The 12th International Trichoderma & Gliocladium Workshop

Innovations and Applications
Programme
& Abstracts
The 12th International Trichoderma
& Gliocladium Workshop
27 30 August 2012
Commerce Building
Lincoln University
New Zealand
TG2012@lincoln.ac.nz
www.bioprotection.org.nz/TG2012
Welcome
On behalf of the Organising Committee we are delighted to welcome you to The 12th International
Workshop on Trichoderma and Gliocladium at Lincoln University, Lincoln, New Zealand. This is the first
time the TG workshop has travelled to the South Pacific, a region rich in cultural diversity and steeped
in tradition. The indigenous people of New Zealand (Mori) named our country Aotearoa, which means
Land of the Long White Cloud and for those of you who have travelled here for the first time we warmly
welcome you to Aotearoa - Haere Mai, Haere Mai, Nau Mai Haere Mai.
The theme for TG2012 is Innovations and Applications and this is reflected in the varied program
which spans from pure fundamental science to real world applications delivering solutions to industry.
We have been impressed by the large number of registrants and submitted abstracts, particularly from
around the Asia-Pacific region a clear indication of the global economic importance of this group
of microorganisms. With many delegates attending for the first time, this event will be an excellent
opportunity for forging new networks and collaborations between Trichoderma researchers.
Lincoln is situated in the heart of Canterbury, a large rural province in the South Island of New Zealand.
It is located 20 minutes south of Christchurch The Garden City, which is New Zealands third largest
city. To the west of Lincoln lie the majestic Southern Alps and National Park Sanctuaries and to the east
is picturesque Banks Peninsula with its numerous harbours, bays and the worlds rarest and smallest
dolphin Hectors Dolphin. While you are here, we hope that you will take some time to enjoy the local
sites and visit some of the many attractions Canterbury has to offer.
During the conference please do not hesitate to contact one of the committee members for further
information and once again welcome and we hope that you enjoy the Workshop and find it a valuable
experience.
Kind regards

Kirstin McLean & Johanna Steyaert
Co-Chairs of The 12th International Trichoderma and Gliocladium Workshop Organising Committee.
27 30 August 2012
Commerce Building
Lincoln University
New Zealand
www.bioprotection.org.nz
Programme
Organising Committee
Monday 27th August 2012 | Commerce 1 for all sessions

3.00
Registration desk open
5.00-5.10
Welcome address | Jo Steyaert and Kirstin McLean
Kirstin McLean
Johanna Steyaert
Alison Stewart
Artemio Mendoza-Mendoza
Anna Heslop
Andrew Holyoake
Janine Johnson
5.10-6.00
6.00-8.00
Agrimm Technologies Ltd Plenary lecture: Distinguished Professor Alison Stewart

Have Trichoderma researchers delivered on their promises?
Mix and mingle with substantial nibbles and drinks
Secretariat
Tuesday 28th August 2012 | Commerce 1 for all sessions

8.55 -9.10
Housekeeping
Jan Latham
Professional Development Group, Lincoln University
Session 1 | Environmental signalling | Chair: Benjamin Horwitz

9.10-9.40
Keynote: Monika Schmoll
Editors
9.40-10.00
Kirstin McLean
Johanna Steyaert
Alison Stewart
Trichoderma communication: environmental communication, sensing and

responding to external cues
Trichoderma-plant communication: are first signals and final interactions related?
Nieto-Jacobo, M. F; Braithwaite, M.; Brown, C.; Greenwood, D.; Hill, R. A.; Steyaert, J. M.; Stewart, A.
and Mendoza-Mendoza, A.
10.00-10.20
The role of H+-pump in nutrient transport during germination of Trichoderma

atroviride conidia
imkovi, M.; Vilimov, V.; Olejnkov, P.; Hudecov, D.; Krytofov, S. and Vareka, .
Cite proceedings as:

The 12th International Workshop on Trichoderma and Gliocladium. Eds. K.L. McLean, J.M. Steyaert,
A. Stewart. 27-30th August, Lincoln University, New Zealand, pp.
10.20-10.30
10.30-11.00
Workshop Photograph - steps of the Lincoln University Library

Morning tea | Commerce Foyer
Session 1 cont. | Environmental signalling | Chair: Monika Schmoll

11.00-11.20
Interactive effects of abiotic factors in the in vitro conidiation of Trichoderma
stromaticum, a biocontrol agent of the witches broom disease in cacao
Leite, S. S.; Corra, F. M.; De Souza, J. T. and Loguercio, L. L.
11.20-11.40
Trichoderma atroviride xylanase regulator 1 (Xyr1) and its role in mycoparasitism

Reithner, B. and Mach, R. L.
11.40-12.00
Over-expression of the Saccharomyces cerevisiae Dolichol-phosphate mannose

synthase (DPM1) gene in Trichoderma hamatum GD12 and its consequences for
biological control and plant growth promotion (PGP)
Le Cocq, K. F. and Thornton, C. R.
12.00-12.20
Terpenes of Trichoderma
Monte, E.; Cardoza, R. E.; Malmierca, M. G.; Hermosa, R. and Gutirrez, S.
12.30-1.30
Lunch | Commerce Foyer
Programme
Session 2 | Biotechnological applications | Chair: Christian Derntl and Rita Gorsche
1.30-2.00
Keynote: Markku Saloheimo
TBA
2.00-2.20
Development of Trichoderma reesei as a host for production of enzymes for
enzymatic hydrolysis of liginocellulosic biomass
Merino, S.
2.20-2.40
Tra1, an adenine deaminase gene participated in copper accumulation in

Trichoderma reesei
3.10-3.40
Biological and molecular investigation of a potential strain of Trichoderma

harzianum against carbendazim
4.40-5.00
Sundram, S.; Seman, I. A. and Naidu, Y.
10.30-11.00
Session 3 cont. | Biocontrol and ecology | Chair: Dean Metcalf

11.00-11.20
Field application of Trichoderma technology in semi-arid eastern plains and humid
south eastern plains of Rajasthan state, India
Shama, P.
Bienkowski, D.; Hicks, E.; Braithwaite, M.; Falloon, R. E.; McLean, K. L. and Stewart, A.
11.40-12.00
Jijakli, M. H.; Phillips, N. and Lahdenper, M.-L.
12.00-12.20
Development of a cold tolerant Trichoderma atroviride LU132 variant for control

of Botrytis cinerea on grapes using protoplast technology
Kandula, J.; Braithwaite, M.; Steyaert, J. M.; Hay, A. J. and Stewart, A.
Derntl, C.; Steiger, M.; Gorsche, R.; Mach-Aigner, A. R. and Mach, R. L.
12.30-1.30
Impacts of a Trichoderma transformant with Metarhizium genes on the bacterial

community in the Asian corn borer larvae midgut
Session 3 cont. | Biocontrol and ecology | Chair: Robert Hill

1.30-1.50
Application of Trichoderma asperellum in the control of wilt of pineapple caused
by Fusarium oxysporum in the field in Costa Rica
Expression of Trichoderma viride endoglucanase in the larvae of silkworm, Bombyx

mori L. and characteristic analysis of the recombinant protein
Lunch | Commerce Foyer
Obregn, M.
1.50-2.10
Wang, M.-X.; Liang, S.; Li, X.-H. and Maio, Y-G.
Colonisation with Trichoderma harzianum and/or Pseudomonas fluorescens

improves quality of farm yard manure
Identification and characterization of cellobiose transporter inTrichoderma reesei
Zaidi, N. W. and Singh, U. S.
Wednesday 29 August 2012 | Commerce 1 for all sessions

8.55-9.00
Housekeeping
Session 3 | Biocontrol and ecology | Chair: Alison Stewart
9.00-9.30
Keynote: Robert Hill
2.10-2.30
Biological control of Botrytis cinerea in grapes in Tasmania

Metcalf, D.
Management of Phomopsis canker in tea plantations using Trichoderma

harzianum under field conditions
Ponmurugan, P.
Factors influencing rhizosphere competence and endophytic colonisation of

Trichoderma atroviride
Guazzone, N.; McLean, K. L.; Hill, R. A. and Stewart, A.
2.30-2.50
Capacity of Trichoderma stromaticum to colonise cacao sapwood

De Souza, J. T.; Loguercio, L. L. and Pomella, A. W. V.
2.50-3.10
Trichoderma root endophytes enhance plant health and vigour
9.50-10.10
Improved potato yields from in-furrow application of Trichoderma
Afternoon tea | Commerce Foyer
Kou, Y.; Zang, W.; Xu, J.; Chen, G. and Liu, W.
9.30-9.50
Morning tea | Commerce Foyer
Applications of Gliocladium catenulatum strain J1446 formulations to control soilborne and foliar pathogens on vegetable, ornamental and aromatic plants
Li, Y.-Y.; Fu, K. and Chen, J.
4.20-4.40
Potential use of Trichoderma spp. as biological control agents in suppressing

Ganoderma basal stem rot in oil palm seedlings
Sharma, P.; Singh, U. S.; Zaidi, N. W.; Kumar, P. V.; Saravanan, K.; Singh, D. V. and Sharma, M.
Session 2 cont. | Biotechnological applications | Chair: Markku Salohemio

3.40-4.00
A novel utilization of the biopolymer chitin: Generation of a pharmaceutically
relevant fine chemical by a whole-cell catalyst approach in Trichoderma
4.00-4.20
10.10-10.30
11.20-11.40
Fu, K.; Fan, L.; Li, Y. and Chen, J.
2.40-3.00
Programme
Development and validation of a species-specific marker for Trichoderma

atroviride
Lange, C.; Hay, A. J.; Kandula, J.; Yardley, J. A.; Braithwaite, M.; Weld, R. J.; Stewart, A. and Steyaert, J. M.
3.20-4.30
6.00-10.00
Afternoon tea | Poster Session
6pm
Coach departs outside Recreation Centre for Conference Dinner at Riccarton Park
10pm
Coach departs Riccarton Park for return to Lincoln University
Conference Dinner
Programme
Posters
Thursday 30 August 2012 | Commerce 1 for all sessions

9.25-9.30
Housekeeping
Effect of temperature and length of incubation period on Trichoderma atroviride LU132 conidial
production, germination and bioactivity. Daryaei, A.; McLean, K. L.; Glare, T. R. and Stewart, A.
Session 4 | Omics | Chair: Enrique Monte

9.30-10.00
Keynote: Scott Baker
Development of a microbial biocontrol agent for control of Sclerotinia sclerotiorum in vegetables. Hicks,
E.; McLean, K. L.; Braithwaite, M. and Stewart, A.
Connecting Trichoderma reesei phenotypes with genotypes

10.00-10.20
Trichoderma virens a tale of two strains

Mukherjee, P. K. and Horwitz, B. A.
10.30-11.00
Morning tea
Session 4 cont. | Omics | Chair: Scott Baker

11.00-11.20
Improving in vivo footprinting finding a more sensitive way to detect proteinDNA interactions
Gorsche, R.; Derntl, C.; Mach, R. L. and Mach-Aigner, A. R.
11.20-11.40
Transcriptomic responses of tomato plants to Trichoderma

Monte, E.; Domnguez, S.; Cardoza, R. E.; Gutirrez, S.; Hermosa, R.; Nicols, C. and Rubio, M. B.
11.40-12.00
Expression of genes encoding small secreted cysteine-rich proteins (SSCPs) in

Trichoderma virens root interactions
Horwitz, B. A.; Moran-Diez, M. E.; Trushina, N. and Kenerley, C. M.
12.00-12.20
Identification of putative effectors through bioinformatics analysis of Trichoderma

proteomes
Lawry, R; Aleman, I.; Delaye, L.; Olmedo, V.; Horwitz, B. A.; Kenerley, C. M.; Herrera-Estrella, A.;
Brown, C.; Greenwood, D.; Stewart, A. and Mendoza-Mendoza, A.
12.30-1.30
Lunch
Session 4 cont. | Omics - Taxonomy | Chair: Leandro Loguerico

1.30-1.50
Biodiversity of Trichoderma species in New Zealand
Braithwaite, M.; Steyaert, J. M.; McLean, K. L.; Johnston, P. R.; Hill, R. A.; Ball, S.; Stewart, A. and
Bissett, J.
1.50-2.10
Molecular diversity of Trichoderma species from groundnut rhizosphere

Srilakshmi, P.; Prasad, K. S. and Rao, M. M.
2.10-2.30
Biodiversity of root-endophytic Trichoderma from Malaysian Borneo

Cummings, N. J.; Steyaert, J. M.; Ambrose, A.; Roslan, H.; Agbayani, F. V.; Okang, A. E.; Valdez, R. B.;
Yatim, M; Hay, A. J.; Ball, S.; Bissett, J.; Chong, L.; Abdullah, J.; Stewart, A. and Hill, R. A.
2.30-3.15
3.20-3.50
3.50-4.30
Conference summary discussion | Jo Steyaert and Kirstin McLean

Afternoon tea
Prize giving, AGM, next conference location, conference close
Jo Steyaert and Kirstin McLean
Effect of a Trichoderma bio-inoculant on emergence and growth of Camelina seedlings. Kandula, D. R. W.;
Alizadeh, H. and Stewart, A.
Incorporation of trehalose into Trichoderma atroviride conidia and its effect on viability of conidia during
storage. Kandula, J.; Braithwaite, M.; Steyaert, J.; Hay, A. J. and Stewart A.
Mycelial injury as a key abiotic factor affecting conidiation of genetically distinct Trichoderma
stromaticum isolates. Leite, S. S.; De Souza, J. T. and Loguercio, L. L.
Heterologous expression characteristics of Trichoderma viride endoglucanase V in the silkworm, Bombyx
mori L. Liang, S.; Wang, M-X.; Li, X.-H. and Miao, Y.-G.
Antimicrobial mechanism of Trichoderma harzianum L-amino-acid oxidase N-terminal sequence derived
peptides. Lin, H.-S.; Liu, S.-Y.; Peng, K.-C. and Lo, C.-T.
The impact of illumination of the production of cellulases and anthraquinones from Trichoderma
harzianum ETS 323. Liu, S.-Y.; Hsu, C.-W.; Chaing, J.-H.; Lo, C.-T. and Peng, K.-C.
Screening strains and mechanisms of Trichoderma spp. for phosphate solubilisation. Lo, C.-T.; Feng, P.-C.
and Peng, K.-C.
Validation of protocols to evaluate the quality of Brazilian commercial products based on Trichoderma
spp. Lucon, C. M. M.; Bettiol, W.; Haddad, P. E.; Pinto, Z. V. and Morandi, M. A. B.
Antagonism of local isolates of Trichoderma spp. on pineapple heart rot disease caused by Phytophthora
cinnamomi var cinnamomi in Costa Rica. Mata, X.; Obregn, M.; Lorito, M. and Monte, E.
Non-target impacts of the biocontrol agent Trichoderma atroviride on native ecosystems in New Zealand.
McLean, K. L.; Dodd, S. L.; Minchin, R. F.; Ohkura, M. and Stewart, A.
ABC transporters in acetic acid resistance of Trichoderma atroviride. Olejnkov, P.; Vyhonsk, Z.;
Palukov, V.; Chromkov, R.; Reichov, J.; imkovi, M. and Krytofov, S.
Effect of chitin-rich residues on the chitinolytic activity of Trichoderma harzianum in in vitro and
greenhouse experiments. Pascual, J. A.; Lopez-Mondejar, R.; Blaya, J. and Ros, M.
Plant growth promotion and induction of resistance against anthracnose in common bean by
Trichoderma spp. Pedro, E. A. S.; Lucon, C. M. M.; Harakava, R. and Guzzo, S. D.
Posters
Agrimm Technologies Ltd Plenary lecture
Rhizosphere competence and survivability of Trichoderma harzianum (Th3) in crops grown in semi-arid
eastern plains of Rajasthan state, India. Sharma, P.; Saini, M.; Patel, A.; Deep, S. and Bhati, D. S.
Have Trichoderma researchers delivered on their promises?
Activation of a glutathione S-transferase and a deoxycytidine deaminase expression in Brassica oleracea

var capitata that correlates with resistance to Rhizoctonia solani challenge during Trichoderma harzianum
ETS 323 association. Shibu, M. A.; Lin, H.-H.; Yang, H.-H.; Liu, S.-Y.; Lo, C.-T. and Peng, K.-C.
Alison STEWART
Delivering Trichoderma spp. spores via seed coating. Swaminathan, J.; Wilson, M.; Wessman, P. and
Jackson, T.
Bio-Protection Research Centre, P.O. Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand.
alison.stewart@lincoln.ac.nz
For decades, Trichoderma researchers have obtained funding for their research on the basis that this
fungal genus shows potential for commercial application in the promotion of plant growth or control
of plant diseases. This paper critiques how well Trichoderma researchers have delivered on these
commercial goals using comparative biometrics that assess a range of research outputs. The data will
highlight how well we translate our research into commercial outcomes compared with other research
disciplines and answer the question of whether we are focusing our research on the right areas or not.
The paper will also address the major constraints to achieving consistent performance of Trichoderma
products in the field environment. It will review the different abiotic and environmental factors that
drive successful early stage colonisation by Trichoderma of plant surfaces (both below and aboveground) and highlight ways in which this information can be used to develop better formulation and
application strategies. The key restrictions, both economic and regulatory, that impede the widespread
commercialisation of Trichoderma biocontrol agents will be discussed and the value of international
research networks highlighted as one means to overcome some of these limitations. The aim of the
paper will be to challenge the Trichoderma research community to find ways to deliver greater impact
and applied outcomes from our work.
27 30 August 2012
10
11
Commerce Building
Lincoln University
New Zealand
SESSION 1
Environmental signalling
KEYNOTE
Trichoderma communication: environmental communication,
sensing and responding to external cues
Monika SCHMOLL
Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology,
Gumpendorfer Strae 1a-1665, A-1060 Vienna, Austria.
Monika.schmoll@tuwien.ac.at
Trichoderma reesei (anamorph of Hypocrea jecorina) is predominantly known for its efficiency in
cellulase production. However, our research revealed that despite decades of cultivation in the lab
this biotechnological workhorse still preserved its evolutionary heritage. The important group of
genes encoding glycoside hydrolases shows considerable regulation by light and the light response
machinery. Accordingly, environmental signals perceived by the pathway of heterotrimeric G-proteins
are transmitted in a light dependent manner to eventually adjust cellulase gene expression to the
current nutritional and light conditions. In this interconnection between nutrient and light signalling, the
phosducin like protein PhLP1 and the light regulatory protein ENV1 act as a mutually regulated pair and
hence constitute an important node between the two pathways. However, the function of ENV1 is not
limited to this transcriptional interaction, but also extends to regulation of transcript levels of G-protein
alpha subunit genes, cAMP levels and development of T. reesei. Investigation of the impact of the light
signalling pathway on sexual development in T. reesei revealed a negative regulation of pheromone
precursor and pheromone receptor genes of the photoreceptors BLR1 and BLR2. The strong upregulation
of these genes caused by a lack of ENV1 even caused female sterility in the respective mutants. In
accordance with the interrelationship of light response with nutrient signalling, also PhLP1, the G-protein
beta and gamma subunit as well as the downstream pathway of cAMP signalling are important for sexual
development, which reflects an interconnected network of signal integration for light, nutrients and
developmental signals in T. reesei.
27 30 August 2012
15
Commerce Building
Lincoln University
New Zealand
Trichoderma-plant communication: are first signals and final

interactions related?
NIETO-JACOBO, M.F.1; Braithwaite, M.1; Brown C.2; Greenwood, D.3; Hill, R. A.1;
Steyaert, J. M.1; Stewart, A.1 and Mendoza-Mendoza, A.1
Bio-Protection Research Centre, P.O. Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand.
maria.nieto-jacobo@lincoln.ac.nz; mark.braithwaite@lincoln.ac.nz; robert.hill@lincoln.ac.nz; johanna.steyaert@lincoln.ac.nz,
alison.stewart@lincoln.ac.nz; artemio.mendoza@lincoln.ac.nz
Biochemistry Department and Genetics Department, University of Otago, 710 Cumberland St, Dunedin 9016, New Zealand.
chris.brown@otago.ac.nz
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
david.greenwood@auckland.ac.nz
Trichoderma spp. are soil-borne filamentous fungi with superior biocontrol capabilities. Many species
of Trichoderma have been shown to enter the roots of higher plants and form symbiotic relationships
(endophytism). Endophytic Trichoderma strains can improve plant growth, confer disease resistance
and abiotic stress tolerance, and, at the same time, obtain nutrients from the plant without causing
disease. To maintain a good relationship between endophytes and their respective hosts requires
constant communication between the organisms involved. Here, we investigated the first steps during
Trichoderma-plant interaction, including the production of the fungal-derived phytohormone indole
acetic acid (IAA) in the signalling dialogue between Trichoderma and the model plant Arabidopsis
thaliana. A range of Trichoderma strains selected for their ability to enhance root production, were
tested for IAA production in synthetic medium and found to produce 0.2 to 1 mg/dried weight of
mycelium. Interestingly, while some isolates produced a 23-fold increase in IAA levels in the presence
of L-Tryptophan, others showed only a 1.5-fold increase. No significant differences were observed
between the isolates in their abilities to promote A. thaliana root growth on Murashige & Skoog (MS)
plates, however, one Trichoderma isolate induced anthocyanin production in the leaves, which may
be an indication of stress. Interestingly, in bioassays conducted in sterile soil, we observed contrasting
results, while four isolates significantly enhanced shoot weight, two had no effect on shoot weight and
one isolate negatively impacted total plant fitness, reducing growth by 50% compared with the untreated
control. Thus, we conclude that although IAA could have an important role in plant root promotion, there
are additional chemical signals or factors which may be even more important for a positive interaction
between these two organisms.
27 30 August 2012
17
Commerce Building
Lincoln University
New Zealand
The role of H+-pump in nutrient transport during germination of

Trichoderma atroviride conidia
IMKOVI, M.; Vilimov, V.; Olejnkov, P.; Hudecov, D.; Krytofov, S. and
Vareka, .
Department of Biochemistry andMicrobiology, Faculty of Chemical and Food Technology, Slovak University of Technology,
Radlinskho 9, 81237 Bratislava, Slovak Republic.
martin.simkovic@stuba.sk; ludovit.varecka@stuba.sk
Germination of fungal conidia starts with a wide spectrum of genetic and metabolic activities, and
morphological changes. One of the most spectacular changes accompanying germination is growth
(swelling) of conidia and the formation of a germ tube. It could be expected, due to the absence of
apparent metabolite stores - precursors of macromolecule biosynthesis in conidia, that the step(s)
preceding germination-linked biochemical changes is the transport of nutrients across the cytoplasmic
membrane. The transportation of nutrients into conidia of filamentous fungus Trichoderma atroviride at
different stages of the germination process were studied by means of radioactively labelled biopolymer
(N-acetyl-D-[1-3H]-glucosamine, [2-14C] uracil, and L-[U-14C]valine) or complex lipid ([methyl-3H]choline)
precursors. The uptake of nutrients was dependent on the developmental status of the conidia. The
most pronounced increase of the nutrient transport activity was observed after approximately 8h from
the beginning of cultivation. Characteristics of nutrient uptake(s) indicate that all selected compounds
are translocated by transport systems that are driven by the H+-motive force. Uncoupler(s) inhibited
nutrient uptake into conidia and the degree of inhibition was increased with the time of germination.
Measurement of the expression of the H+-pump gene (PMA1) showed that this enzyme is not expressed
in non-germinated conidia. Its expression starts between 6-10 h of germination, which correlates with
the onset of both nutrient transport and formation of germ tubes. Results demonstrate that the H+pump may work as a universal trigger of nutrient uptake during the T.atroviride conidia germination thus
serving as accelerator of their metabolism.
This work was supported by the Science and Technology Assistance Agency under the contract Nrs. APVV0282-10, and APVV-0642-07.
27 30 August 2012
19
Commerce Building
Lincoln University
New Zealand
Interactive effects of abiotic factors in the in vitro conidiation

of Trichoderma stromaticum, a biocontrol agent of the witches
broom disease in cacao
LEITE, S. S.1; Corra, F. M.2; De Souza, J. T.3 and Loguercio, L. L.1
Post-graduation Programme in Genetics & Molecular Biology (PPG-GBM),
Dept. Exact & Techonological Sciences (DCET); State University Santa Cruz (UESC), Rod. BR 415, Km 16, Salobrinho, 45992-900
Ilhus-BA, Brazil.
Recncavo da Bahia Federal University, CCAAB; R. Rui Barbosa, 710, Centro, 44380-000 Cruz das Almas-BA, Brazil.
leandro@uesc.br
Biological control of the witches broom of cacao by Trichoderma stromaticum (Ts) is a promising
alternative for management of this disease in cocoa-producing areas of south eastern Bahia (Brazil).
However, some Ts isolates with strong biocontrol activity have shown to be recalcitrant for mass
production of conidia, which is required for development of commercial products. Studies on the
effect of abiotic factors influencing in vitro sporulation of these isolates is a first step in an attempt to
establish more suitable conditions for conidiation at a large-scale level. In this study, four Ts isolates
labelled as ALF-15, ALF-1092 (genetic group I), ALF-64 and ALF-113 (group II) were subjected to three
replicates of different abiotic treatments in culture media, which were established in a factorial design.
These treatments included different C and N amounts and sources (BDA, minimal medium with urea or
with KNO3), two light conditions and four acid pHs (2.8, 3.6, 4.4 or 5.2). Isolates were grown at 28C in
solid media for 5 days under total darkness; half of all treatment combinations received a single 20min white-light pulse after 48-h culture. Results from colony morphologies and spore-counting overall
demonstrated that different factors tested simultaneously showed larger or smaller effects on conidiation
in an interactive manner. Despite pertaining to the same Trichoderma species, the isolates individually
displayed specific qualitative and quantitative patterns of sporulation. This suggests that each isolate
will likely demand very particular combinations of these factors to produce higher levels of conidiation.
Consequences of these findings to large-scale production of more efficient Ts isolates for biocontrol
purposes of witches broom of cacao will be discussed.
27 30 August 2012
21
Commerce Building
Lincoln University
New Zealand
Trichoderma atroviride xylanase regulator 1 (Xyr1) and its role in

mycoparasitism.
REITHNER, B and Mach, R. L.
Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrae 1a/1665, A-1060 Wien, Austria.
barbara.reithner@tuwien.ac.at
Comparative transcriptome analysis of the mycoparasitic fungus Trichoderma atroviride IMI206040 in

interaction with different host fungi revealed the presence of binding sites for the transcription factor
Xyr1 in the promoter region of various differentially expressed genes (Reithner et al., 2011). Hence,
the gene transcription of xyr1 under the same conditions was determined, showing a distance and
confrontation partner dependent relative gene expression. Consequently, a Xyr1-negative strain was
generated. As in T. reesei, the T. atroviride xyr1 deletion strain showed no phenotypic difference, no
changed radial growth on potato dextrose agar and only a slight reduction of growth rate on minimal
medium supplemented with glucose compared with the parental strain. The addition of D-xylose as
sole carbon source led to a 2/3 reduction in growth rate in the mutant strain. To monitor the influence
of the transcriptional activator of major hydrolytic enzymes on the expression of genes involved in
mycoparasitism of T. atroviride, plate confrontation assays were conducted. The obtained results will be
presented in relation to the mycoparasitic and biocontrol ability of this fungus.
Reithner et al. (2011) AEM 77:4361-4370.
27 30 August 2012
23
Commerce Building
Lincoln University
New Zealand
Over-expression of the Saccharomyces cerevisiae Dolicholphosphate mannose synthase (DPM1) gene in Trichoderma
hamatum GD12 and its consequences for biological control
and plant growth promotion
LE COCQ, K. F. and Thornton, C. R.
College of Life and environmental Sciences, Geoffrey Pope Building, Stocker Road, University of Exeter, EX4 4QD, UK.
kfl202@exeter.ac.uk; C.R.Thornton@exeter.ac.uk
Trichoderma species are ubiquitous soil saprotrophs and well-characterised biological control agents.
Certain strains have also been shown to stimulate plant growth through the production of bioactive
secondary metabolites, and are therefore receiving increased attention as natural plant growth
stimulants. Previous research at Exeter University (Ryder et al., 2012) has shown that the Plant-GrowthPromoting (PGP) activity of a biocontrol strain of T. hamatum (strain GD12) can be dramatically enhanced
by targeted mutation of the N-acetyl--D-glucosaminidase-encoding gene NAG. The chitinase-deficient
mutant Thnag::hph has an altered cell wall structure, with abnormal chitin deposition at the hyphal
tip, and hyper-secretes water-soluble PGP compounds that leads to dramatically increased PGP activity
in the absence of root pathogens. However, due to the loss in chitinase activities, Thnag::hph displays
loss of saprotrophic competitiveness and reduced fitness as a biocontrol agent. Here, we set out to
investigate whether hyper-secretion and enhanced PGP activities could be engineered in GD12 without
impairing biocontrol activity. Over-expression of the S. cerevisiae gene Dolichol-phosphate mannose
synthase (DPM1) in T. reesei leads to altered cell wall architecture and increased secretory potential.
Using the constitutive promoter ToxA, we over-expressed the DPM1 gene in T. hamatum GD12 and
assessed its effects on the biocontrol and PGP activities of the fungus. Here we present data to show
that single copy insertions of ToxA-DMP1 leads to improved PGP activities, while biocontrol fitness is
unaffected. However, while multiple copy insertions similarly lead to enhanced PGP, such strains display
impaired biocontrol of soil-borne pathogens such as the plurivorous damping-off pathogen Sclerotinia
sclerotiorum. This work demonstrates that while significant improvements in crop productivity can be
achieved through genetic modification of the beneficial rhizosphere fungus T. hamatum GD12, it can have
important consequences for other aspects of its biology and ecology and competence as a soil-borne
microorganism.
Ryder et al. (2012) Microbiology 158: 84-97.
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New Zealand
Terpenes of Trichoderma
MONTE, E.1; Cardoza, R.E.2; Malmierca, M.G.2; Hermosa, R.1 and Gutirrez, S.2
Centro Hispano-Luso de Investigaciones Agrarias (CIALE). Departamento de Microbiologa y Gentica, University of Salamanca.
37185, Salamanca, Spain.
emv@usal.es; rhp@usal.es.
Area of Microbiology. Universitary School of Agricultural Engineers. University of Len. Campus de Ponferrada. Avda. Astorga
s/n. 24400 Ponferrada, Spain.
re.cardoza@unileon.es; mgomh@unileon.es; s.gutierrez@unileon.es.
Terpenes are one of the largest groups of natural products comprising numerous secondary metabolites
that are involved in biocontrol, due to their antifungal properties (ergokonins and viridins), and cell
membrane structure (ergosterol). Trichoderma produce a large series of terpenes with a common
mevalonic acid biosynthetic origin. Several different enzymes participate in the synthesis of terpenes
although only three genes have been characterised in T. harzianum: hmgR, erg1 and erg7, encoding an
HMG-CoA reductase (Cardoza et al., 2007) and a squalene epoxidase and an oxidosqualene lanosterolcyclase (Cardoza et al., 2006). The trichothecene biosynthesis pathway has been studied in two
Trichoderma species from the Brevicompactum clade (Cardoza et al., 2011). Depending on the nature
and concentration of the Trichoderma trichothecenes, these can act as antimicrobial compounds with
biocontrol activity at a high concentration or as plant development signaling molecules when they are
normally produced at a low concentration. A curious example is the case of trichothecenes produced
by T. brevicompactum (trichodermin is antifungal and toxic to plants) (Tijerino et al., 2011) or T.
arundianceum (far from being toxic, harzianum A induces plant defences) (Malmierca et al., 2012).
Cardoza et al. (2006). FGB 43: 164-178.
Cardoza et al. (2007). FGB 44: 269-283.
Cardoza et al. (2011). AEM 77: 4867-4877.
Malmierca et al. (2012). AEM 78: 4856-4868.
Tijerino et al. (2011). FGB 48: 285-296.
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SESSION 2
Biotechnological Applications
KEYNOTE
Molecular and omics approaches for development of Trichoderma
reesei as a protein production cell factory
Markku SALOHEIMO; Pakula, T.; Arvas, M.; Hkkinen, M.; Westerholm-Parvinen,
A.; Joensuu, J.; Aro, N. and Penttil, M.
VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland.
markku.saloheimo@vtt.fi.
Trichoderma reesei is among the protein production cell factories most widely used by the enzyme
industry. The protein production levels obtained from T. reesei are not surpassed by any other protein
production organism, and this fungus has a central role as a producer of enzymes for the second
generation biofuel and chemical production from lignocellulosic biomass. T. reesei has also served as
an important model system in studies on enzymatic degradation of cellulose and hemicellulose and the
underlying regulatory mechanisms governing this process. The main topic of the presentation will be
transcriptional profiling and proteomics experiments completed to understand the enzyme expression
and protein production processes in T. reesei. Transcriptional profiling was performed from T. reesei
mycelia after adding simple compounds like cellulose, xylan or sophorose or complex substrates like
pre-treated wheat straw, spruce or bagasse into the culture. This allowed identification of enzyme groups
induced by each of the substrates. Interestingly, it also indicated that sets of neighbouring genes are
co-regulated in these conditions, suggesting chromatin-level regulation of the hydrolytic enzyme system.
The dataset let us identify a set of novel putative regulatory factors of the glycosyl hydrolase genes,
and their over-expression showed that they are indeed involved in the regulatory process. In a second
experiment, we studied the effect of growth rate and cell density on protein production and physiology
of T. reesei in chemostat cultures. As shown before, protein production rate was highest at low growth
rates around 0.03 h-1. Our data showed that major biosynthetic activities had a negative correlation with
the specific protein production rate, whereas secreted protein expression and secondary metabolism
are positively correlated with it. Interestingly, this dataset also revealed chromosomal clusters of genes
whose expression had a positive correlation with the specific protein production rate.
27 30 August 2012
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Development of Trichoderma reesei as a host for production of

enzymes for enzymatic hydrolysis of lignocellulosic biomass
MERINO, S.
Novozymes, 1445 Drew Ave., Davis, CA, USA.
same@novozymes.com
The majority of commercial enzyme products for biomass hydrolysis are produced by the saprophytic
mesophilic fungus Trichoderma reesei. Trichoderma produces two cellobiohydrolases (CBHI and CBHII),
five endoglucanases (EGs), and two -glucosidases (BGs). Although this mixture of enzymes is highly
efficient at degrading cellulose we have developed new technologies for improving upon this base
mix of components. Over the years Novozymes research has contributed significantly to reducing
the cost of enzymes for hydrolysis of lignocellulosic biomass by optimising the mixture of cellulase
enzymes. To significantly reduce the enzyme loading required and thus reduce cost, one may replace
Trichoderma components with more efficient candidates or augment the enzyme system with additional
components to improve the overall enzyme performance. In addition, proteases have been identified
and deleted resulting in improved product stability. In this presentation we will show that the expression
of recombinant proteins from other species in conjunction with native proteins and protease deletions
results in a Trichoderma strain with a superior mixture of enzymes for hydrolysis of lignocellulosic
biomass.
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Tra1, an adenine deaminase gene, was participated in copper

accumulation in Trichoderma reesei
FU, K.1; Fan, L.-L.2; Li, Y.-Y.3 and Chen, J.4
Department of Resource and Environmental Science Room 2-409, School of Agriculture and Biology, Shanghai Jiaotong
University, Dongchuan Road 800, Shanghai 200240, The Peoples Republic of China.
fkh0112@sina.com
fogfan_31@sina.com
liyingying16@sjtu.edu.cn
Corresponding author: Department of Resource and Environmental Science Room 2-401, School of Agriculture and Biology,
Shanghai Jiaotong University, Dongchuan Road 800, Shanghai 200240, The Peoples Republic of China.
jiechen59@sjtu.edu.cn
A modified Agrobacterium tumefaciens-mediated transformation method was established for the

construction of mutants with improved copper accumulation capability in Trichoderma reesei. One
mutant, AT01, exhibited the highest copper accumulation capability. AT01 reached the maximum copper
removal capability with a 12.73 mg copper/g biomass, whereas the wild-type strain had 5.97 mg copper/g
biomass (removal rate of 49.6%). The optimal initial pH was 5.0, and the optimal incubation temperature
was 28C. The pigment change of Trichoderma mycelia was a potential indicator of copper accumulation.
AT01 grown in copper-containing medium appeared to be verdigris in color. Electron microscope
observations revealed that copper was mainly accumulated in cell vacuoles. The high ability of copper
accumulation of AT01 was regulated by the Tra1 gene, which encoded a member of amidohydrolase
superfamily. It contains 511 amino acids and belongs to COG0402. The best substrate for the enzyme
activity was adenine. The gene function was investigated by overexpression and RNAi-mediated silencing.
Results showed that copper accumulation increased when the gene was overexpressed, and decreased
when the gene was silenced. Meanwhile, the mRNA expression levels of the copper transport chaperones
were also found to be increased in the overexpression mutant, and decreased in the RNA interference
mutant. Furthermore, the gene expression profile of mutant AT01 was compared with wild-type strain
of T. reesei in a medium containing 1.0 mM Cu2+. A total of 627 genes were up-regulated and 305 genes
were down-regulated under copper stress. The expression analysis showed that the amino acid transport
and metabolism related genes were up-regulated, which was associated with the metabolism of adenine.
Furthermore, the inorganic ion transport and metabolism associated genes and copper transporter were
also up-regulated. This indicated that Tra1 encoded an adenine deaminase, which may participate in
copper accumulation in T. reesei.
27 30 August 2012
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New Zealand
Biological and molecular investigation of a potential strain of

Trichoderma harzianum against carbendazim
SHARMA, P.1; Singh, U. S.2; Zaidi, N. W.2; Kumar, P. V.1; Saravanan, K.1; Singh, D. V.3
and Sharma, M.1
Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110012.
pratibha@iari.res.in; vigneshkumar236@gmail.com; snrsaro88@gmail.com; manikapathology29@gmail.com
International Rice Research Institute, India Office, New Delhi-110012.

u.singh@irri.org, n.zaidi@irri.org
Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012.

dv_singhpp@rediffmail.com
Carbendazim is a benzimidazole fungicide used by farmers in their agricultural practices. The -tubulin
gene has been identified as a primary key binding site for this group of fungicides. An attempt was made
using molecular genetic techniques to identify sites of interaction of -tubulin receptors of biocompetent
Th3 with benzimidizole anti-microtubule agents. The isolate Trichoderma harzianum (Th3) was selected
through rigorous screening of isolates which confer resistance to the benzimidazole agent including
benomyl and its active breakdown product, carbendazim, which became the centre of our research.
-tubulin sequences were obtained through cloning of a wide range of collected competent isolates
from different eco-zones of the country. qPCR results showed higher expression in Th3 than other
counterparts. An increased demand for food and fibre has led to the chemicalisation of agriculture and
we have reached the stage where we are totally dependent on fertilizers and chemicals which in a real
sense are polluting our life and ecology by entering the food-chains. An experiment was conducted under
in vitro conditions to determine the breakdown potential of biocompetent Th3 to mitigate the effect
of carbendazim, through HPLC analysis. The isolate was grown in mineral salt medium that contained
different concentrations of carbendazim ranging from 10 ppm, 50 ppm, 100 ppm, 150 ppm and 200 ppm
respectively. The metabolite detected through HPLC was benzimidazole after 2 days of shaker incubation.
There was a steep decrease in carbendazim content observed in all concentrations of carbendazim
tested. The absence of carbendazim was also observed in 10 ppm, 50 ppm and 100 ppm concentrations
after 3 days of treatment. Results determined that strain Th3 showed a maximum breakdown percentage
of up to 98%. These results indicate that Th3 has promise as a tool for soil bioremediation studies.
27 30 August 2012
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A novel utilisation of the biopolymer chitin: Generation of a

pharmaceutically relevant fine chemical by a whole-cell catalyst
approach in Trichoderma
DERNTL, C.1; Steiger, M.2; Gorsche, R.1; Mach-Aigner, A. R.1 and Mach, R. L.1
Gene Technology Group, Inst. of Chemical Engineering, Vienna University of Technology, Gumpendorferstr. 1a, A-1060
Vienna, Austria.
christian.derntl@tuwien.ac.at; rgorsche@mail.zserv.tuwien.ac.at; aaigner@mail.zserv.tuwien.ac.at;
rmach@mail.zserv.tuwien.ac.at
Institute for Applied Microbiology, University of Natural Ressources and Life Sciences Vienna, Muthgasse 107, A-1190
Vienna, Austria.
matthias.steiger@boku.ac.at
Annually, 109 to 1011 tons of chitin are produced worldwide making chitin the second most abundant
renewable organic source in nature after cellulose. The polymer is composed of -(1,4)-linked units of
the amino sugar N-acetylglucosamine (GlcNAc). Only a small proportion of this compound is exploited
for industrial and agricultural applications. N-acetylneuraminic acid (NeuNAc), a C9 monosaccharide,
is the most predominant exponent of sialic acids. In nature more than 50 derivatives of sialic acids
are known today. NeuNAc is believed to serve as a precursor of all these derivatives as all biochemical
pathways proceed via this substance. In biological systems, sialic acids are mostly terminal components
of glycoproteins presented at the respective cell surface. Many viruses use sialic acids as docking stations
to infect their host cells due to the exposed position of these compounds. Important and well known
examples are influenza viruses A and B. As a result, sialic acid derivatives are successfully applied in the
therapy of such virus-borne diseases. Preparations on the market are e.g. Relenza by GlaxoSmithKline
functioning as neuraminidase inhibitor and produced from the precursor NeuNAc. We will present an
alternative strategy of NeuNAc production based on a genetically engineered Trichoderma strain. This
application is the first introduction of a bacterial multi-step enzyme cascade into a filamenous fungus as
heterologous host. This strategy involves the application Trichoderma as a whole-cell catalyst for direct
synthesis of NeuNAc from the cheap, renewable biopolymer chitin.
27 30 August 2012
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New Zealand
Impacts of a Trichoderma transformant with Metarhizium genes

on the bacterial community in the Asian corn borer larvae midgut
LI, Y.-Y.1, Fu, K.2 and Chen, J.3
liyingying16@sjtu.edu.cn
fkh0112@sina.com
Corresponding author: Department of Resource and Environmental Science Room 2-401, School of Agriculture and Biology,
Shanghai Jiaotong University, Dongchuan Road 800, Shanghai 200240, The Peoples Republic of China.
jiechen59@sjtu.edu.cn
Trichoderma has been extensively studied in the application to control various crop soil-borne diseases.
However, Trichoderma is not commonly recognised in interactions with insects, therefore it is valuable to
study the relationship of Trichoderma particularly when insect pests and diseases occur simultaneously
in the same space. We have previously transformed chit42 cloned from Metarhizium anisopliae, ligated
with genes of chitin binding domains (chBD), and demonstrated that overexpression of M. anisolpiae
chit42 gene with chitin-binding domain in T. koningii displayed an increased biocontrol activity, especially
against the Asian corn borer larvae. However we lacked an understanding of the transformants impact
on the midgut when present during insect larvae feeding. Results revealed that the diversity of the
total bacterial community was significantly changed in the fourth instar larvae midgut of the Asian corn
borer after feeding on diets containing Trichoderma chitinase fermentation liquids. Proteobacteria
and Firmicutes were the highest represented phylum, which were present in all midgut bacterial
communities. The observed species richness was simple, ranging from 4 to 5 among all 16S rRNA clone
libraries. The Shannon-Weaver and Simpson indexes of larval midgut bacterial clone library treated by
Trichoderma transformants chitinase fermentation liquids was the lowest when compared with the
other three treatments. The populations of the genera Ochrobactrum extensively declined relative to
populations in the controls, while Enterococcus populations increased. These results provide the first
description of bacterial diversity of the Asian corn borer larvae midgut and have demonstrated that
Trichoderma chitinase fermentation liquids could influence the midgut bacterial community structure.
It was concluded that the imbalance of total bacterial community structures might be an indicator to
increased mortality of corn borer larvae.
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New Zealand
Expression of Trichoderma viride endoglucanase in the larvae

of silkworm, Bombyx mori L. and characteristic analysis of the
recombinant protein
WANG, M.-X.#; Liang, S.#; Li, X.-H. and Miao, Y.-G.
College of Animal Sciences, Zhejiang University, 866YUHANGTANG Road, Hangzhou 310058, P. R. China.
wmxzihang2008@126.com; Corresponding author: Y.-G. Miao; miaoyg@zju.edu.cn
#
contributed equally to this research
Cellulases catalyze the hydrolysis of cellulose. There are three main types of cellulase: endoglucanases,
cellobiohydrolases and -glucosidases. In this paper, we cloned a 1380 bp endoglucanase I (EG I) gene
from the mycelium of the filamentous fungus T. viride strain AS 3.3711 using PCR-based exon splicing
methods. We expressed the recombinant EG I mature peptide protein in both silkworm BmN cell
line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression
system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori
nucleopolyhedrovirus (BmNPV). An approximately 49-kDa protein was visualised after mBacmid/
BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The
ANOVA showed the enzymes from recombinant baculoviruses infected silkworms exhibited a significant
maximum enzyme activity at pH 7.0 and at 50C. It was stable at a pH range from 5.0 to 10.0 and at a
temperature range from 50C to 60C, and increased 24.71% and 22.84% compared with that from wild
baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities
of EG I that the silkworm provides maybe greatly facilitate the future research and the potential
application in industries.
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New Zealand
Identification and characterization of cellobiose transporter in

Trichoderma reesei
Kou, Y.; Zang, W.; Xu, J.; Chen, G. and Liu, W.
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, P.R. China.
Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures
the rapid launching of cellulase mixtures in cellulolytic Trichoderma reesei. Cellobiose or its derivative
is a reasonable candidate for such a signal. A putative cellobiose permease has been proposed to exist,
although its identity has been elusive. In the present research we have identified a transporter, TrCdt1,
from T. reesei which could support the growth of yeast on cellobiose when engineered together with a
-glucosidase into yeast. The identified transporter also enables the growth of yeast deleted for all of its
hexose transporters on glucose. Deletion of TrCdt1 almost ceased the induced production of cellulases
on cellobiose, whereas simultaneous absence of the major extracellular -glucosidase Bgl I restored the
induction by cellobiose. Nevertheless, the absence of TrCdt1 increased rather than alleviated the induced
production of cellulases on cellulose. These preliminary results suggest that cellobiose transporters
including TrCdt1 may participate in delivering cellobiose intracellularly to invoke cellulase formation in
T. reesei.
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SESSION 3
Biocontrol and ecology
KEYNOTE
Trichoderma root endophytes enhance plant health and vigour
Robert HILL
Bio-Protection Research Centre, PO Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand.
robert.hill@lincoln.ac.nz
Plant health depends on associated fungi. Symbiotic Trichoderma root endophytes promote crop growth,
activate defences, improve nutrition, increase tolerance to abiotic stresses and control pathogens
through antibiosis and mycoparasitism. Control of Armillaria disease in New Zealand (Kiwifruit and
Pinus radiata) with selected Trichoderma mixtures led to the development of the commercial products
DRH and ArborguardTM (AG). Inoculum application rate, timing and mode of application for AG were
optimised in commercial forest nurseries. Seed coating was the most cost-effective system for both
containerised and soil bed nurseries. In forest plantation trials, AG treatment reduced mortality from
Armillaria disease by 20-50% after two years. It was found that ectomycorrhizal colonisation of P. radiata
roots was not reduced by AG and that a close symbiotic relationship between the roots and Trichoderma
was associated with increased seedling vigour. Increased resistance to Diplodia pinea was induced in P.
radiata seedlings by particular Trichoderma isolates. Root initiation and subsequent growth and health
were stimulated and enhanced by specific Trichoderma isolates in cuttings of a variety of plant species.
These successes resulted in an invitation in 2008 to evaluate the utility of Trichoderma bio-inoculants
in the Planted Forest Zone of Sarawak, Malaysia. Several constraints led to a decision to only isolate
Trichoderma from within the roots of very healthy local plants. These isolates were trialled in a large
commercial nursery growing containerised Acacia mangium. The best isolates gave a 66% increase in
the number of seedlings meeting nursery specification and large scale validation trials gave identical
results. Plantation trials resulted in a 15% increase in growth and a 30% decrease in disease mortality
by Trichoderma-inoculated trees. A purpose-built Trichoderma mass production facility now supplies
the whole nursery and operational practice has changed from multiple fungicide sprays to Trichoderma
inoculation at seeding resulting in multi-million dollar economic benefits annually.
27 30 August 2012
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New Zealand
Biological control of Botrytis cinerea in grapes in Tasmania

METCALF, D. A.
Biocontrol Australia Pty Ltd, 211 Wyre Forest Rd, Molesworth, Tasmania 7140. Australia.
metcalf@tassie.net.au
Botrytis cinerea causes extensive losses in grapes in temperate vineyards of Tasmania, where the wine
industry initiated research to develop tools for sustainable production, including a biological control
agent for B. cinerea. To find suitable candidate biological control agents, a survey was undertaken of the
micro flora associated with developing grape flowers and fruit. Antagonistic fungi such as Trichoderma
spp. had a low frequency in developing flowers and were not commonly found in the grape phyllosphere
until around the stage of veraison. One interpretation of this observation is that there would be potential
to reduce the infection of grape flowers by B. cinerea by introducing an antagonistic Trichoderma species
at the time of flowering. Trichoderma koningii (Td67) collected from grape flowers, was applied to
grapes twice during flowering and the fungus reduced the incidence of B. cinerea at veraison by 26.8%
(sig P=0.05). Over the following four seasons, experiments were conducted using this isolate and the
use pattern was refined to the stage where an 80% reduction in infection at veraison was repeatably
obtained. A prototype spore powder containing 1010 CFU/g was developed in 2007. To optimise the
use pattern, a series of experiments were conducted to compare application at 0, 50, 100 & 500 g/
ha from which 100 g/ha was selected as the optimum rate of application. To optimise the timing of
application, untreated controls were compared with 20%, 80% and both 20 & 80% capfall application.
The combination of 20 & 80% resulted in the greatest percentage of flower colonisation by Td67, and
the lowest percentage of B. cinerea in the experiment, but most vineyards would only wish to apply
on one occasion, and 80% capfall was found to be the most effective timing for a single spray. Td67 is
commercially available in Australia as the Colonizer biological fungicide.
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New Zealand
Management of Phomopsis canker in tea plantations using

Trichoderma harzianum under field conditions
PONMURUGAN, P.
Department of Biotechnology, K.S.Rangasamy College of Technology, Tiruchengode 637 215, Namakkal District, Tamil Nadu,
India. Phone : +91 4288 274741 44 (4 lines), Mobile : +91 9865857816, Fax : +91 4288 274745.
drponmurugan@gmail.com
Phomopsis canker caused by a deuteromycetes fungus Phomopsis theae Petch is one of the most
important stem diseases of tea in all the tea growing countries of the world. The disease is prevalent
in young tea plantations planted with clonal tea and to a certain extent in mature tea fields also. The
disease has been a serious problem in southern India since 1958 and is of great importance, as the area
under replanting and new clearings with clonal tea has increased in recent years. Crop loss due to this
disease is substantial as it leads to capital losses. Despite its importance, effective control measures
are not available other than pruning to healthy wood and application of copper fungicides on prune
cuts. Avoidance of pre-disposing factors and uproot and burning of the bushes in severe cases are also
recommended as a control measure. There are no effective control measures available for the control
of Phomopsis canker under field conditions. An experiment was conducted to study the efficacy of
certain fungicides including contact and systemic and biocontrol agents like Trichoderma harzianum and
Gliocladium virens in controlling Phomopsis canker disease of tea under field conditions. Among the
various treatments, soil application and wound dressing of biocontrol agents was found to be superior
to fungicides in controlling Phomopsis canker. Wound dressings of contact fungicides such as copper
oxychloride and mancozeb were found ineffective whereas soil drenching with systemic fungicides
such as carbendazim and contaf were moderately effective. Various treatments healed the wound to
varying degrees and improved plant health. The bud break, tipping weight and green leaf yield were also
increased in plants to some extent after treatments. Among the biocontrol agents tested, Gliocladium
virens was better than Trichoderma harzianum in terms of curing cankers and increasing yield potential.
Moreover, there was an improvement in tea quality parameters after imposing various treatments
especially in the Trichoderma treated plots.
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Potential use of Trichoderma spp. as biological control agents in

suppressing Ganoderma basal stem rot in oil palm seedlings
SUNDRAM, S.; Seman, I. A. and Naidu, Y.
Malaysian Palm Oil Board, No 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia.
shamala@mpob.gov.my; idris@mpob.gov.my; yuvarani@mpob.gov.my
Two potential Trichoderma spp namely T. asperellum (T9) and T. virens (T29) were found to be effective
biocontrol agents of basal stem rot disease in oil palm caused by Ganoderma boninense. This was
based on in vitro and in vivo assessments. In vitro assessment found the biocontrol agents were able to
suppress the radial growth of the pathogen through dual culture and culture filtrate assays. Application
of the biocontrol agents utilises a natural biowaste that is produced in the oil palm mills that extract
oil from the fresh fruit bunches. The oil palm seedlings were treated with Trichoderma-enriched palm
press fibre (PPF), applied as surface mulch. Both isolates suppressed the disease significantly better as
a single application compared with a mixed application and control treatment. Disease assessment was
carried out based on disease severity based on sequential disease manifestation, area under the disease
curve (AUDPC) and epidemic rate. The investigation continued further to assess the induced systemic
resistance (ISR), a biochemical activity that is often triggered by the plant which is due to the presence of
nonpathogenic microorganisms. Enzyme activity of peroxidase (POX), polyperoxidase (PPO) and chitinase
was analyzed in five time intervals after the application of Trichoderma isolates as soil drench on the oil
palm seedlings. The activity of PPO and chitinase in the leaf were found to be almost two fold higher
and continued to increase in the initial 3 sampling intervals after which it started to decline. PAL showed
increased activity with no significant difference. This study suggests that the preliminary successful use
of oil palm biowaste using an integrated approach of microbial enrichment is positive but requires an
extensive field trial for the reproducibility of disease suppression to be assessed.
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New Zealand
Field application of Trichoderma technology in semi-arid eastern

plains and humid south-eastern plains of the Rajasthan state, India
SHARMA, P.
Biocontrol Lab, Plant Pathology, Indian Agricultural Research Institute, New Delhi-12. India.
pratibha@iari.res.in.
In the present study, bioformulation of the antagonistic fungus Trichoderma harzianum (Th3) as both a
powder and a liquid was developed under DST-TIFAC funded project entitled On Farm Demonstration
and Commercial Production of Trichoderma as Biopesticide and Growth Promoter 2008-2012). The
bioformulation was introduced into the fields of 20 villages in the Rajasthan state, India (V- Raholi,
Thali, Jaiypura, Samode, Takarda, Khejroli, Dhodhasar, Vijaysinghpura, Sultanpura, Kaladera, Singodh
and Hanutia in Jaipur District and Digodh, Shauli, Devpura, Kasampura, Dagaria, Mundla, Parlia and
Nyagaon in Kota District). There were two districts of different soil conditions and agroclimatic zones
Rajasthan state (viz., Jaipur and Kota) which covered 22 crops (Barley, Brinjal, Cauliflower, Chilli, Garlic,
Fenugreek, Wheat, Mustard, Onion, Tomato, Pea, Coriander, Potato, Groundnut, Rice, Tilli, Maize, Perl
millet, Chickpea, Okra, Watermelon and Soyabean). One hundred and eighty eight farmers applied
the bioformulations in their fields. The Trichoderma technology has been successfully demonstrated
in winter crops grown in the sandy soils of Jaipur district of Rajasthan. The plant protection was made
more effective and sustainable by the farmers participatory approach. The field performance of the Th3
bioformulation was evaluated in terms of its rhizosphere competence, survivability (root colonisation
behaviour) and non-host specificity in different crops. The field performance of Trichoderma harzianum
(Th3) was previously evaluated in terms of its rhizosphere colonisation and competence, survivability
(root colonisation behaviour) and non-host specificity in additional different crops including cereals,
legume, oilseed crops, vegetables, ornamental crops and fruit (Watermelon) of both Rabi and Kharif
cropping seasons (Sharma, 2008, 2009; Sharma et al, 2011). This project is a success story of a tripartite
interaction between Farmers- NGOs- Scientists leading to the understanding of the use of an introduced
strain of Trichoderma harzianum (Th3) and growth promotion of crops in the field. This encourages the
technologists to deliver their products to the farmers level.
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Improved potato yields from in-furrow application of Trichoderma

BIENKOWSKI, D.; Hicks, E.; Braithwaite, M.; Falloon, R. E.; McLean, K. L. and
Stewart, A.
damian.bienkowski@lincolnuni.ac.nz
Rhizoctonia diseases of potato are caused by the soil-borne fungal pathogen Rhizoctonia solani (Khn).
Symptoms include cankers (lesions) on below-ground shoots, stolons, and tubers, which can cause
delayed emergence and poor yield and quality of tubers, and black scurf, pathogen sclerotia on the
surfaces of daughter tubers. These diseases are global problems in potato cultivation, and their control
by synthetic fungicides is not always sufficient to prevent economic loss. Biological control of Rhizoctonia
diseases could play an important role in disease management, and there have been many studies which
suggest that the fungal genus Trichoderma can be employed in this context. Thirty Trichoderma strains
from New Zealand agricultural soils, as well as selected strains from the Biocontrol Microbial Culture
Collection, Bio-Protection Research Centre, Lincoln University, were evaluated for their capacity to reduce
Rhizoctonia diseases of potato in greenhouse bioassays. Individual Trichoderma strains provided only
moderate (40-50%) and inconsistent disease reductions. However, six strains were selected, four capable
of reducing disease parameters and two that increased number and weight of tubers, for evaluation in
a pot trial. The four best strain combinations were also assessed in a field trial, with artificial R. solani
inoculation. The results of two harvests (one post-emergence, one at plant maturity) found no evidence
that treatments reduced emergence or deformed tubers (disease suppression). However, two treatments
significantly increased yield of marketable tubers by up to 12%, similar to that achieved with fungicide
treatment. This suggests that the Trichoderma strains have growth promotion effects rather than disease
suppression.
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Applications of Gliocladium catenulatum strain J1446 formulations

to control soil-borne and foliar pathogens on vegetable,
ornamental and aromatic plants
JIJAKLI M. H.1, Phillips N.2 and Lahdenper M.-L.3
Lallemand Group, 233 chausse de Waterloo, 1060 Bruxelles, Belgium.
hjijakli@lallemand.com
Lallemand Group, 10 Faraday Rise, Hope, Richmond, Nelson 7002, New Zealand
Lallemand Group, Verdera Oy, Kurjenkellontie 5B, FI-02270 Espoo, Finland
Prestop and Prestop Mix are two formulated biofungicides based on Gliocladium catenulatum strain
J1446. This strain was isolated from Finnish field soil and is able to colonise both the root system and
the canopy of numerous plants. Thanks to that property, it controls the development of both soil borne
pathogens (e.g. Pythium, Phytophthora, Rhizoctonia and Fusarium) and foliar pathogens (e.g. Botrytis
and Didymella) on vegetable, small fruit, ornamental and aromatic plants. The main modes of action
of G. catenulatum strain J1446 are competition for nutrients and space and hyperparasitism. Prestop
is a wettable powder that can be applied by drenching of seedlings, foliar spraying (including LVM
system) and also through irrigation system. Prestop Mix contains larger sized particles and is used by
incorporating it into the growing media to control soil borne diseases. It can also be delivered by bees on
strawberry flowers to inhibit the development of B. cinerea. G. catenulatum strain J1446 can be used in
IPM systems. For instance; the chemical Previcur can be applied on the same substrate at the same time
as strain J1446. In foliage, chemical fungicides may decrease the efficacy of strain J1446, but integrated
use is still possible by choosing harmless fungicides for that strain. Furthermore, most of the chemical
insecticides are compatible with strain J1446 and it is not harmful to beneficial insects, nematodes or
pollinators. Gliocladium catenulatum strain J1446 is registered as plant protection product at European
level (listed in annex 1). Prestop and Prestop Mix are now commercialised in several European
countries (including Finland, Sweden, Holland and Belgium). We will present recent efficacy trials of
Prestop and Prestop Mix in several European countries and describe the parameters influencing the
efficacy (concentration, mode of application, compatibility with other treatments, etc).
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Development of a cold tolerant Trichoderma atroviride LU132

variant for control of Botrytis cinerea on grapes using protoplast
technology
KANDULA, J.; Braithwaite, M.; Steyaert, J. M.; Hay, A. J. and Stewart, A.
Bio-Protection Research Centre, PO Box 84, Lincoln University, Lincoln 7647, New Zealand.
janaki.kandula@lincoln.ac.nz
Grey mould or bunch rot, caused by Botryotinia fuckeliana (de Bary) Whetzel, anamorph Botrytis cinerea
Pers., is an economically important disease of grapes worldwide. The commercial product Sentinel (a.i.
Trichoderma atroviride isolate LU132) provides an alternative to fungicides for grey mould control. To
improve the performance range of this product, protoplasts were generated and selected under cold
conditions (6C) to produce a new strain of T. atroviride LU132 biologically active at cooler temperatures
(LU806) and more suitable for the European market. The variant LU806 produced T. atroviride colonies on
PDA that were greater in diameter by 8.4% at 10C compared with the parent strain. In detached necrotic
strawberry leaf assays, LU806 significantly supressed B. cinerea growth and sporulation statistically
equivalent to the parent strain. In two shade-house experiments, variant LU806 significantly suppressed
B. cinerea symptoms on grape flowers and fruit, to levels statistically equivalent or better than the parent
strain. In an additional cool temperature controlled growth room experiment, only LU806 provided
significant control of latent B. cinerea infection of grape berries. However, LU132 and LU806 both
significantly reduced B. cinerea berry infections compared with the infected control. Incorporation of this
variant strain into the Sentinel product may facilitate use of this product in cool temperature viticulture.
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Application of Trichoderma asperellum in the control of wilt of

pineapple caused by Fusarium oxysporum in the field in Costa Rica
OBREGN, M.
Dr. Obregon Laboratories, Heredia, Costa Rica.
m.obregon@doctor-obregon.com
The hypocrea Fusarium oxysporum was identified through Kochs postulates, as the causal agent of wilt of
pineapple plants. Fusarium oxysporum can persist as chlamydospores for many years in the soil and can
infect the roots of host plants by direct penetration. The fungus can grow in the xylem vessels and cause
blockages in the xylem. The infected stem bases show a browning of the vascular bundles. The objective
of this research was to select the best Trichoderma species from in vitro research and then evaluate their
potential to control wilt of pineapple caused by F. oxysporum in vivo. Dual culture tests showed than all
Trichoderma species tested inhibited the mycelial growth of F. oxysporum. However T. asperellum was the
best species: after 48 hours, 63% of the F. oxysporum colony was colonised and after 120 hours, 100% of
the colony was covered. Also, T. asperellum produced substances with activity against F. oxysporum. This
species was produced in solid substrate and liquid fermentation with both formulations containing 109
conidia/mL and a mixture of toxic metabolites. These formulations were tested under field conditions by
applying to pineapple plants grown in soil highly contaminated with F. oxysporum. The results obtained
confirmed the ability of T. asperellum to control F. oxysporum. Positive effects on root development were
observed and no disease symptoms were found during the trial. These results suggest the possibility
of using a mixture of conidia and toxic metabolites of T. asperellum for the biological control of wilt of
pineapple.
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Colonisation with Trichoderma harzianum and/or Pseudomonas

fluorescens Improves Quality of Farm Yard Manure
ZAIDI, N. W. and Singh, U. S.
International Rice Research Institute, India Office,1st floor, NAAS Complex, D. P. S. Marg, New Delhi-12, India.
n.zaidi@irri.org, u.singh@irri.org
Trichoderma harzianum Rifai (TH) and Pseudomonas fluorescens Migula (PsF) are two most widely used
biocontrol agents for the management of plant diseases. They are applied to soil, seed, or roots of
agricultural crops for the suppression of seed, root and foliar pathogens. Both Trichoderma harzianum
and Pseudomonas fluorescens not only suppress plant diseases but can also improve plant growth.
Farm yard manure (FYM) proved to be an excellent substrate for the mass multiplication of Trichoderma
harzianum and/or Pseudomonas fluorescens. FYM colonised with Trichoderma harzianum and/or
Pseudomonas fluorescens significantly improved the growth of tomato {Solanum lycopersicum (L) (=
Lycopersicon esculentum Mill)} and okra (Abelmoschus esculentus (L) Moench) compared with noncolonised FYM. This growth-promoting effect of colonised FYM was retained even when such composts
were made free from microbes by autoclaving before application to the soil indicating that the growth
promoting effect of colonised FYM was not solely due to disease suppressing and/or direct growth
promoting effect of bioagents. The water soluble components of P, K, S, Zn, Cu and Fe were significantly
higher in Trichoderma harzianum and/or Pseudomonas fluorescens colonised FYM as compared to noncolonised FYM. There was a 3 to 7 fold increase in water-soluble humic matter content in colonised FYM
compared with non-colonised FYM. The highest proportion of dissolved organic matter (DOM) in water
extract of FYM was present in a non-complexed form followed by complexes of Mg and Ca. Less than
0.1 % of DOM was complexed by micronutrient cations like Zn and Cu. A higher content of water soluble
humic matter and an increased availability of macro and micronutrients as a result of colonisation of
organic manure by Trichoderma harzianum and/or Pseudomonas fluorescens are primarily responsible
for the improved plant health. This study adds a new dimension to the growth promotion effects of
biocontrol agents.
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Factors influencing rhizosphere competence and endophytic

colonisation of Trichoderma atroviride
CRIPPS-GUAZZONE, N.; McLean, K. L.; Hill, R. A. and Stewart, A.
natalia.guazzone@lincolnuni.ac.nz, kirstin.mcLean@lincoln.ac.nz, hillr3@lincoln.ac.nz, alison.stewart@lincoln.ac.nz
Rhizosphere competence is the ability of a microorganism to propagate and function in the rhizosphere
of developing roots. Rhizosphere competence and endophytic colonisation has been shown to be
important for successful biological control. The current research was conducted to understand the
mechanisms underlying rhizosphere competence and subsequently enable improvements in the efficacy
of Trichoderma biological control systems. The three most and three least rhizosphere competent
Trichoderma isolates from a preliminary study on sweet corn were selected to investigate the host plant
specificity of rhizosphere competence. Conidial suspensions of these isolates were coated onto seeds of
sweet corn (Zea mays) variety Chieftain, onion (Allium cepa), cauliflower (Brassica oleracea var. botrytis
cymosa), perennial ryegrass (Lolium perenne) variety Impact, white clover (Trifolium repens) variety
Nomad and carrot (Daucus carota). After 3 weeks, ryegrass was the most receptive plant to Trichoderma
irrespective of the Trichoderma isolate. Trichoderma atroviride LU132 and Trichoderma harzianum LU151
were the most rhizosphere competent strains across the six plants. The effect of soil moisture, pH and
nitrogen content on rhizosphere competence was studied using T. atroviride LU132 on ryegrass and
sweetcorn. T. atroviride LU132 was rhizosphere competent across a range of soil moisture contents (16,
20 and 24% w/w), pH (5, 6 and 7) and nitrogen concentrations (75, 150 and 225 kg soil available N/ha)
irrespective of plant species. Endophytic colonies of T. atroviride LU132 were recovered from the shoots
and the roots of both plant species. However, T. atroviride LU132 was dominant in ryegrass rather than
sweet corn, irrespective of the soil environment. This research indicates that the success of rhizosphere
and endophytic colonisation of Trichoderma primarily depends on the isolate and the plant species rather
than the soil environmental conditions.
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Capacity of Trichoderma stromaticum to colonise cacao sapwood

DE SOUZA, J. T.1; Loguercio, L. L.2 and Pomella, A. W. V.3
Recncavo da Bahia Federal University, CCAAB; Rua Rui Barbosa, 710, Centro, 44380-000 Cruz das Almas-BA, Brazil.
jgeteodoro@gmail.com
Post-graduation Programme in Genetics & Molecular Biology (PPG-GBM); State University Santa Cruz (UESC), Rod. BR 415, Km
16, Salobrinho, 45992-900 Ilhus-BA, Brazil.
leandro@uesc.br
Farroupilha Seeds Co.; Av. Julia Fernandes Caixeta 555, bairro Cidade Nova, 38706-900 Patos de Minas-MG, Brazil.
alan@sementesfarroupilha.com.br
The witches broom disease caused by Moniliophthora perniciosa is the main constraint for cocoa
production in Bahia State, Brazil. Trichoderma stromaticum is known to be the most promising species of
this genus for biological control of the witches broom pathogen. Previous investigations have shown that
some isolates of T. stromaticum can be found inside cacao tissues and this endophytic life-style may be
exploited to produce cacao seedlings treated with this biocontrol agent. To investigate the capacity of T.
stromaticum to colonise cacao endophytically, two field experiments were conducted with four isolates
from two distinct genetic groups. It was observed that all four isolates were recovered at levels higher
than 50% from cacao trunks 30 days after application, but only isolates from group II were recovered
120 days after spraying, at levels between 10-20%. In order to determine whether T. stromaticum is
able to colonise the sapwood of adult trees, we sampled the trunk of 300 cacao plants grown in fields
sprayed one year earlier with T. stromaticum for the control of witches broom disease. A total of 100
isolates of Trichoderma were obtained and identified by sequencing fragments of the ITS region of the
RNA polymerase and the EF1-alpha subunit. Several species, including T. harzianum, T. reesi, T. orientalis,
T. brevicompactum, T. longibrachiatum, T. asperellum, T. amazonicum, T. virens, T. koningiopsis, T.
atroviride and T. pseudokoningii were identified in cacao sapwood, but no T. stromaticum was found. T.
harzianum was the species most frequently isolated. These results indicate that the endophytic capacity
of T. stromaticum is limited and differs between the genetic groups. This differential response should be
further investigated to understand the genetic basis of endophytic colonisation in this fungus.
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Development and validation of a species-specific marker for

Trichoderma atroviride
LANGE, C.1; Hay, A. J.1; Kandula, J.1; Yardley, J. A.1; Braithwaite, M.1; Weld, R. J.2;
Stewart, A.1 and Steyaert, J. M.1
Bio-Protection Research Centre, P O Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand.
claudia.lange@lincolnuni.ac.nz; amanda.hay@lincoln.ac.nz; janaki.kandula@lincoln.ac.nz; jessica.yardley@lincoln.ac.nz;
mark.braithwaite@lincoln.ac.nz; alison.stewart@lincoln.ac.nz; johanna.steyaert@lincoln.ac.nz.
Lincoln Ventures Limited, PO Box 133, Lincoln University, Lincoln 7647, New Zealand.
richard.weld@lvl.co.nz.
Biocontrol agents can use a range of strategies to control pathogens, such as plant growth promotion,
induced resistance, mycoparasitism, antibiosis and competition. The mechanisms of biocontrol used
by any particular agent vary depending on the conditions they experience. In recent years, it has
become more apparent how complex these mechanisms are in respect to a combination of abiotic
and biotic factors such as soil type, pH, temperature, host plant and pathogen. In order to study
biocontrol activity by Trichoderma atroviride in a natural environment, it is important to distinguish it
from other Trichoderma species present and to track and quantify the strains in the environment. Here
we present the development and validation of a species-specific molecular marker for T. atroviride.
The Trichoderma endochitinase-42 encoding gene chit42 is a single-copy gene and was selected as it
has only low sequence homology to chitinases in other fungi. We looked for sequence variations in
chit42 that distinguished T. atroviride from other Trichoderma species. Genomic sequences of chit42
from 66 Trichoderma isolates, representing 9 species, were compared to identify T. atroviride-specific
polymorphisms. Based on these sequence polymorphisms, primers were designed that only amplify T.
atroviride. A species-specific PCR amplification of chit42 using these primers was optimised to find the
most stringent PCR conditions. Using these primers in real-time PCR, standard curves were generated to
quantify the amount of T. atroviride chit42 DNA in natural non-sterilised soil inoculated with spores or
mycelial tissue. This species-specific marker will allow us to study T. atrovirides mode of action in more
detail in pot and field trials and natural ecosystems.
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SESSION 4
Omics
KEYNOTE
Connecting Trichoderma reesei phenotypes with genotypes
Scott E. BAKER
Pacific Northwest National Laboratory, Richland, WA 99354, US.
scott.baker@pnnl.gov
Trichoderma reesei is an industrially important producer of plant biomass degrading enzymes. The
genome of T. reesei strain QM6a was sequenced by the US DOE Joint Genome Institute (JGI). In addition,
the high cellulose producing T. reesei strain RUT-C30 and as well as one of its precursor strains, NG14 was
resequenced in collaboration with the JGI. Analysis of the mutant strain genomic sequences indicated
point mutations in 43 genes as well as several large and small deletions. We have continued resequencing
analysis of two additional T. reesei mutant strains QM9136 and QM9978 which do not secrete cellulases.
Moreover, post-genomic analyses are ongoing using advanced analytical instrumentation at the DOE
Environmental Molecular Science Laboratory (EMSL) user facility at the Pacific Northwest National
Laboratory. These analyses include helium ion microscopy and laser ablation mass spectrometry on
T. reesei strains. In summary, a range of analyses are focused at connecting T. reesei phenotypes with
genotypes.
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Trichoderma virens - a tale of two strains

MUKHERJEE, P. K.1 and Horwitz, B. A.2
Central Institute for Cotton Research, Nagpur, India.
Prasunmukherjee1@gmail.com.
The Technion-Israel Institute of Technology, Haifa, Israel.

horwitz@tx.technion.ac.il
Unlike most other species in this genus, the mycoparasitic and plant root symbiont Trichoderma virens
population is comprised of two well defined groups - the Q strains that produce gliotoxin and the P
strains that produce gliovirin. In this study, we compared two extensively studied strains each belonging
to either of these groups - the GVW (IMI 304061, P strain) isolated from India and the Gv 29-8 (Q
strain) isolated from Texas, USA. GVW does not produce gliotoxin (we could not detect gliovirin either),
but Gv 29-8 rapidly produces copious amounts of gliotoxin. GVW is fast growing and produces dispersed
conidiophores, unlike Gv 29-8, which is slow growing and produces conidiophores in clusters. Both the
strains are antagonistic and effective against the pathogen Rhizoctonia solani, but most interestingly,
Gv29-8 is ineffective against Sclerotium rolfsii in seedling assays. The inability of Gv 29-8 to control S.
rolfsii can be attributed to its inability to parasitise the sclerotia of this pathogen, which are readily
colonised and destroyed by GVW. We have sequenced GVW by using Illumina platform and a first analysis
of the NGS sequencing data indicates the gliotoxin cluster to be missing in this strain, thus providing a
plausible explanation for lack of gliotoxin production in GVW.
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Improving in vivo footprinting finding a more sensitive way to

detect protein-DNA interactions
GORSCHE, R.; Derntl, C.; Mach, R. L. and Mach-Aigner, A. R.
Department of Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology,
Vienna, Austria.
rita.gorsche@tuwien.ac.at; christian.derntl@tuwien.ac.at; robert.mach@tuwien.ac.at; astrid.mach-aigner@tuwien.ac.at
In order to understand the mechanisms responsible for transcriptional regulation in a cell it is necessary
to know which bases of a promoter region are targeted by proteins during induction or repression of a
gene. Therefore we altered traditional in vivo footprinting via LM-PCR (Rauscher et al., 2006) to obtain
a more sensitive method to detect condition-dependent Protein-DNA interactions. Our enhanced
method is based on fluorescent 5-[6-FAM]-labeling of DNA fragments and analysis via capillary gel
electrophoresis (CGE) (Zianni et al., 2006). In addition, we created a graphical user interface that is
essential in the automation of data processing and visualisation of results. This improved protocol
could be essential in obtaining a better insight in the regulation of gene expression and supporting the
systematic manipulation of expression patterns. This condition-dependent approach allows a detailed
analysis of the switching of protein-DNA interaction events in regulons, consequently supporting a better
understanding of regulatory networks within an organism, tissue or cell. The use of this refined method
has already led to new insights into the xyr1-regulon responsible for (Hemi-)Cellulase-expression in the
industrially important fungus Trichoderma reesei.
Rauscher et al. (2006). Eukaryotic Cell 5: 447-456.
Zianni et al. (2006). J Biomol Tech. 17: 103-113.
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Transcriptomic responses of tomato plants to Trichoderma

MONTE, E.1; Domnguez, S.1; Cardoza, R.E.2; Gutirrez, S.2; Hermosa R.1; Nicols C.1
and Rubio, M.B.1
Centro Hispano-Luso de Investigaciones Agrarias (CIALE). University of Salamanca. 37185, Salamanca, Spain.
emv@usal.es; sara126_5@usal.es; rhp@usal.es; cnicolas@usal.es; belenru@usal.es.
Area of Microbiology. Universitary School of Agricultural Engineers. University of Len. Campus de Ponferrada. Avda. Astorga
s/n. 24400 Ponferrada, Spain.
re.cardoza@unileon.es; s.gutierrez@unileon.es
The fungal genus Trichoderma includes species that have the ability to antagonise, parasitise or even
kill other fungi. Some strains of Trichoderma are used as biocontrol agents in agriculture and can
establish themselves in the plant rhizosphere, stimulate plant growth and elicit plant defence reactions
against pathogens (Hermosa et al., 2012). Several studies have analyzed transcriptomic and proteomic
interactions of Trichoderma spp. with plants (Lorito et al., 2010). Similarly, other studies have analyzed
the transcriptomic, proteomic and metabolomic responses of plants to Trichoderma colonisation. In this
study, we have investigated the gene expression patterns of T. harzianum seed-coated tomato plants at
four weeks of interaction using GeneChip Tomato Genome Arrays (Affymetrix) including 9.254 transcripts.
After a triplicate chip experiment, nine genes (6 up- and 3 down-regulated) were differentially expressed
in respect to the uninoculated tomato control (FC>2 and FDR <0.1). Among the six up-regulated genes,
four were related to defense [a protein involved in response to the ethylene induced xylanase (EIX) of
Trichoderma, an extensin protein, a cathepsin inhibitor protein and a peroxidase], one was involved in
terpene metabolism and the other one was a precursor of amino acid biosynthesis. The three downregulated genes were the typical marker gene for the jasmonic acid defense cascade PDF1.2, a nonsymbiotic class I haemoglobin that plays a role in the defense responses against pathogen invasions,
possibly by modulating the nitrogen metabolism, and a phosphoenolpyruvate carboxykinase, a junction
enzyme between glycolysis and the Krebs cycle. Results indicate that after the initial Trichoderma-plant
interaction process, when the root colonisation is well established, Trichoderma induces minor changes
in plant transcriptome and only few genes related to plant metabolism and defense responses have
shown a significant modification of their expression pattern.
Hermosa et al. (2012). Microbiology 158: 17-25.
Lorito et al. (2010). Ann. Rev. Phytopathol. 48: 395417.
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Expression of genes encoding small secreted cysteine-rich proteins

(SSCPs) in Trichoderma virens root interactions
HORWITZ, B. A.1; Morn-Diez, M. E.2; Trushina, N.1 and Kenerley, C. M.2
Department of Biology, Technion-IIT, Haifa, Israel.
horwitz@tx.technion.ac.il.
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, USA.
c-kenerley@neo.tamu.edu
Small secreted cysteine-rich proteins (SSCPs) are often part of the fungal-plant dialog in host-pathogen
interactions. Trichoderma genomes encode large sets of these proteins, which might act as effectors in
establishment of the interaction with plant roots. Interestingly, the mycoparasitic species T. atroviride
and T. virens have a more diverse set of SSCPs than the saprophyte T. reesei. The Trichoderma-root
interaction can be hosted by a wide range of plant species, including both monocots and dicots. To test
the hypothesis that SSCP gene expression is modulated by the plant, we cocultured T. virens with maize
or tomato in a hydroponic system allowing interaction with the roots. The transcriptomes for T. virens
alone and with tomato or maize roots were compared by hybridisation on Agilent custom oligonucleotide
microarrays designed from the predicted protein-coding gene models ((Kubicek et al., 2011), http://
genome.jgi-psf.org/cgi-bin/searchGM?db=Trive1). At the chosen cutoff for statistical significance, 9 SSCP
genes were upregulated at least 2.5 fold in co-culture with tomato, and 7 with maize. This supports our
hypothesis that specific SSCPs are regulated in response to interaction with plant roots. The 11645 unique
probes (defined by JGI T. virens v1.0 protein ID numbers) on the array do not necessarily correspond to
all transcribed genes, but they do provide a genome-wide sampling. We also found that the expression
of many genes (including some SSCPs) is modulated by interaction with the roots. The tomato and
maize induced transcriptomes differ from each other and from the saprophytic growth transcriptome.
Reporter genes chosen from the microarray data will be useful to follow, in time and space, how the
fungal cell detects plant signals, and will provide an entry point to the signaling pathways. SSCPs released
in response to plant signals might have a role in inducing systemic resistance in the plant.
Kubicek et al. (2011). Genome Res. 12(4):R40
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Identification of putative effectors through bioinformatics analysis

of Trichoderma proteomes
LAWRY, R.1; Aleman, I.2; Delaye, L.2; Olmedo, V.3; Horwitz, B. A.4; Kenerley, C. M.5;
Herrera-Estrella, A.6; Brown, C.7; Greenwood, D.8; Stewart, A.1 and MendozaMendoza, A.1
Bio-Protection Research Centre, P O Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand.
robert.lawry@lincolnuni.ac.nz; artemio.mendoza@lincoln.ac.nz; alison.stewart@lincoln.ac.nz.
Cinvestav-Irapuato Department of Genetic Engineering, Km. 9.6 Libramiento Norte 36822 Irapuato Guanajuato, Mexico.
alemanmd@gmail.com; ldelaye@ira.cinvestav.mx.
Universidad de Guanajuato Biology Department. Noria Alta s/n; C.P. 36050; Guanajuato, Mexico. vg.olmedo@ugto.mx.
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. horwitz@tx.technion.ac.il.
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843.
c-kenerley@neo.tamu.edu.
National Laboratory of Genomics for Biodiversity, Km. 9.6 Libramiento Norte 36822 Irapuato Guanajuato, Mexico.
aherrera@langebio.cinvestav.mx.
Biochemistry Department and Genetics, University of Otago P.O. Box 56, 710 Cumberland St, Dunedin 9054, New Zealand
chris.brown@otago.ac.nz.
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
david.greenwood@auckland.ac.nz.
Trichoderma are a widely distributed group of fungi, several of which are capable of forming endophytic
relationships with commercially important plants such as maize and tomato. Despite forming these
beneficial interactions, Trichoderma species face many of the same challenges that pathogens face
during colonisation of the plant host. Effector proteins are thought to be a significant mechanism by
which fungi overcome host defenses in order to colonize plants. Effector proteins are generally small
cysteine rich proteins that contain a signal peptide directing secretion and often have little homology
to known proteins. Using these criteria, a range of bioinformatic analyses were carried out to identify
effectors present within the proteomes of T. virens, T. reesei and T. atroviride available from the Joint
Genome Institute. SignalP was used to identify signal peptides from this dataset and to create a database
of predicted secreted proteins. This dataset was then analyzed with TMHMM to detect potential
transmembrane domains and remove those proteins from the dataset. MEME, BLAST, PROTCOMP and
ELM were used to identify novel domains, key features and cellular localisation of the remaining proteins.
XSTREAM was used to detect tandem repeat proteins. Manual searches were carried out to separate
proteins by size, cysteine richness and comparison to known effector motifs. Combining these analyses,
we show that on average, 9% of T. atroviride, T, virens and T. reseei proteomes appear to contain a signal
peptide, with approximately 58% of these proteins being non membrane bound. Notably, a number of
large (500 amino acids or more) proteins were detected by this method. A database of the secretome
was created, linking these proteins to localisation, tandem repeat and BLAST data, allowing a relatively
robust prediction of putative effector proteins to be made. Future work in this area should focus on
laboratory analysis of candidate proteins to test the function of the putative effector proteins.
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Biodiversity of Trichoderma species in New Zealand

BRAITHWAITE, M.1; Steyaert, J. M.1; McLean, K. L.1; Johnston, P. R.2; Hill, R. A.1;
Ball, S.3; Stewart, A.1, and Bissett, J.4
mark.braithwaite@lincoln.ac.nz; johanna.steyaert@lincoln.ac.nz; kirstin.mclean@lincoln.ac.nz; robert.hill@lincoln.ac.nz;
alison.stewart@lincoln.ac.nz.
Landcare Research, Private Bag 92170, Auckland Mail Centre, Auckland 1142, New Zealand.
JohnstonP@landcareresearch.co.nz.
Natural Resources Canada, 580 Booth Street, Ottawa, Ontario K1A 0E4, Canada.
Shelley.Ball@nrcan.gc.ca.
Agriculture and Agri-Food Canada, 960 Carling Ave, Ottawa, Ontario K1A 0C6, Canada.
John.Bissett@AGR.GC.CA.
A taxonomic study of New Zealand Trichoderma/Hypocrea species in the Bio-Protection Research Centre
and Landcare Research fungal culture and herbarium collections was completed. Sixty-six species of
Trichoderma were identified, including 14 species which appear to represent undescribed taxa. The
New Zealand species are positioned within nearly every major Trichoderma clade, with terminal lineages
of the Harzianum clade, Citrina clade, and Trichoderma section well represented. This wide diversity
of species suggests that a broad spectrum of ancestral Trichoderma species from the super-continent
Gondwana may have survived the marine transgression of the New Zealand land mass. Subsequently,
populations may also have been supplemented and maintained by trans-oceanic dispersal mechanisms,
such as ocean currents and animal migrations from Australia and the sub-tropical land areas north of
New Zealand. The New Zealand Trichoderma species were isolated from above ground (e.g. wood, bark,
leaves) and below ground (roots, soil) sources. From above ground substrates, ten Hypocrea species
were reported only from New Zealand. A further seven species were known only from New Zealand
and Australia. Below ground, the most common Trichoderma species isolated in New Zealand was an
undescribed sister species to the cosmopolitan T. atroviride, which also occurs in New Zealand. The
Trichoderma below-ground isolates had a much higher proportion of species occurring outside
New Zealand with a broad, pan-temperate or subtropical distribution. This pattern of distribution is
indicative of frequent introductions either by natural mechanisms within the local region or by the
activities of humans such as colonisation and the movement of soil and plant material. The diversity of
Trichoderma species in New Zealand, their taxonomic relationships, distribution, ecology, and possible
origins will be discussed.
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Molecular diversity of Trichoderma species from groundnut

rhizosphere
SRILAKSHMI, P.1; Prasad, K. S.1; M. and Rao, M. M.2
Department of Botany, Osmania University, Hyderabad 500 007, Andhra Pradesh, India.
kattasp@gmail.com.
Department of Botany, Government College for Women, Khammam, Andhra Pradesh, India.
Biological control is one of the potential options to reduce preharvest aflatoxin contamination in
groundnut. Aflatoxin contamination is an important problem in groundnut affecting quality, trade and
its product. Trichoderma species offer considerable potential for controlling aflatoxin contamination
in groundnut. However, targeted and rapid identification of useful isolates is hampered by an array
of practical classification issues. In this study, Two hundred and twelve Trichoderma isolates from the
groundnut rhizosphere soil were collected from groundnut growing areas of four districts (Anantapur,
Chitoor, Cuddapah and Kurnool) of Andhra Pradesh and two districts (Kolar and Tumkur) of Karnataka.
Over 40% of the samples yielded Trichoderma, with a maximum of 64% from soil samples of Anantapur.
Trichoderma isolates were at maximum of 11% from the samples of Kolar while Trichoderma population
density was maximum in Chittoor soil samples. Experiments for antagonism or mycoparasitism in
vitro against A. flavus (Af 11-4) revealed that 150 isolates were antagonistic. Among the 150 isolates
antagonistic to A. flavus (Af 11-4), 38 isolates grew until the colony met the A. flavus colony, 48 isolates
showed an inhibition zone when paired with A. flavus and 69 parasitised A. flavus. Forty eight isolates
which showed both antagonism and mycoparasitism were subjected to molecular characterisation using
AFLP markers and ITS Primers. Results showed a high level of genetic diversity among the 48 isolates. ITS
primers 1 and 4 were used to evaluate their relationship with geographical distribution and taxonomic
correlation. Twenty-five Trichoderma isolates only showed amplification producing 17 polymorphic
bands. Dendrogram clusters correlated with species designations based on morphological characteristics
and geographical areas. The rDNA regions amplified by ITS 1 and 4 clearly divided the isolates into two
major groups which showed a distinct distribution of species. A close correlation of molecular characters
with morphological identification was seen in this research. The genetic similarity coefficients obtained
using the Jaccard algorithms were used to generate an UPGMA dendrogram. Based on MDS analysis
of the ITS dataset it was possible to separate isolates into 5 different groups. Antagonistic properties
observed in different species of Trichoderma distributed all over the groundnut growing areas of Andhra
Pradesh and Karnataka and exhibited molecular variation among the antagonistic isolates. rDNA-ITS
analysis revealed a broad relationship with geographical distribution.
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Biodiversity of root-endophytic Trichoderma from Malaysian

Borneo
CUMMINGS, N. J.; Steyaert, J. M.; Ambrose, A.; Roslan, H.; Agbayani, F. V.; Okang,
A. E.; Valdez, R. B.; Yatim, M.; Hay, A. J.; Ball, S.; Bissett, J.; Chong, L.; Abdullah, J.;
Stewart, A. and Hill, R. A.
nicholas.cummings@lincoln.ac.nz; robert.hill@lincoln.ac.nz
Trichoderma root endophytes confer a number of benefits to their host plants including enhanced
growth and disease resistance. However, few researchers have examined the taxonomy of Trichoderma
species associated with plant roots in tropical regions. Trichoderma is currently used on a wide scale
as a replacement for fungicides in plantation forestry in South East Asia. During research evaluating
Trichoderma isolates for Acacia mangium bioprotection, root samples were collected from indigenous
plants and a variety of exotic ornamental and crop species at sites throughout the Malaysian states of
Sarawak and Sabah in Borneo. Collections included over 57 species in 27 plant families. Trichoderma was
isolated from surface sterilised roots and isolates identified to species level based on analysis of partial
translation elongation factor-1 (tef1) sequences. Species present included Trichoderma asperelloides,
Trichoderma asperellum, Trichoderma harzianum, Trichoderma reesei, Trichoderma strigosum, and
Trichoderma virens. Members of the T. harzianum species complex and T. virens were the most frequently
isolated taxa. Analysis of sequence data also indicated that several isolates are likely to represent
undescribed Trichoderma species in two main clades. These results suggest that tropical plants are a
useful source of novel root-associated Trichoderma that may have potential applications in agriculture
and plantation forestry.
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POSTER SESSION
Wednesday 29th August
Effect of temperature and length of incubation period on

Trichoderma atroviride LU132 conidial production, germination
and bioactivity
DARYAEI, A.; McLean, K. L.; Glare, T. R. and Stewart A.
Bio-Protection Research Centre, Lincoln University, Lincoln, 7647 Canterbury, New Zealand.
amir.daryaei@lincolnuni.ac.nz; kirstin.mcLean@lincoln.ac.nz; travis.glare@lincoln.ac.nz; alison.stewart@lincoln.ac.nz
Trichoderma atroviride LU132 has been commercialised in New Zealand as Tenet and Sentinel for the
control of Sclerotium cepivorum and Botrytis cinerea, respectively. To determine the effect of culturing
conditions on efficacy of T. atroviride LU132, the present research examined the influence of incubation
temperature (20, 25, and 30C) on the production of conidia under constant light over a 25 day period.
Two measures of quality of the conidia produced were also assessed - germination and bioactivity of
the subsequent colony against Rhizoctonia solani. Maximum conidial production occurred at 25C and
after 20 days of growth. A decline in conidial production at 25 days was observed. The conidia produced
at 30C, germinated significantly faster than those produced at 25 and 20C, respectively. Incubation
temperature did not have a significant effect on subsequent colony bioactivity against R. solani. However,
a significant increase in bioactivity was seen with colonies arising from spores produced after 25 days
incubation compared with the shorter incubation periods. To determine if the decline in conidial
production continued after 25 days, the experiment was repeated at 25C for 50 days. Extending the
incubation period resulted in a second peak in conidial production after 45-50 days. The produced conidia
in the extended experiment had an optimum germination after 20 days incubation and the optimum
bioactivity was achieved with conidia harvested after 15 days. This study suggests that T. atroviride
formulations based on conidia produced to optimise quantity may not be the most bioactive.
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Development of a microbial biocontrol agent for control of

Sclerotinia sclerotiorum in vegetables
Effect of a Trichoderma bio-inoculant on emergence and growth

of Camelina seedlings
HICKS, E.; McLean, K. L.; Braithwaite, M. and Stewart, A.
KANDULA, D. R. W.; Alizadeh, H. and Stewart, A.
emily.hicks@lincoln.ac.nz; kirstin.mclean@lincoln.ac.nz; mark.braithwaite@lincoln.ac.nz; alison.stewart@lincoln.ac.nz
diwakar.kandula@lincoln.ac.nz; hossein.alizadeh@lincoln.ac.nz; alison.stewart@lincoln.ac.nz
Sclerotinia sclerotiorum (Lib.) de Bary (white mould) is an important soil-borne plant pathogen which
causes significant rots in maturing vegetable crops worldwide, resulting in costly yield losses for
commercial growers. The aim of this research was to identify potential biocontrol agent(s) that could
control white mould in vegetable crops. Microorganisms (sixty eight isolates) from both New Zealand
agricultural soils, and the Biocontrol Microbial Culture Collection, Bio-Protection Research Centre,
Lincoln University were screened against S. sclerotiorum using a detached cauliflower leaf assay. Nine
isolates significantly reduced S. sclerotiorum lesion area by more than 85% compared with the pathogen
control treatment when applied to wounds 24 hours before pathogen application. These nine isolates
were also assessed in pot trials using dwarf bean (Phaseolus vulgaris) where five Trichoderma spp. and
two Gliocladium spp. significantly reduced the plant disease score by 32-60%. In an additional pot trial
Trichoderma sp. 7 and Gliocladium sp. 2 significantly reduced the percentage of infected beans by 47%
and 53%, respectively. This result was statistically equivalent to the control achieved with the fungicide.
Trichoderma sp. 7 and Gliocladium sp. 2 will be commercially formulated and further evaluated in field
trials.
Camelina (Camelina sativa) or false flax has been the subject of research in New Zealand as an alternative
oilseed crop that can be used as renewable biodiesel feedstock. While being tested in agronomy trials,
damping-off and root rot by Rhizoctonia solani was observed to constrain seedling establishment and
seed-coating with fungicides was required. The effectiveness of a Trichoderma strain mixture applied
in granule or prill formulation on two varieties of Camelina (Suneson and 4164) was evaluated in three
glasshouse experiments. Soil from a field with known history of R. solani infestation was used. The
bio-inoculant was applied either with or without fungicide (Vitaflo) coated seeds and the numbers of
emerged seedlings, diseased and healthy plants were recorded. For the Suneson variety, application with
either formulation increased seedling emergence compared with the bare seed control and seedling
emergence with the granule formulation was equal to the fungicide treatment (p<0.05). For the 4164
variety, both bio-inoculant formulations increased seedling emergence significantly compared with
the bare seed control. In the prill treatment for both Camelina varieties, shoot and root dry weights
were equal to the fungicide treatment. With the granule formulation, root dry weight in both Camelina
varieties as well as shoot dry weight in 4164 gave significantly higher values than the fungicide
treatment. When fungicide coated seeds were sown with the bio-inoculant formulations, significantly
higher healthy seedling emergence was observed in Suneson but not 4164. The results from this
study show that Trichoderma bio-inoculants can be used alone or in conjunction with a fungicide seed
treatment to promote seedling emergence.
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Incorporation of trehalose into Trichoderma atroviride conidia

and its effect on viability of conidia during storage
Mycelial injury as a key abiotic factor affecting conidiation

of genetically distinct Trichoderma stromaticum isolates
KANDULA, J.; Braithwaite, M.; Steyaert, J. M.; Hay, A. J. and Stewart, A.
LEITE, S. S.1; De Souza, J. T.2; Loguercio, L. L.1
janaki.kandula@lincoln.ac.nz; mark.braithwaite@lincoln.ac.nz; johanna.steyaert@lincoln.ac.nz; amanda.hay@lincoln.ac.nz;
alison.stewart@lincoln.ac.nz
Biocontrol agents are living organisms that require stabilisation, usually through product formulation,
to attain an acceptable shelf life of 1-2 years. Trehalose (a storage carbohydrate in a wide variety of
organisms) is an osmo-protectant that is stable under hot and acidic conditions and is able to protect
fungal spores against freezing or excess heat. Trehalose has also been shown to increase spore viability
in fungi. In the current research, the ability of trehalose to prevent loss of Trichoderma atroviride
conidial viability during storage under conditions of accelerated aging to simulate long-term storage was
evaluated. T. atroviride conidia were produced by solid substrate fermentation, with the initial broth
amended with varying amounts of trehalose (ranging from 0-24.4 mM). Conidia were harvested in sterile
water and allowed to air-dry before storage at either 4 or 30C with uniform water activity. Condial
viability was assessed at monthly intervals by counting percentage germination. At harvest, trehalose
amendment of the growth media resulted in a significant increase in trehalose content of T. atroviride
conidia of between 18 and 42%, when compared with the untreated controls. The 0.8 mM trehalose
treatment significantly increased viability of conidia, as measured by the area under the conidia viability
progress curve, in both the 4 and 30C storage treatments by 5 and 12%, respectively. However, at this
concentration, trehalose did not significantly extend shelf-life of T. atroviride. The addition of higher
concentrations of trehalose significantly reduced spore viability.
100
Post-graduation Programme in Genetics & Molecular Biology (PPG-GBM), State University Santa Cruz (UESC); Rod. BR 415,
Km 16, Salobrinho, 45992-900 Ilhus-BA, Brazil.
Recncavo da Bahia Federal University, CCAAB; Rua Rui Barbosa, 710, Centro, 44380-000 Cruz das Almas-BA, Brazil.
leandro@uesc.br
Trichoderma stromaticum (Ts) is a promising biocontrol agent against the witches broom disease of
cacao in the cocoa-producing region of southeastern Bahia (Brazil). Despite showing strong biocontrol
activity in the field, some Ts isolates from two previously defined genetic groups (I and II) have shown
to be recalcitrant for production of conidia at levels needed for development of commercial products.
Previous in vitro studies on the interactive effects of medium types, acid pHs and light exposure
demonstrated isolate-dependent sporulation responses to a specific combination of factors. Here, we
advanced our investigation toward the effect of mycelial injury on the in vitro conidiation of these isolates
(labelled ALF-15 and ALF-1092 from genetic group I; ALF-64 and ALF-113 from group II). Isolates were
subjected to the optimum specific abiotic conditions in culture media previously defined, varying only the
injury and light treatments with three replicates each. They were grown at 28C in solid media dispensed
in large 15-mm Petri dishes for 5 days, in two sets of experiments: one with injury applied alone or
combined with a light-pulse treatment (growth in total darkness served as control), and another with
different amounts of mycelial cuts imposed to the Ts colonies, during growth in the dark. Morphology of
colonies and spore counting per plate were the qualitative and quantitative variables assessed. Results
indicated a positive effect of injury in increasing in vitro conidiation, although the sporulation patterns
obtained appeared to be isolate-specific. This suggests that mycelial injury is a special physical factor with
relevant effect on T. stromaticum. This factor has the potential to be further explored to provide higher
levels of conidiation, thereby helping with large-scale production of more efficient isolates for biocontrol
purposes.
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Heterologous expression characteristics of Trichoderma viride

endoglucanase V in the silkworm, Bombyx mori L.
Antimicrobial mechanism of Trichoderma harzianum L-amino-acid

oxidase N-terminal sequence derived peptides
LIANG, S.#; Wang, M.-X.#; Li, X.-H. and Miao, Y.-G.
LIN, H.-S.1; Liu, S.-Y.2; Peng, K.-C.1 and Lo, C.-T.3
College of Animal Sciences, Zhejiang University, 866YUHANGTANG Road, Hangzhou 310058, P. R. China.
liangshuang0307@163.com. Corresponding author: Y.-G. Miao: miaoyg@zju.edu.cn
Department of Life Science and the Institute of Biotechnology, National Dong Hwa University, No. 1, Sec. 2, Da Hsueh Rd.,
Shoufeng, Hualien 97401, Taiwan, R.O.C.
H.-S. Lin: m9513008@ems.ndhu.edu.tw; kcpeng@mail.ndhu.edu.tw.
Department of Molecular Biotechnology, Da-Yeh University, No.168, University Rd., Dacun, Changhua 51591, Taiwan R.O.C.
syliu@mail.dyu.edu.tw.
contributed equally to this research
Department of Biotechnology, National Formosa University, No.64, Wunhua Rd., Huwei, Yunlin 63201, Taiwan, R.O.C.
ctlo@sunws.nfu.edu.tw.
Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include
exoglucanases, endoglucanases and -1,4-glucosidase. In this study, we used an improved Bac-to-Bac/
BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes
of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V gene from T. viride in
silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in
silkworm larvae. The results showed that an around 36-kDa protein was visualised in BmN cells at 48 h
after the 2nd generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The enzymes from
recombinant baculoviruses infected silkworms exhibited significant maximum activity at pH 5.0 and
temperature 50C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV
baculovirus infected silkworms and normal silkworms, respectively. It was stable at a pH range from 5.0
to 10.0 and at a temperature range from 40C to 60C. The availability of large quantities of EG V that the
silkworm provides may greatly facilitate future research and potential application in industries.
102
Due to misuse of antibiotics, the number of multi-drug resistant microorganisms has increased. Instead
of traditional chemical compounds, an antimicrobial peptide has potential to be a candidate of new
generation antibiotics. L-amino acid oxidase from Trichoderma harzianum (ThLAAO) demonstrated
inhibition of bacteria and fungi growth via mitochondria-dependent apoptosis and cell leakage. This cell
leakage of two bacteria was induced by cultivation with the synthesised N-terminal sequence of ThLAAO
(TLN). Since the hydrophobic moment was considered as an important parameter of antimicrobial
activity, seven modified peptides derived from the TLN sequence were created. The antimicrobial activity
of these synthesized peptides against bacteria and fungi pathogens was measured. The toxicity of these
peptides to mammalian cell was performed as well as the haemolysis activity of red blood cell. To realise
the relationship between function and structure as well as sequence content, the TLN derived peptides
were applied on the far-UV circular dichroism. Here, we present the peptides modified by the partial
sequence of an enzyme which effectively antagonised pathogenic microorganisms. This could be a new
strategy for the discovery of antimicrobial peptides.
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The impact of illumination on the production of cellulases

and anthraquinones from Trichoderma harzianum ETS 323
Screening strains and mechanisms of Trichoderma spp.

for phosphate solubilisation
HSU, C.-W.1; Liu, S.-Y.2; Chiang, J.-H2; Lo, C.-T.3 and Peng, K.-C.1
LO, C.-T.1; Feng, P.-C.1 and Peng, K.-C.2
Department of Life Science and Institute of Biotechnology, National Dong Hwa University
C.W. Hsu: glamp40@hotmail.com; kcpeng@mail.ndhu.edu.tw.
Department of Biotecnology, National Formosa University; Yunlin 63208, Taiwan. ctlo@nfu.edu.tw.
Department of life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, 97401, Taiwan.
kcpeng@mail.ndhu.edu.tw
Department of Molecular Biotechnology, Da-Yeh University.

syliu@mail.dyu.edu.tw; J.-H. Chaing: jmpeng@yahoo.com.tw.
Department of Biotechnology, National Formosa University.

ctlo@nfu.edu.tw
Trichoderma harzianum is a major industrial source for cellulases which degrade cellulose into glucose
and are considered the most important enzymes in second generation bio-ethanol production.
In addition, T. harzianum can also produce versatile bio-active secondary compounds, such as
anthraquinones. The secretion of either proteins (including hydrolytic enzymes) or secondary metabolites
is modulated by various environmental factors, light is one of these. In the present trial, treatment
with bright light, dark, plus custom-designed light sources with defined wavelengths, mainly red640,
green520, and blue465 on minimal media supplemented with 1% CMC was used to determine the impact
on secretion of enzymes and secondary metabolites of T. harzianum. The anthraquinone production
was quantitated by O.D. 436 (extinction coefficient at 436 nm equals to 11.8 for anthraquinones).
Cellulase secretion was also measured by enzymatic activity as well as RNA expression in the mycelia.
The results showed that the highest cellulase activity was observed with blue light treatment followed
by green, red, bright light and dark treatment when cultured on an agar plate. The production of various
anthraquinones was altered by different light treatment as well. The possible rationale and correlation
with the biocontrol mechanism against phytopathogens will be addressed.
Phosphorus (P) plays a vital role in plant nutrition and is also an essential macronutrient for biological
growth and development. Despite its wide distribution in nature, it is deficient in most soils due to
its high fixation capacity such as phosphates of iron, aluminum and calcium. Many soil bacteria and
fungi have been reported to solubilise phosphate minerals and make them available to plants through
acidification, chelation and exchange reactions. Trichoderma spp. are one of the most common fungi
used for biological control of plant pathogens and they have been recorded to degrade the chitin,
cellulose and other natural materials. However, there is very little literature available on the effect
of Trichoderma spp. on plant growth through the release of immobilised nutrients. Therefore, in this
study we try to determine how many strains of Trichoderma spp. in Taiwan have the ability to solubilise
phosphate minerals and make them available to plants. The results revealed that only 1.54% of tested
Trichoderma strains had the ability to solubilise phosphate as determined by the use of Tricalcium
phosphate medium (TCP). The results showed that a positive correlation between the clear zone on TCP
and phosphate concentration in liquid culture. pH analysis of the liquid cultures of various Trichoderma
strains indicated that there are no significant difference among strains and imply that the potential
mechanism for phosphate solubilisation might not only be acidification. On the other hand, the strains of
Trichoderma that have secreted the acid phosphatases and phytases have an important role in phosphate
solubilisation. In green house tests, these strains were also used to determine the effect of Trichoderma
on plant growth. The preliminary results revealed that some strains of Trichoderma could promote the
seedling growth of cabbage and Chinese cabbage and reduce the symptoms of phosphorus deficiency.
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105
Validation of protocols to evaluate the quality of Brazilian

commercial products based on Trichoderma spp.
Antagonism of local isolates of Trichoderma spp. on pineapple

heart rot disease caused by Phytophthora cinnamomi var
cinnamomi in Costa Rica
LUCON, C. M. M.1; Bettiol, W.2; Haddad, P. E.1; Pinto, Z. V.2 and Morandi, M. A. B.2
Instituto Biolgico, CP 12898, 04010-970, So Paulo, SP, Brazil.
mantova@biologico.sp.gov.br; bettiol@cnpma.embrapa.br; patriciaehaddad@yahoo.com.br; zvpinto@yahoo.com.br.
Embrapa Meio Ambiente, Jaguarina, SP, Brazil. mmorandi@cnpma.embrapa.br
Escuela de Agronomia, Instituto Tecnolgico de Costa Rica, Alajuela, San Carlos. xmata@itcr.ac.cr.
Dr. Obregon Laboratories, Heredia, Costa Rica. M.obregon@doctor-obergon.com.
In the last decade many companies have started producing and selling biofungicides in Brazil. However,
the companies and the Government have no standard methodology to evaluate the commercial products
based on Trichoderma spp. to ensure the quality of biofungicides. The goal of this paper was to test
several protocols used by private companies and public institutions to establish an efficient and low cost
methodology to evaluate the quality of Brazilian commercial products based on Trichoderma spp. The
tested protocols contemplated the enumeration of total conidia, number of viable conidia and forming
colony units of biofungicides formulations based on Trichoderma. Briefly, the established protocol was
based on a serial dilution, the estimation of the number of conidia was performed in Neubauer chamber
under an optical microscope (250x magnification), spreading 100 L of the appropriate suspensions on
the surface of PDA medium plus Triton X100 (0.1% v:v) to enumerate the colony forming-units (CFU)
after 48 h of incubation at 25C and on PDA without Triton X100 to enumerate the geminated conidia
after incubation for a specific period (usually 14-18h). The fungal identification colonies were done on
the basis of morphological characteristics of the colony, conidia and conidiogenous cells. The proposed
protocols have proven to be efficient and practical to be used by private and Government companies to
guarantee the quality of commercial bioproducts based on Trichoderma spp. Six Government institutions
and several private companies approved the methodologies, and more than 60 people were trained to
perform them correctly.
106
MATA, X.1; Obregn, M.2; Lorito, M.3 and Monte, E.4
Department of Ar. Bo. Pa. Ve., Plant Pathology Section, University of Naple and CNR IPP-Institute for Plants Protecction,
Portici Section, Via Universita 100, Portici (Napoli) 80055 Italy;
Centro Hispano-Luso de Investigaciones Agrarias (CIALE), University of Salamanca, 37185 Salamanca, Spain. emv@usal.es
Pineapple (Ananas comosus) is the second most important agricultural product and the fourth in
national export trade in Costa Rica, with an accelerated growth in the late 70s. This led to intensive
use of chemicals, in some cases they were highly toxic and left significant residue levels, which caused
social, economic and environmental problems including, biodiversity losses and appearance of resistance
pest populations. The Oomycete Phytophthora cinnamomi var cinnamomi, identified using Kochs
postulates, is considered the most important pathogen for pineapple as it causes heart rot disease and
reduces fruit yield and quality. The main method for control is the use of synthetic agrochemicals, but
both the European Union and the US Environmental Protection Agency (EPA) has decided to reduce
the maximum level of residue accepted (LMR) in order to reduce the application of pesticides used
to control this disease. The scope of this research was to evaluate the ability of Trichoderma isolates
to inhibit the mycelial growth of P. cinnamomi var cinnamomi in vitro. We evaluated 38 isolates of
Trichoderma collected from soil samples from different locations in the Costa Rica provinces of Alajuela,
Cartago, Limn, Guanacaste, Heredia and Puntarenas (35 isolates), as well as 2 isolates obtained from
Minas Gerais in Brazil and 1 isolate from Italy. In the dual culture test, an agar plug of P. cinnamomi var
cinnamomi mycelium was placed onto a PDA plate and after 72 h another agar plug of Trichoderma
mycelium was placed on the opposite side of the plate. The evaluation of the pathogen growth inhibition
was performed every 48 h for 8 days. The data collected showed that P. cinnamomi var cinnamomi
had grown 27 to 32.2% after 72 h. However, this percentage decreased to 8.5 to 23.5% after 48 h
in dual cultures of Trichoderma. Trichoderma isolates showed pathogen colonisation percentages
of 32.2 to 91.9%. The colonisation percentage after 96 h ranged from, 34.7 to 100% and after 144 h
from 39.9 to 100%. The data were analysed by an ANOVA and a Tukey test using the software JMP of
the SAS INSTITUTE. After 48 and 96 h, the antagonistic behaviour was statistically different (p<0.05),
with the best Trichoderma showing a mean of 100% colonisation. After 144 h, only five isolates were
statistically different (p<0.05), with mean values between 39.9 and 90.5%. In addition, after 48 h, five
isolates showed a fungistatic effect and six isolates showed both a fungistatic and mycoparasitic effect
towards the pathogen. Isolates also differed in phenotypic features (such as; growth rate and mycelium
colouration), and medium pigmentation. Our results demonstrated that 37 isolates were effective and
that those isolates showing both a fungistatic and mycoparasitic abilities were the most effective in
controlling P. cinnamomi var cinnamomi, in vitro.
27 30 August 2012
27 30 August 2012
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Lincoln University
New Zealand
Commerce Building
Lincoln University
New Zealand
107
ABC transporters in acetic acid resistance of Trichoderma

atroviride
Non-target impacts of the biocontrol agent Trichoderma atroviride
on native ecosystems in New Zealand
McLEAN, K. L.1; Dodd, S. L.2; Minchin, R. F.1; Ohkura, M.1 and Stewart, A.1
1
Bio-Protection Research Centre, PO Box 84, Lincoln University 7647, Canterbury, New Zealand.
kirstin.mclean@lincoln.ac.nz; alison.stewart@lincoln.ac.nz
Landcare Researh, Private Bag 92170, Auckland Mail Centre, Auckland 1142, New Zealand.
dodds@landcareresearch.co.nz
The Environmental Protection Agency (EPA) assesses the risks versus the benefits associated with
the importation and release of new organisms into the New Zealand environment. In order to make
decisions on whether to release an organism, the EPA needs to understand what potential impacts
introduced species may have on New Zealands unique native ecosystems. To understand such risks,
a commercialised Trichoderma atroviride strain LU132 (Tenet, registered for the control of onion
white rot) was used as a model to assess the impacts of an introduced biocontrol agent on native
Podocarp forest species and a native grassland environment. Seven physical markers and photosynthetic
pigments were utilised to assess plant health of seven plant species from the Podocarp forest and four
plant species from the grassland environment. Results showed T. atroviride treatment had no effect
on plant health with the exception of a significant increase in trunk diameter and root:shoot ratio for
Plagianthus regius and Pittosporum eugenioides, respectively compared with the untreated control.
Some significant photosynthetic pigment differences also occurred between the T. atroviride treated
and the control plants however, these were not supported by physical changes in plant health. Cultural
data analysis of arbuscular mycorrhizae (AM) species found no significant difference in colonisation or
viable spore numbers in the rhizosphere for all plant species except Dacrycarpus dacrydioides where
T. atroviride treatment increased the number of viable spores compared with the untreated control.
Denaturing gradient gel electrophoresis data indicated that the addition of T. atroviride had no effect
on the diversity of AM and Pseudomonas species in the plant roots and rhizosphere. The plant health
assessment protocols developed from these studies will provide a useful tool for assessing biosecurity
risks associated with Trichoderma importation applications in New Zealand.
108
OLEJNKOV, P.; Vyhonsk, Z.; Palukov, V.; Chromkov, R.; Reichov, J.;
imkovi, M. and Krytofov, S.
Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology,
Radlinskho 9, 81237 Bratislava, Slovak Republic.
petra.olejnikova@stuba.sk; svetlana.krystofova@stuba.sk
Weak organic acids such as acetic, formic and levulinic acid are the most common acids produced during
hydrolysis of lignocellulose. Acetic acid is released during de-acetylation of hemicelluloses, while formic
and levulinic acids are degradation products of 5-hydroxymethyl-2-furaldehyde (HMF). Depending on
plant substrate the acetic acid concentration often exceeds the tolerance level of T. atroviride (less than
20 25 mM). T.atroviride can use acetate as a sole carbon source very efficiently as confirmed by its
growth in medium with low acetate supply as well as by measured inward acetate transport. We have
taken interest in studying T. atroviride sensitivity toward higher acetic acid concentrations which is clearly
in contrast with observations in other fungi including yeast. T.atroviride genome sequence revealed
that it contains nine genes encoding full length and one half size PDR transporters. Three of T. atroviride
PDR transporter genes share close similarity with yeast Pdr5p and Pdr12p of which Pdr12p is known to
be responsible for resistance against organic acids. Two T. atroviride genes encoding PDR transporters
Tapdr5 and Tapdr12 clearly showed an increase in their expression during growth in a medium containing
acetate. Analysing promoter regions of Tapdr5 and Tapdr12 revealed multiple consensus sequences for
FacB whose homolog was found in T. atroviride genome TafacB. Precise functional characterisation
of Tapdr5 and Tapdr12 is currently underway in order to identify their possible substrate(s) as well as
physiological functions during resistance and development.
27 30 August 2012
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Lincoln University
New Zealand
Commerce Building
Lincoln University
New Zealand
109
Effect of chitin-rich residues on the chitinolytic activity of

Trichoderma harzianum at in vitro and greenhouse experiment
Plant growth promotion and induction of resistance against

anthracnose in common bean by Trichoderma spp.
PASCUAL, J. A.1; Lopez-Mondejar, R.1; Blaya, J.1 and Ros, M.1
PEDRO, E. A. S.; Lucon, C. M. M.; Harakava, R. and Guzzo, S. D.
Department of Soil and Water Conservation and Organic Management. (CEBAS-CSIC). Campus Universitario de Espinardo N25,
30100 Murcia (Spain).
jpascual@cebas.csic.es; rubenlopezmondejar@gmail.com; pepabf@gmail.com; margaros@cebas.csic.es
Instituto Biolgico, CP 12898, 04010-970, Brazil, So Paulo, SP.

erica_spedro@hotmail.com; mantova@biologico.sp.gov.br; harakava@biologico.sp.gov.br; guzzo@biologico.sp.gov.br
One of the most used microorganisms for biological control (BCAs) in agriculture is the fungus
Trichoderma harzianum, which has shown good results against Fusarium oxysporum f. sp melonis. Its
beneficial effect is due to mode of action mechanisms such as mycoparasitism where hydrolytic enzymes
as chitinases are a key factor. These enzymes are encoded by genes whose expression is activated by
the presence of chitin or the products of its degradation. Chitin and its products, such as chitosan and
oligomers, have also been shown to act as potent agents that elicit defence reactions in plants and
inhibit the growth of pathogenic fungi and bacteria. In this study, three different chitin-rich residues
Chitosan (CHIT), shrimp shell powder (SSP) and mushroom wastes (MW) were evaluated with regard to
enhancement of the chitinolytic activity of T. harzianum used as a BCA against F. oxysporum f. sp melonis
in greenhouse nurseries. These chitin-rich residues activated the expression of the T. harzianum genes
encoding the NAGase and chitinase activities (exc1, exc2, chit42, chit33 and chit37), the highest in vitro
activities of T. harzianum being seen with MW. Under greenhouse nursery conditions, the treatments
involving amendment with these residues and with T. harzianum increased the NAGase activity (between
5- and 20-times higher than peat) and the chitinase activity (between 3- and 6-times higher than peat)
of the growing media and maintained the shoot dry weight of plants infected with F. oxysporum f. sp
melonis. Moreover, the incorporation of these residues into the growing media enhanced the growth
(first leaf length, stem length and shoot dry weight) of muskmelon seedlings. The use of these residues
with T. harzianum as amendments of growing media enhanced the growth of muskmelon seedlings and
decreased the weight loss due to the pathogen, giving an added-value to these residues and enhancing
their management.
110
The goals of the present study were to evaluate the ability of Trichoderma spp. to promote growth
of common bean plants, to verify their ability to induce systemic resistance against Colletotrichum
lindemuthianum, to evaluate the effect of different concentrations of Trichoderma spp. in the severity
of the disease, and to identify, by DNA sequencing, the best performing isolates. The growth promotion
was evaluated for sixty Trichoderma spp. isolates by measuring the increase in the dry mass matter
(DMM). Resistance induction by seven Trichoderma spp. isolates applied to the substrate was evaluated
by inoculation of leaves with a suspension of C. lindemuthianum conidia. The effect of different
concentrations of the isolate IB28/07 on disease severity was verified by adding it to the substrate at
concentrations of 0.5%, 1%, 1.5%, and 2% (w:v). Seven Trichoderma spp. isolates promoted increase in
DMM above 30%, in at least two assays. These isolates reduced disease severity between 62.73% and
97.54%. The isolate IB 28/07, applied in increasing concentrations, caused reduction in disease severity of
41.51%, 55.15%, 81.82%, and 96.06%. DNA sequencing identified the isolates as Trichoderma harzianum
(5), Trichoderma strigosum (1), and Trichoderma theobromicola (1).
27 30 August 2012
27 30 August 2012
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Lincoln University
New Zealand
Commerce Building
Lincoln University
New Zealand
111
Rhizosphere competence and survivability of Trichoderma

harzianum (Th3) in the crops grown in semi-arid eastern plains
of Rajasthan state, India
Activation of a glutathione S-transferase and a deoxycytidine

deaminase expression in Brassica oleracea var capitata that
correlates with resistance to Rhizoctonia solani challenge during
Trichoderma harzianum ETS 323 association
SHARMA, P.; Saini, M.; Patel, A.; Deep, S. and Bhati, D. S.

Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110012.
pratibha@iari.res.in; maheshkumarsai09@gmail.com; amarnath312@gmail.com; swatideep24@gmail.com;
dinesh.bhati.ncr@gmail.com
SHIBU, M. A.1; Lin, H.-H.1; Yang, H.-H.2; Liu, S.-Y.3; Lo, C.-T.4 and Peng, K.-C.1
Department of Life Science and the Institute of Biotechnology, National Dong Hwa University, Hualien, 97401, Taiwan.
shibu.m.a@gmail.com; H.-H. Lin: m9513008@ems.ndhu.edu.tw; kcpeng@mail.nahu.edu.tw.
Department of Research, Buddhist Tzu Chi General Hospital, General Education Center, Tzu Chi College of Technology,
Hualien, Taiwan.
hhyang@tzuchi.com.tw.
Field performance of a bioformulation of Trichoderma harzianum (Th3) was evaluated in terms of its
rhizosphere competence, survivability (root colonisation behaviour) in different winter (Rabi) crops
including cereals, legumes, oilseed crops, vegetables at three different crop growth stages (seedling,
flowering and pre-harvesting) at different villages of Jaipur district of Rajasthan (semi-arid eastern plains),
India). Populations of Trichoderma harzianum (Th3) were isolated on Trichoderma Selective Medium
(TSM), showed rhizosphere colonisation behaviour. Survivability of T. harzianum was maximum at R.C.
Index value: 4.384 at the flowering stage and c.f.u. value: 75 x 106/g at pre-harvesting stage. Minimum
T. harzianum survival was at R.C. Index value: 0.3 and c.f.u.: <40x 106/g at the seedling stage for most of
the crops. Trichoderma harzianum (Th3) was detected in large quantities with every crop tested when
rhizosphere (roots with adhering) soil were assayed. Rhizosphere population densities were consistently
higher in treated soils compared with the untreated.
112
Department of Molecular Biotechnology, Da-Yeh University, Changhua 51591, Taiwan.

syliu@mail.dyu.edu.tw.
Department of Biotechnology, National Formosa University, Yunlin 63208, Taiwan.

ctlo@sunws.nfu.edu.tw
Plant interactions with bio-control associated microbes are often used as experimental models to
understand the development of resistance-related molecular adaptations, developed in the host. In a
hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism
enabled resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. Rhizoctonia
solani challenge on leaves initiated an increase in lipoxygenase activity and associated hypersensitive
tissue damage with characteristic PCD like features that facilitated the infection. The plants that were
briefly (6 h) root colonised by T. harzianum ETS 323, developed resistance to R. solani infection by
significantly reducing the host hypersensitive tissue damage that is essential for R. solani infection. The
resistance that developed in the distal leaf tissue was associated with the expression of a H2O2 inducible
glutathione S-transferase (BoGST) that scavenges cytotoxic reactive electrophiles and a deoxycytidine
deaminase (BoDCD) that modulates the host molecular expression and potentially neutralises the DNA
adducts and maintains DNA integrity. The cDNA of BoGST and BoDCD were cloned and both BoGST and
BoDCD mRNAs expressions were verified by RT-PCR analysis and found to be transcriptionally activated
during the three-way interaction.
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Lincoln University
New Zealand
Commerce Building
Lincoln University
New Zealand
113
Delivering Trichoderma spp. spores via seed coating

SWAMINATHAN, J.1; Wilson, M.2; Wessman, P.1 and Jackson, T.1
AgResearch, Lincoln Research Centre, Christchurch, New Zealand
jayanthi.swaminathan@agresearch.co.nz; per.wessman@agresearch.co.nz; trevor.jackson@agresearch.co.nz.
AgResearch, Ruakura Research Centre, Hamilton, New Zealand.

michael.wilson@agresearch.co.nz
Trichoderma species have been reported to enhance seedling growth and provide protection from
diseases in the soil. Many Trichoderma products are simply mixed into soil or compost, but seed
treatment offers the potential to reduce spore application rates by directly targeting the developing
rhizosphere where efficacy is realised. In this study, we evaluated the ability of eight contrasting
formulations, including oils, polysaccharides and biopolymers, to assist Trichoderma spores to adhere
to seeds using wheat seed as a model. The best formulations provided a loading of >1.00 x 107 spores
per seed. The overall efficiency of the formulation process as well as the applicability of different
formulations to commercial systems will be discussed.
27 30 August 2012
114
Commerce Building
Lincoln University
New Zealand
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Innovations and Applications

Innovations and Applications

Innovations and Applications

Innovations and Applications

Monday 27th August 2012 | Commerce 1 for all sessions

Agrimm Technologies Ltd Plenary lecture: Distinguished Professor Alison Stewart

Tuesday 28th August 2012 | Commerce 1 for all sessions

Session 1 | Environmental signalling | Chair: Benjamin Horwitz

Trichoderma communication: environmental communication, sensing and

The role of H+-pump in nutrient transport during germination of Trichoderma

Cite proceedings as:

Workshop Photograph - steps of the Lincoln University Library

Session 1 cont. | Environmental signalling | Chair: Monika Schmoll

Trichoderma atroviride xylanase regulator 1 (Xyr1) and its role in mycoparasitism

Over-expression of the Saccharomyces cerevisiae Dolichol-phosphate mannose

Lunch | Commerce Foyer

Innovations and Applications

Innovations and Applications

Tra1, an adenine deaminase gene participated in copper accumulation in

Biological and molecular investigation of a potential strain of Trichoderma

Sundram, S.; Seman, I. A. and Naidu, Y.

Session 3 cont. | Biocontrol and ecology | Chair: Dean Metcalf

Jijakli, M. H.; Phillips, N. and Lahdenper, M.-L.

Development of a cold tolerant Trichoderma atroviride LU132 variant for control

Derntl, C.; Steiger, M.; Gorsche, R.; Mach-Aigner, A. R. and Mach, R. L.

Impacts of a Trichoderma transformant with Metarhizium genes on the bacterial

Session 3 cont. | Biocontrol and ecology | Chair: Robert Hill

Expression of Trichoderma viride endoglucanase in the larvae of silkworm, Bombyx

Lunch | Commerce Foyer

Wang, M.-X.; Liang, S.; Li, X.-H. and Maio, Y-G.

Colonisation with Trichoderma harzianum and/or Pseudomonas fluorescens

Identification and characterization of cellobiose transporter inTrichoderma reesei

Zaidi, N. W. and Singh, U. S.

Wednesday 29 August 2012 | Commerce 1 for all sessions

Biological control of Botrytis cinerea in grapes in Tasmania

Management of Phomopsis canker in tea plantations using Trichoderma

Factors influencing rhizosphere competence and endophytic colonisation of

Capacity of Trichoderma stromaticum to colonise cacao sapwood

Trichoderma root endophytes enhance plant health and vigour

Improved potato yields from in-furrow application of Trichoderma

Afternoon tea | Commerce Foyer

Kou, Y.; Zang, W.; Xu, J.; Chen, G. and Liu, W.

Morning tea | Commerce Foyer

Li, Y.-Y.; Fu, K. and Chen, J.

Potential use of Trichoderma spp. as biological control agents in suppressing

Session 2 cont. | Biotechnological applications | Chair: Markku Salohemio

Fu, K.; Fan, L.; Li, Y. and Chen, J.

Development and validation of a species-specific marker for Trichoderma

Afternoon tea | Poster Session

Coach departs Riccarton Park for return to Lincoln University

Innovations and Applications

Innovations and Applications

Thursday 30 August 2012 | Commerce 1 for all sessions

Session 4 | Omics | Chair: Enrique Monte

Connecting Trichoderma reesei phenotypes with genotypes

Trichoderma virens a tale of two strains

Session 4 cont. | Omics | Chair: Scott Baker

Transcriptomic responses of tomato plants to Trichoderma

Expression of genes encoding small secreted cysteine-rich proteins (SSCPs) in

Identification of putative effectors through bioinformatics analysis of Trichoderma

Session 4 cont. | Omics - Taxonomy | Chair: Leandro Loguerico

Molecular diversity of Trichoderma species from groundnut rhizosphere

Biodiversity of root-endophytic Trichoderma from Malaysian Borneo

Conference summary discussion | Jo Steyaert and Kirstin McLean

The 12th International Trichoderma & Gliocladium Workshop

The 12th International Trichoderma & Gliocladium Workshop