 109
SSCP, 341, 342, 349, 353, 356, 360 Suberin, 7, 16, 21
ssDNA fragments, 341 Subiculum, 142
Stagonospora nodorum, 334, Sucrose glucose ratios, 252
346, 357 Sucrose, 219, 220
Staphylococcus aureus, 354, 380 Sugar beet, 229
Starch accumulation, 44 Sugarcane, 273
Stargrass, 382 Sulfonamides, 320
Steam disinfestation, 261 Sulfur, 17, 29
Steam, 151, 250, 258, 261, 263 Suppression, 243, 253, 255, 258, 259
416 INDEX
Suppressive mechanisms, 314 Teak, 383

Suppressiveness, 152 Tebuconazole, 10, 278
Surface wetness, 248 Tectona grandis, 383
Surface-like receptors, 232 Telial horns, 37
Surgery, 61, 71 TEM, 43, 44, 46
Susceptible tissues, 246, 255 Temperature, 5, 7, 11, 17–19, 64–68,
Sustainability, 87 70, 105, 122, 130, 140, 145, 152,
Sustainable management, 243 183, 185, 191, 229, 230, 233, 244,
Sweden, 187 248–250, 256, 258, 259, 262, 263,
Sweet cherries, 6, 8, 20, 149 277–278, 281–284, 286–288, 291,
Sweet clover, 229 293–295, 297
Sweet orange, 68, 72, 78, 80 Template DNA, 201
Sweet potato, 283 Terminalia arjuna, 371, 386
Switzerland, 88, 96, 98, 99, 112, Terminalia morobensis, 376
165, 318 Terpenoid, 373–374
Symbiosis, 305, 348, 359 Texas, 195
symptom, 27, 28, 33, 34, 37–39, 43, Text messaging, 179
44, 46, 48, 49, 51, 53–56, 62, 67, Thai medicinal plant, 371, 387
68, 72, 74, 79, 119–124, 126, 128, Thailand, 64, 139, 216
149, 192, 194, 196–198, 200, 213, Thanatephorus cucumeris, 282, 292
218, 226, 228, 230–232, 235, 271, Thaumatin-like proteins, 253
272, 276, 277, 280, 281, 283, 284, Theobroma gileri, 382
289, 291–295, 297, 334, 367 Thermal PCR cycling, 354
Symptomatic plants, 129 Thermal programed capillary
Synnemata, 142, 143 electrophoresis (TP-CE), 353
Synthetic fungicides, 29 Thiabendazole, 234, 235
Systemic acquired resistance, 48, Thiophanate-methyl, 11, 234,
211, 219 255, 294
Systemic compounds, 168 Thiram, 10
Systemic fungicide, 61, 74, Thrips, 281
77–79, 321 Thymol, 8, 20
Systemic infection, 290 Thymus mastichina, 231
Thysanoptera, 281
T7 polymerase RNA Tiger stripes, 283
amplification, 339 Tillage, 225, 228, 230, 245, 251, 262
Taiwan, 143, 195 Tillering, 288, 290, 292
Talaromyces flavus, 230 Tissues, 120, 121, 126
Talaromyces, 236 Tobacco Streak Virus, 271, 281
Tall fescue, 367, 376 Togninia minima, 121
Tannase, 99 Tolerance, 232
Taphrina deformans, 4 Tomato, 161, 162, 177, 211, 225,
Taphrina pruni, 4 283, 359
Taxus mairei, 375, 386 Tomato bacterial diseases, 213
Tea, 138, 139, 382 Tomato big bud, 49
INDEX 417
Tomato production, 212 Trunk, 61, 62, 65–67, 72,

Tomato ringspot virus, 4 74–78, 80
Topoisomerase II, 338, 358 Trunk gummosis, 61, 62
Torreya grandis, 375, 386 Tryfloxystrobin, 10
Torula, 89 Tuber(s), 151
Total RNA, 339 Tuber infection, 179, 180
Toxins, 312 Tuberculina costaricana, 307
Tracheomycosis, 120, 126–128, 133 Tuberculina maxima, 307
Tracheomycotic fungi, 119, 120, Tuberculina persicina, 307
127, 128 Tuberculina sbrozzii, 307
Transcriptomes, 348, 357, 361 Tuberculina, 306, 307
Transmission Electron Tungro Virus, 271, 295
Microscopy, 43 Tunisia, 227
Transmission, 47, 51–53, 56, 85, Turgor, 284
100–102, 200, 202, 203 Turkey, 64, 88, 96, 195
Transmission rates, 100 Tuscany, 97, 99, 101, 319
Transovarial transmission, 47 Twigs, 8, 13, 15, 61, 62, 66–68, 77,
Transpiration, 127, 304 78, 148
Transplant, 191, 193, 196, 199, 200, Twig dieback, 62
202, 204, 206 Tyloses, 226
Transplanting, 94
Tranzschelia pruni-spinosae, 307 Udine, 87
Tree species, 309, 316, 319, 323 UK, 139, 251, 255
Tree, 316 Ulocladium alternariae, 371
Trematosphaeria, 369 Uncinula, 370
Triademefon, 29 United States, 3, 4, 6, 7, 9, 20, 38, 62,
Trichoderma atroviride, 8 96, 149
Trichoderma harzianum, 106, 153, Urea, 284, 289
236, 282, 283, 293 Uredia, 280
Trichoderma koningii, 236, 254 Urediniomycetes, 304, 306, 310,
Trichoderma reesei, 334 315, 320
Trichoderma viride, 8, 106, 236, Uredo cyclotrauma, 309
282, 283 Uromyces dianthi, 308
Trichoderma, 106, 131, 150, 153, Uromyces, 307–309, 319
230, 236, 253, 254, 278, 290, 291, Urtica dioica, 52
293, 295, 312 USA, 119, 123, 161, 165, 166,
Tricyclazole, 289 191–193, 195, 196, 206, 211, 217,
Triflumazole, 10 228, 318, 319, 323, 337, 357, 358,
Trifluralin, 235 360, 361
Triforine, 10, 321 Ustilago maydis, 334
Trimming, 251
Tripterygium wilfordii, 376, 378 Validation, 333
Triticum aestivum, 369, 385 Vascular browning, 226
Tropical tree, 3 Vascular pathogens, 119
418 INDEX
Vascular tissues, 283 Wageningen, 179

v-c groups, 100–102 Walnut, 94, 95, 112
v-c loci, 100 Washington, 15, 18, 251
Ve gene, 227, 232 Water capacity, 145
Vectors, 45, 47, 49, 51–53, 55, 56 Water management, 289
Vegetables, 273 Water mold, 381
Vegetative hyphae, 282 Watermelon, 191–198, 202–206
Vegetative propagation, 43, 47 Waxes, 11, 109, 110
Veins, 49 Weather data, 179, 181
Venturia inaequalis, 28 Weather forecast, 29, 179–181, 186
Vertical cordon, 132 Weather stations, 179
Verticillium, 371 Weather, 161–163, 167–169, 172–174,
Verticillium albo-atrum, 225, 228, 211, 213, 216–218, 252, 272,
229, 232, 233, 236 276–278, 287, 289, 291–293,
Verticillium album-minimum, 307 298, 317
Verticillium coccorum, 307, 308 Weed control, 225, 231
Verticillium compactiusculum, 307 Weed seeds, 230, 234
Verticillium dahliae race, 2, 227 Weed species, 231
Verticillium dahliae, 225–236, Weed, 43, 52, 56, 61, 71, 76, 137,
275, 283 138, 143, 145, 149, 150, 152,
Verticillium hemileiae, 308 195, 197
Verticillium lecanii, 308 WGS analysis, 350
Verticillium malthousei, 308 Wheat, 250, 343, 345, 357, 358, 363,
Verticillium wilt, 4, 225, 227, 228, 376, 378, 382
230, 376 White mold, 253
Verticillium, 225–228, 230–236, 271, White root rot, 137–139, 141, 142,
275, 283, 307 146, 148–150, 152
Verticillium-free soil, 230 White swallow wort, 317, 319
Vf cluster, 32 Whole genome shotgun sequencing
Vf gene, 31 (WGS), 351
Vigna aconitifolia, 282 Wild apples, 28
Vinca major, 307 Wild bur gherkin, 195
Vincetoxicum hirundinaria, 317 Wild relatives, 322
Vinclozolin, 255 Wilting, 91, 108, 119, 127
Vine decline, 120 Wind speed, 186
Vine diseases, 132 Wisconsin, 227
Vine yield, 119 Witches’ broom, 53, 90
Virginia, 96, 99, 102 Witches’ brooming, 44
Viroids, 346, 361 Wood pulping process, 218
Virulence, 88, 91, 100, 102, 139, 146, Wood, 119–128, 130, 132
153, 304, 310, 314, 335, 367, 382 Wood-rotting fungi, 348
Viruses, 336, 346, 351, 356 Woody plants, 52, 378
Volatile organic compounds, 381 Wound response, 16
Volatile substances, 152 Wounding, 8, 94
INDEX 419
Wounds, 13, 15, 20, 119, 125, Xanthomonas, 212–215

127–129, 132 X-disease, 4
Xylaria, 367, 382
Xanthomonad races, 216 Xylariaceae, 138, 367
Xanthomonads, 214, 215, 217 Xylariomycetidae, 138
Xanthomonas axonopodis pv. Xylem vessels, 283–285
vesicatoria, 214 Xylem, 120, 121, 226
Xanthomonas campestris f. sp.
malvacearum, 275, 285 Yeast genes, 347
Xanthomonas campestris pv. Yeast, 339
malvacearum, 276 Yellowing, 140
Xanthomonas campestris pv. Yugoslavia, 88
oryzae, 291
Xanthomonas campestris pv. pruni, 4 Zambia, 205
Xanthomonas campestris pv. Zea mays, 369, 373
vesicatoria, 214, 216, 219 Zinc sulfate, 291
Xanthomonas malvacearum, 276 Zinc-deficiency, 287
Xanthomonas oryzae pv. oryzae, Ziram, 10, 278, 279
290, 291 ZnSO4, 282
Xanthomonas oryzae pv. Zonation, 173
oryzicola, 291 Zoospores, 65, 66, 74, 75
Xanthomonas perforans, 212, 215 Zymomonas mobilis, 342, 356
Xanthomonas vesicatoria, 212, 215

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Integrated Management of Diseases Caused by Fungi, Phytoplasma

Cover Illustration: Bacterial Spot Fruit lesions. (Courtesy Jeffrey B. Jones)

ISBN: 978-1-4020-8570-3 e-ISBN: 978-1-4020-8571-0

Library of Congress Control Number: 2008927634

Printed on acid-free paper

SECTION 1 - Diseases of Perennial Crops

2 Towards a Sustainable, Integrated Management of Apple Diseases .......... 27

2.3.4. Cultivars ...................................................................................... 35

3 Management and Ecology of Phytoplasma Diseases of Grapevine

4 Management of Citrus Diseases Caused by Phytophthora spp.................... 61

2.4. Symptomatic Diagnosis......................................................................... 67

5 Biological Control and Management of Chestnut Diseases......................... 85

4.3. Mixed Inoculum .................................................................................. 101

6 The Esca Disease Complex........................................................................... 119

7 Integrated Management of Rosellinia necatrix Root Rot on Fruit

6. Control ....................................................................................................... 146

SECTION 2 - Diseases of Annual Crops

9 An example of Integrated Forecasting System for Phytophthora

2.8. Sub Model 3: Combination of Unprotected Leaf Area

10 Integrated Pest Management of Bacterial Fruit Blotch of Cucurbits ...... 191

11 Integrated Management of Tomato Bacterial Spot ................................... 211

3.3. Distribution of Pathogen Groups ........................................................ 216

12 Integrated Management of Verticillium Wilt of Tomato .......................... 225

13 New Progress in the Integrated Management of Sclerotinia Rot

3.2. Storage Practices ................................................................................. 258

14 Integrated Management of Key Diseases of Cotton and Rice ................... 271

5.8. Stem Rot ............................................................................................. 295

15 Biological and Integrated Means to Control Rust Diseases ...................... 303

SECTION 3 - Advances in Management Tools

2.1.1. Ribotyping................................................................................. 337

17 Endophytic Fungi for Pest and Disease Management ............................... 365

Jorge L. Andrade-Piedra Santa Olga Cacciola

O. M. Bambawale Ruangelie Edrada-Ebel,

Greg J. Boland G. A. Forbes

Giovanni Bubici W. E. Fry

Jan Hadders Susheel Kumar

Cezarina Kora Keith R. Mitchelson

M. T. Momol Aleksa Obradovic

Rita Musetti Leonardo Schena

Franco Nigro Peter Sholberg

D. Shtienberg Tullio Turchetti

Giuseppe Surico Ron R. Walcott

In the following review of biological control and management of chestnut

analysis of resistance components, comparative epidemiology, development of

able to provide a further tool, useful in the comprehension and sustainable

Diseases of Perennial Crops

INTEGRATED MANAGEMENT OF STONE FRUIT

Table 1. Common diseases of stone fruit.

Disease Causal agent Comments

Armillaria root rot Armillaria spp. Occasional problem

2.1. Pathogen Identification and Disease Biology

2.2. Integrated Management of Brown Rot

Table 3. Susceptibility of cherry cultivars to brown rot in controlled laboratory tests

Cherry, sweet Susceptibility to Monilinia fructicola

Bing Medium to High

3.1. Pathogen Identification and Disease Cycle

3.2. Integrated Management of Bacterial Canker

Figure 2. Bacterial canker of peach with gumming from cankers.

Olson and Jones (1983) reduced P. syringae pv. morsprunorum populations to

4.1. Pathogen Identification and Disease Cycle

4.2. Integrated Management of Leucostoma Canker

canker surfaces such as underground drip or microsprinkler irrigation. Common

5.1. Pathogen Identification and Disease Cycle

Figure 3. Powdery mildew of nectarine, showing white mildew growth on fruits.