MORPHOLOGICAL AND MOLECULAR

CHARACTERIZATION OF ROOT-KNOT NEMATODE

(MELOIDOGYNE SPP.) DIVERSITY IN FIJI

By

Sunil Kumar SINGH

A Thesis Submitted in Partial Fulfilment of the

Requirements for the degree of

Master in Science in Biology

School of Biological and Chemical Sciences

Faculty of Science, Technology and Environment

The University of the South Pacific

2009

i
 STATEMENT OF ORGINALITY

I, Sunil Kumar Singh, hereby declare that this thesis is my own work and that,

to the best of my knowledge, it contains no material previously published, or

substantially overlapping with material submitted for the award of any other

degree at any institution, except where due acknowledgement is made in the

text.

................................................ …………………
Sunil Kumar Singh Date

The research in this thesis was performed under my supervision and to my knowledge

is the sole work of Mr. Sunil Kumar Singh.

................................................ …………………
Dr. Uma Khurma Date
(Principal Supervisor)

ii
DEDICATION

To my parents

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 ACKNOWLDEGMENTS

I would like to thank the Faculty of Science, Technology and Environment and Allan

Wilson Centre for Molecular Ecology and Evolution for jointly funding my research. I

am also grateful to the Office of Research and Graduate Affairs for providing me with

a graduate assistantship for part of my studies.

I would like to acknowledge the following people for making my learning experience

rewarding and enjoyable. My greatest thanks go to Dr Uma Khurma, (Principal

supervisor, USP) for being a great teacher who always took her time out to provide

guidance, feedback and encouragement from the start of my research career. I am

extremely grateful to Professor Peter Lockhart, (Co-supervisor, Massey University) for

his constructive feedback and all his assistance from the beginning of my M. Sc

research. My sincere thanks go to Dr Richard Winkworth, (Molecular lab supervisor,

USP) and Trish McLenachan, (Molecular lab technician, Massey University) for

helping with the standardization of the molecular protocols. I would also like to thank

Professor Arthur Whistler (former professor in plant biology, USP), for verifying weed

specimen identifications.

My gratitude goes to the farmers visited during the sampling trips for their hospitality,

lively discussions and giving permission to take soil and plant samples from their

farms. Special thanks, to my parents, brother and sisters for their love, guidance and

support through the tough and challenging times in life which has helped me to learn

and better understand life. My sincere thanks to all persons who assisted me in anyway

while doing my research and writing my thesis.

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 TABLE OF CONTENTS

Contents Page No.

Statement of Originality ii

Dedication iii

Acknowledgements iv

Contents v

List of Tables vii

List of Figures vii

List of Appendices x

Abstract xi

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 Agriculture and Nematology in Fiji 1

1.1.1 Agriculture in Fiji 1

1.1.2 Nematology in Fiji 4

1.2 Root-knot nematodes (Meloidogyne Göldi 1892) 8

1.2.1 Meloidogyne spp. 8

1.2.2 Identification of Meloidogyne species 11

2.0 METHODS

2.1 Sample collection and screening 17

2.1.1 Sample collection 17

2.1.2 Screening soil samples for Meloidogyne spp. 20

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 2.2 Identification of Meloidogyne spp. 23

2.2.1 Molecular characterization 23

2.2.1.1 Selection of Molecular Markers 23

2.2.1.2 Extraction of DNA 23

2.2.1.3 Polymerase Chain Reaction (PCR) protocol 26

2.2.1.3 Gel Electrophoresis 27

2.2.2 Morphological characterization 29

2.2.2.1 Female 30

2.2.2.2 Juvenile and Male 31

3.0 RESULTS

3.1 Distribution and Hosts 33

3.1.1 Meloidogyne spp. distribution on Viti Levu 33

3.1.2 Crop hosts of Meloidogyne spp. 37

3.1.3 Weed hosts of Meloidogyne spp. 41

3.2 Meloidogyne spp. characterization 45

3.2.1 Description of Meloidogyne spp. 47

4.0 DISCUSSION

4.1 Root-knot nematode diversity and distribution on Viti Levu, Fiji 81

4.2 Identification of root-knot nematode species 85

4.3 Current and future implications for agriculture in Fiji 90

REFERENCES 92

APPENDIX 105

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LIST OF TABLES

Table 2.1: List of primers 22

Table 2.2: PCR programs 25

Table 3.1: Incidence of root-knot nematodes on Viti Levu 31

Table 3.2: Crop hosts of root-knot nematodes 37

Table 3.3: Good weed hosts of root-knot nematodes 40

Table 3.4: Poor weed hosts of root-knot nematodes 41

Table 3.5: Weed non hosts of root-knot nematodes 42

Table 3.6: Measurements from juveniles and females of M. arenaria,

M. incognita and M. javanica 77

Table 3.7: Measurements from males of M. arenaria and M. incognita 78

Table 3.8: Measurements from juveniles and females of

Meloidogyne sp. 1, 2 and 3 compared with closest sp. from literature 79

Table 3.9: Literature values from M. enterolobii, M. mayaguensis, and

M. brasilensis for comparison 80

LIST OF FIGURES

Figure 1.1: Map of Fiji Islands 2

Figure 1.2: Life cycle of root-knot nematode 10

Figure 2.1: The 10 areas sampled from Viti Levu 18

Figure 2.2: Sketch of sampling route on a rectangular farm 18

Figure 2.3: Soil core (Locally made) 19

Figure 2.4: Microscope (Olympus model BX 51) with imaging system 32

Figure 3.1: Ginger farm heavily infested with M. incognita 35

Figure 3.2: Eggplant (Solanum melongena) roots with distinct root galls
infected by M. incognita from field sample 35

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Figure 3.3: Weed (Vernonia cinerea) and okra (Abelmoschus esculentus) roots with
coalescing root galls infected by M. javanica and M. arenaria respectively collected
from two different fields 36

Figure 3.4: Grass (Eleusine indica) roots with small root galls infected by
M. incognita collected from bioassay pot 36

Figure 3.5: Root gall with protruding egg mass produced by M. incognita on tomato
root 37

Figure 3.6: Meloidogyne species composition on Viti Levu 45

Figure 3.7: Gel photograph showing bands obtained from M. incognita

populations using species specific primers MIF/MIR 47

Figure 3.8: M. incognita LM photograph of (A) Female

(B) Eggs with juveniles ready to hatch 48

Figure 3.9 M. incognita LM photograph of (A, 1-3) Perineal patterns

(B) Drawing of perineal pattern 49

Figure 3.10 LM photographs of M. incognita perineal patterns showing variations

from the typical 50

Figure 3.11 M. incognita male LM photograph of (A) Head region,

(B) Mid body region, (C) Tail region 51

Figure 3.12: M. incognita LM photograph of (A) Juvenile

(B) Juvenile head
(C) Tail region 52

Figure 3.13: Gel photograph showing bands obtained from M. javanica

populations using the primers Fjav/Rjav 53

Figure 3.14: M. javanica LM photograph of (A) Female

(B, 1-3) perineal patterns
(C) Drawing of perineal pattern 55

Figure 3.15: (A, 1-2) LM photographs of M. javanica perineal patterns

showing variation from typical 56

Figure 3.16: Gel photograph showing bands obtained from a pure M. incognita
population and two mixed population of M. incognita and M. javanica 57

Figure 3.17: LM photograph of perineal pattern from mixed population

(A) M. javanica
(B) M. Incognita 58

Figure 3.18: Gel photograph showing the bands characteristic of

M. arenaira with primers Far/Rar 59

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Figure 3.19: M. arenaria LM photograph of (A) Female
(B, 1-3) Perineal patterns
(C) Drawing of perineal pattern 61
Figure 3.20: (A, 1-3) LM photographs of M. arenaria perineal patterns
showing variations from typical 62

Figure 3.21: LM photograph of M. arenaria male (A 1-2) Head region with stylet
(B) Mid-body region with lateral lines
(C) Posterior end
(D) Tail region with spicules 63

Figure 3.22: LM photograph of M. arenaria (A) Juvenile

(B) Juvenile tail 64

Figure 3.23: Gel photograph showing Meloidogyne sp. 1, 400bp fragments

with primer C2F3/Mel450R 65

Figure 3.24: LM photograph of Meloidogyne sp. 1 (A) Female

(B, 1-3) Perineal patterns 67

Figure 3.25: Gel photograph showing Meloidogyne sp. 2 PCR results

with primers C2F3/1108 and C2F3/Mel450R 69

Figure 3.26: Gel photograph showing PCR results with SCAR primers MIF/MIR
for Meloidogyne sp. 2 compared with M. incognita 70

Figure 3.27 LM photograph of Meloidogyne sp. 2 (A) Female

(B, 1-2) Perineal patterns 71

Figure 3.28: LM photograph of Meloidogyne sp. 2 (A) Juvenile

(B) Juvenile tail 72

Figure 3.29: Gel photograph showing Meloidogyne sp. 3 PCR results

with primers C2F3/1108 and C2F3/Mel450R 73

Figure 3.30: LM photograph of Meloidogyne sp. 3 perineal patterns (A, 1-2) 74

Figure 3.30: LM photograph of Meloidogyne sp. 3 perineal patterns (A, 3-4) 75

Figure 3.31: LM photograph of Meloidogyne sp. 3 (A) Juvenile

(B) Tail region 76

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 LIST OF APPENDICES

Appendix 1: Some weed hosts of root-knot nematodes from Fiji 105

Appendix 2: Statistical analysis of measurements of M. incognita, M. javanica and

M. arenaria measurements 106

Appendix3: DNA Hyper Ladder I used for estimating band size of PCR products
visualised on agarose gel 109

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 ABSTRACT

Root-knot nematodes are economically important pests of a wide range of crops

worldwide and several species are known to cause widespread damage to crops. A

thorough and systematic survey of 10 different agricultural areas from Viti Levu, in

Fiji was undertaken from April 2007- April 2008 to determine the percentage

incidence, host range and species diversity of root-knot nematodes. Root-knot

nematodes were found to be widely distributed in agricultural areas throughout Viti

Levu with a total incidence of 41% recorded from 675 farms. The incidence of root-

knot nematodes was determined using direct examination method and bioassay of soil

samples using tomato cv. Moneymaker as the host plant. Direct examination for the

presence or absence of root galls indicated presence of root-knot nematodes in 28% of

the samples and the bioassay results recorded infections from an additional 13% of the

samples. The hosts included 33 crop plants and 45 weed hosts. Root-knot nematode

species were identified based on morphological examination and molecular analysis of

nematode specimens obtained from infected tomato host grown in bioassay pots.

Morphological study included examination of female perineal patterns and

measurements of female, juvenile and male specimens. Molecular analysis included

PCR using species specific SCAR molecular markers and visualisation of

amplification products by electrophoresis on 1.5% agarose gel. Three species of root-

knot nematodes which also happen to be the most common Meloidogyne species

worldwide were found to be widespread in Fiji. Meloidogyne incognita was the most

commonly occurring species on 56% of the infected farms followed by M. javanica on

28% and M. arenaria on 10%. Mixed populations of M. incognita and M. javanica

were found on 5% of the infected farms. Another 1% of the populations could not be

identified up to the species level and require further investigation.

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 CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 AGRICULTURE AND NEMATOLOGY IN FIJI.

1.1.1 AGRICULTURE IN FIJI.

Fiji islands covers a land area of 18 333 km2 and is located in the tropical region

between 174o East and 178o West of Greenwich and latitudes 12o S and 22o South (Fiji

Islands Bureau of Statistics, 2008). There are two major islands: Viti Levu, which

covers an area of 10,429 sq km and Vanua Levu, which covers an area of 5,556 sq km

(Figure 1.1). The climatic conditions are tropical with two seasons. The cool and dry

season is from May to October with average temperature range of 19 oC to 22 oC while

the hot and wet season is from November to April with average temperature range of

31 oC to 34 oC (Fiji Islands Bureau of Statistics, 2008). The Fijian land ownership units

(Mataqali’s) own 87.9% of the land in Fiji and lease parts of their land through the

Native Lands Trust Board (NLTB) for agricultural and commercial purposes. A small

percentage of land is owned by state (3.9%) while 7.9% is freehold land (Fiji Islands

Bureau of Statistics, 2008).

Agriculture is an important part of the Fijian economy and is the major source of

income for people living in rural areas. Agriculture has also played an important role in

Fiji’s history and development with sugar being the major agricultural industry.

Sugarcane is grown in the western parts of Viti Levu and in some parts of Vanua Levu.

However, sugarcane farmers now have started to diversify to cash crop farming of

fruits and vegetables due to better prices and increasing demand for fruit and

vegetables from export markets. The climatic conditions in Fiji allow the farmers to

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grow a wide range of tropical crops and an increasing number of farmers have started

growing horticultural crops in Fiji.

Figure 1.1: Map of Fiji Islands.

The most commonly grown crops in Fiji, apart from sugarcane include beans,

eggplant, okra, bele, dalo, cassava, kumala, yams, tobbaco, lettuce, cabbage, tomatoes,

cucumber, pumpkin, watermelon, pawpaw, banana, coconuts, ginger and kava. These

crops are an important source of food and income for the locals.

The local tourism industry and overseas countries such as Australia, New Zealand,

United States, Canada, Japan and China are good markets for high quality horticultural

crops. The “weak capacity of quarantine services” to negotiate access to export

markets has limited fresh produce exports (ADB 2003). The ability of farmers to

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produce good quality crops as required by the local tourism industry and the export

market depends on a number of factors such as awareness about pest and disease,

quarantine requirements, climatic condition and technical skills required in production

and post harvest handling. Pests and diseases have been one of the major limiting

factors in improving the quality of crop production in Fiji. The taro beetle, rhinoceros

beetle, fruit fly, nematode, fungal, bacterial and viral diseases are prevalent in Fiji and

are the cause of major losses in crop production. The presence of pests and disease also

has important implications for trade of agricultural produce. A pest list database for

Fiji and other Pacific island countries has been developed by the Secretariat of the

Pacific Community (http://wwwx.spc.int:8088/pld/index.jsp) to help these countries

with international trade of agricultural produce in accordance to the international trade

regulations and bilateral quarantine agreement.

The first documented plant parasitic nematode was Anguina tritici, discovered in 1743

by T. Needham from wheat galls (Weischer and Brown, 2000). Plant parasitic

nematodes can infect various different parts of a plant, for example, Aphelenchoides

spp. infects leaves, Meloidogyne spp. infects roots causing galls, Pratylenchus spp.

cause root lesions, Heterodera spp. form cyst on roots, Bursaphelenchus spp. infect

stems, Radopholus spp. infect corms, Ditylenchus spp. infect bulbs, Globodera spp.

infect tubers, and Anguina spp. infect seeds (Wiescher and Brown 2000; Brown et al.,

2004). Most of the crop damage is caused by a relatively small number of nematode

genera which include root-knot (Meloidogyne spp.), cyst nematodes (Globodera and

Heterodera spp.) and migratory nematodes (Pratylenchus and Radopholus spp.)

(McK Bird and Kaloshian, 2003). Plant parasitic nematodes cause an estimated loss of

$157 billion (USD) annually in crop production worldwide (Abad et al., 2008).

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 1.1.2 NEMATOLOGY IN FIJI.

Plant parasitic nematodes from the Pacific region are relatively less studied but are

known to parasitize important food crops such as sweet potato, cassava, taro, yam,

banana, citrus, ginger, rice, sugarcane, passion fruit, pawpaw, tobacco and other

vegetables (Orton Williams, 1980; Bridge, 1988). Fiji has a history for the presence of

economically important nematodes, starting with the discovery of Radopholus similis

(burrowing nematode) by Cobb in 1893, from Fijian soil samples taken from farms

with diseased banana plants (Orton Williams, 1980; Siddiqi, 2000; Luc et al., 2005).

A number of renowned nematologists such as N.A. Cobb, A.L. Taylor, M.F. Kirby,

M.R. Siddiqi and K.J. Orton Williams have worked on nematodes from Fiji (Orton

Williams, 1980). N.A. Cobb analysed the soil or nematode samples sent from Fiji to

Australia while M.R. Siddiqi studied nematode samples sent from Fiji to the

Commonwealth Institute of Helminthology (Orton Williams, 1980). Orton Williams

(1980) was involved with the general survey of plant parasitic nematodes in the Pacific

from 1977 – 1980. Most of the soil or nematode samples were usually sent abroad

either to Australia or to the Commonwealth Institute of Helminthology for nematode

identifications due to lack of expertise available locally (Orton Williams, 1980).

The quarantine regulations are getting stricter amidst growing concern over food

security and threats posed by pest and diseases. For instance the New Zealand

quarantine has recently started focusing on plant parasitic nematodes found on root

crops exported from Fiji (Grandison cited in McGregor, 2007). The detection of

quarantined nematodes on any export root crops requires mandatory fumigation which

increases the exportation cost, the shelf price and reduces the shelf life of the produce

44
(McGregor 2007). Similarly the quarantine requirements of other overseas countries

also need certification of crops or growing areas to be free of nematode pests such as

Radopholus spp., Meloidogyne spp. and Pratylenchus spp. before accepting ginger and

other root crops.

Several economically important nematode species have been reported from Fiji.

During a general survey of plant parasitic nematodes in the Pacific, Orton Williams

(1980) found the following economically important nematodes: Radopholus spp. (7%),

Meloidogyne spp. (31%), Pratylenchus spp. (43%), Rotylenchulus spp. (55%) and

Helicotylechus spp. (70%) from approximately 700 Fijian soil samples. Three species

of root-knot nematodes Meloidogyne spp. (M. incognita, M. javanica and M. arenaria)

and over 80 plant hosts of root-knot nematodes were recorded by Orton Williams in

his report (1980). He suggested that root-knot nematodes could possibly be associated

with wilt disease and recommended further investigation on the subject of root-knot

nematodes. A literature review of plant parasitic nematodes from the Pacific by Bridge

(1988) listed the presence of 63 nematode species out of which Meloidogyne spp. were

considered as important pests of a wide range of crops. The Pacific Island Pest list

database (http://wwwx.spc.int:8088/pld/index.jsp) also lists the occurrence of root-knot

nematodes in 12 out of the 19 countries surveyed, with Fiji having the highest number

(207) of root-knot nematode incidence, as of 30th November 2008.

Majority of the literature published on root-knot nematodes in Fiji is from the late

1970s and early 1980s period, arising from the general nematode survey sponsored by

Food and Agriculture Organization (Orton Williams, 1980) and from the work done by

nematologists then working for Ministry of Agriculture in Fiji (Kirby, 1977;

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Kirby et al., 1978; Kirby et al., 1980; Vilisoni and Kirby, 1980). Fiji was also included

in “The International Meloidogyne Project” (1975-1984) during which soil samples

were collected and analysed for the presence of root-knot nematodes and various field

trials on root-knot nematode management were conducted (Davide, 1985). The more

recent publications on root-knot nematodes from Fiji include a revised pest advisory

leaflet (Gowen et al., 2005) and a report on a preliminary investigation on the

incidence of root-knot nematodes (Khurma et al., 2008). Root-knot nematodes along

with another nematode group Radopholus spp. have been cited as the cause of decline

in ginger production in Fiji (ADB 2003).

Crop plants infected by root-knot nematodes have poor growth, reduced vigour, lower

quality and quantity of yields. Damage caused by root-knot nematodes to crops such as

ginger is a good example since the rhizomes are directly affected by root-knot

nematode infection. The damage caused by plant parasitic nematodes is difficult to

assess as they also facilitate the invasion of plants by other plant pathogens such as

bacteria, fungi, insects and viruses which gain entry through feeding or entry wounds

or by acting as specific vector for certain viruses (Manzanilla-Lopez et al., 2004). The

fact that root-knot nematodes can infect over 20 of the commonly grown crops in

Pacific island countries (Gowen et al., 2005) is likely to pose threat to growing good

quality horticultural crops.

The species from the genus Meloidogyne are economically important pests of

agriculture worldwide. Root-knot nematode species identification requires

specialised skills but is of fundamental importance to a wide range of scientific

studies on virulence and nematode management. In addition to species identification,

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information on the distribution and host range of root-knot nematodes is equally

important. Considering the importance of the genus Meloidogyne for agriculture in

Fiji, this study was undertaken with the following objectives:

1. Determine the incidence and host range of root-knot nematodes in

agricultural areas from Viti Levu, Fiji.

2. Identify Meloidogyne populations based on morphological and molecular

characteristics.

3. Standardise a diagnostic protocol for identification of root-knot nematodes.

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1.2 ROOT-KNOT NEMATODES (MELOIDOGYNE GÖLDI 1892).

1.2.1 MELOIDOGYNE SPP.

The presence of root-knot nematodes was first noted on plants in the early 1800’s

when Englishman M.J. Berkerly in 1855 correlated the galls on cucumber roots with

nematodes (Hartman and Sasser, 1985). Cornu in 1879 first described root-knot

nematodes from the roots of Onobrychis sativa Lam. and named them as Anguillula

marioni. Then in 1884, Muller synonymised Anguillula marioni with Heterodera

radicicola based on a detailed morphological study and this name was generally used

for root-knot nematodes until 1932. Goodey reviewed the nomenclature and renamed

root-knot nematodes to Heterodera marioni using Cornu’s earlier specific name

(Hirschmann, 1985).

In the early period of nematode taxonomy (1879-1948), root-knot nematodes were

mostly placed in the same genus as cyst nematode, Heterodera until 1949 when

Chitwood separated root-knot nematodes from cyst nematodes. He reassigned these

species to the genus Meloidogyne which was first named by Göldi in a paper published

in 1887 but reprinted in 1892 (Hirschmann, 1985; Karssen, 2002). There are 95

nominal species of root-knot nematodes described so far (Skantar et al., 2008) and

over 5500 plant hosts (Trudgill and Blok, 2001).

The genus Meloidogyne has been of interest to nematologists worldwide probably due

to their widespread distribution and success as parasites of economically important

crops and is considered as one of the most important genera of plant parasitic

nematodes (Dong et al., 2001; Trudgill and Blok, 2001; McK Bird and Kaloshian,

2003). Recently the whole genome of one of the most common species,

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Meloidogyne incognita has been sequenced to provide insights into the adaptations

required by metazoans to successfully parasitize and counter defenses of

immunocompetent plants (Abad et al., 2008). The importance of Meloidogyne is also

reflected through the large volume of literature with several books published on the

genus itself (Taylor and Sasser, 1978; Lamberti and Taylor, 1979; Sasser and Kirby,

1979; Barker et al., 1985; Sasser and Carter, 1985; Karssen, 2002). One of the

extensive initiatives on root-knot nematodes was “The International Meloidogyne

Project” (1975-1984), a unique worldwide scientific research focusing on Meloidogyne

biology, taxonomy, ecology, management and technology transfer in agricultural

systems (Sasser and Carter 1985). There is ongoing research on root-knot nematodes

worldwide and records of new species and hosts are being added to the existing

literature.

Meloidogyne spp. are endoparasites and their lifecycle varies from three weeks to

several months depending on environmental factors such as temperature, moisture and

availability of suitable host (Taylor and Sasser, 1978). The infective second stage

juveniles (J2) penetrate the roots of the host plant using the piercing action of their

stylet. Once inside the roots, juveniles release esophageal secretions which cause the

formation of multinucleate feeding cells. The juveniles (J2) become sedentary, feed

and undergo three molts (J3, J4, adult). Occasionally vermiform males develop and

migrate out of the roots while the females remain sedentary and are pear to globose in

shape. The females feed and produce eggs in a gelatinous matrix. The embryogenesis

begins inside the egg and after the first molt, second stage juveniles (J2) hatch out

(Abad et al., 2008). A diagrammatic representation of the life cycle of root-knot

nematodes is given in Figure 1.2.

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Root-knot nematode juveniles have sufficient stored energy to survive for a month in

soil to locate and penetrate the roots of a host plant (Riga, 2004). The optimum

temperature for survival and reproduction varies according to the species of root-knot

nematode (Griffin et al., 1990; Karssen and Moens, 2006; Wang and McSorely, 2008).

Some species (e.g. Meloidogyne hapla), are dominant in cooler countries with specific

climate type and cropping history while other Meloidogyne species are dominant in the

tropical and subtropical countries (Karssen and Moens, 2006). The temperature range

of 25 oC to 30 oC is optimum for reproduction and survival of the most common root-

knot nematode species, M. incognita, M. javanica, and M. arenaria (Taylor and

Sasser, 1978) which thrive in tropical and subtropical countries.

50µm

Figure 1.2: Life cycle of root-knot nematode (Source: Abad et al., 2008).

10
10
 1.2.2 IDENTIFICATION OF MELOIDOGYNE SPECIES.

Identification of Meloidogyne species is becoming increasingly important for the

design of effective nematode management practices such as crop rotation and plant

resistance which require precise species identification and for quarantine purposes

(Hussey, 1990; Zijlistra 2000; Zijlistra and Van Hoof, 2006). The need for reliable

Meloidogyne species identification has also increased due to the reduced availability of

broad spectrum chemical nematicides and increased reliance on non-chemical

nematode management strategies. Traditionally, identification and description of

Meloidogyne species was based mainly on perineal pattern morphology and supported

by other morphological characteristics of female, juveniles (J2) and males.

Chitwood, in 1949 used detailed morphological observations (perineal pattern, stylet

morphology and distance from the stylet knobs to the dorsal esophageal gland opening

(DEGO)) to describe M. hapla and M. incognita var. acrita and redescribe

M. arenaira, M. exigua, M. incognita, and M. javanica (Karssen, 2002). The perineal

pattern of Meloidogyne species comprises of tail terminus, phasmids, lateral lines, anus

and vulva surrounded by cuticular folds or striae (Hirschmann, 1985). Perineal patterns

have been widely used in species identification and descriptions since 1949 when

Chitwood used it as an identification character (Karssen and van Aelst, 2001).

The perineal pattern remains as one of the important characters in species

identifications due to the following reasons. Firstly, the perineal pattern is stable and

does not change significantly over an extended period of culturing (Eisenback, 1985;

Hirschman, 1985). The females are relatively larger in size, often numerous, easy to

find from infected tissues and prepare for light microscopic examination than males

11
11
which are difficult to find, or juveniles which are smaller, vermiform and difficult to

prepare for microscopic examination (Eisenback, 1985). The invention of scanning

electron microscope and improved light microscopy techniques now allows more

accurate representation of morphological features which can be used to support line

drawings. Line drawings however are still useful in representing internal features of

the nematode which can not be focused and captured in a single image view.

However, the presence of some variability in the perineal patterns between individuals

of the same species (Eisenback, 1985; Hirschmann, 1985, Hussey, 1985; Karssen and

van Aelst, 2001) and the varied expertise of the persons describing perineal patterns

(Karssen, 2002) limit the accuracy of species identification based only on perineal

patterns. Furthermore, species identifications based on morphological and

morphometrical characters requires a lot of skill (Hooper et al., 2005) and is time

consuming. When using morphological characterization, mixed populations are not

easily detected as large numbers of specimens need to be examined for reliable

identifications.

Soon after Chitwood’s species descriptions, a number of nematologists and plant

breeders noted inconsistencies in host responses for the same species which led to the

development of North Carolina differential host test and the discovery of host races

(Hartman and Sasser, 1985). The test involves the inoculation of six standard host

plants: Cotton ‘Deltapine 61,’ tobacco ‘NC 95,’ pepper ‘California wonder,’

watermelon ‘Charleston Gray,’ peanut ‘Florunner,’ and tomato ‘Rutgers’ and is able to

distinguish the four commonly occurring Meloidogyne spp. (M. incognita, M.

arenaria, M javanica and M. hapla) and races for M. incognita and M. arenaria on the

12
12
basis of host susceptibility or resistance (Taylor and Sasser, 1978). The North Carolina

differential host test can be used for finer differentiation of the species based on

morphological descriptions (Hartman and Sasser, 1985). However, the North Carolina

differential host test is time consuming and is not sufficient to determine mixed

populations or rare populations (Sasser and Carter 1985).

The limitations of morphological characterization and differential host test led to

search for other means to assist in the identification of Meloidogyne species.

Reproductive and cytological characters of Meloidogyne species were studied for their

use in identification and have been used to distinguish the different races based on the

mode of reproduction and chromosome numbers (Triantaphyllou, 1985). Another

method for species identifications includes the use of protein and enzyme composition

patterns, visualized through gel electrophoresis methods to determine Meloidogyne

species identity (Hussey, 1985; Esbenshade and Triantaphyllou, 1985). Out of the four

commonly studied enzyme patterns (non-specific estrases, malate dehydrogenase,

superoxide dismutase and glutamate-oxaloacetate transaminase), esterases have been

the most useful for differentiating the major Meloidogyne species (Esbenshade and

Triantaphyllou, 1985; Cofcewicz et al., 2004).

The accuracy of species identification increases when morphological features are

supplemented by other kinds of taxonomic characters such as cytological, biochemical

and physiological data (Hirschman, 1985). Biochemical, cytological and reproductive

characters have also been useful in studies focusing on host response and parasitism

mechanisms (Williamson and Hussey 1996; Abad et al., 2003).

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13
Ideally, a diagnostic technique should not be limited to the availability of a particular

developmental stage (eggs, juveniles or adults), require small number of individuals or

small amount of tissue and be able to provide reliable identification of a species within

a short period of time. The introduction of molecular techniques has helped nematode

taxonomists to distinguish and identify nematode species which were otherwise

difficult to identify only on the basis of morphological characteristics and host

preferences. Technical advances, starting with the development of polymerase chain

reaction (PCR), use of molecular markers and DNA sequencing (Harris et al., 1990;

Powers and Fleming, 1998) has allowed faster and more reliable nematode species

identification.

Unlike protein and enzyme based characterization, DNA-based diagnostic methods do

not rely on the expressed products of the genome therefore are independent of

environmental influence and independent of stage in the nematode life cycle (Hooper

et al., 2005). The PCR process allows amplification of minute quantities of DNA

which can be extracted from single nematodes, eggs or juveniles and be subjected to

further analysis (Harris et al., 1990). DNA based methods include the use of

mitochondrial DNA (Harris et al., 1990; Powers and Harris, 1993), satellite DNA

(Castagnone-Sereno et al., 1995), ribosomal DNA (Zijlstra et al., 1995; Petersen and

Vrain, 1996; Petersen et al., 1997; Zijlstra, 1997) and randomly amplified polymorphic

DNA fragments (RAPDs).

Various different methods based on DNA and PCR (RAPD, RFLP, SCAR, Multiplex

PCR, and AFLP) have been developed and successfully used for identifying a large

number of Meloidogyne species. The PCR mediated amplification of specific regions

14
14
of the nematode genome offers a highly effective means of detecting inter and intra

specific variation. PCR amplification is directed to the targeted gene using a pair of

specific oligonucleotides (forward and reverse primers). Consistent variations in size

or nucleotide sequence of the amplified PCR product can be used for nematode

identification and characterization. Powers and Harris (1993) demonstrated this by

distinguishing between Meloidogyne species based on amplification of mitochondrial

DNA genes. The PCR fragment band size differences between the different species

became apparent when PCR products were separated on agarose Gels. The variation

in nucleotide sequence of the different species can be further detected by sequencing or

by restriction enzyme digestion of the PCR product (PCR-RFLP).

Several genomic regions have been successfully used in the identification of

Meloidogyne species. The ribosomal DNA repeat units (rDNA) which consists of the

internal transcribed spacers (ITS 1 and ITS 2), located between the repeating array of

nuclear 18S and 28S ribosomal RNA genes and separated by the 5.8S ribosomal RNA

gene, have been used to characterize Meloidogyne species (Blok et al., 1997;

Williamson et al., 1997; Zijlistra, 1997; Zijlistra et al., 1997). The sequences of the

18S, 5.8S and 28S genes tend to be conserved amongst a wide range of organisms

while the ITS region is more variable and taxonomically informative. In addition to

ITS, other regions of mitochondrial DNA which include the intergenic region between

the Cytochrome Oxidase II (COII) and the large subunit of the RNA gene (lrRNA)

have also been widely used for Meloidogyne species diagnostics (Harris et al., 1990).

The sequences of genes coding for other proteins can also be used in species

identification if they are known (Tesarova et al., 2003).

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15
More recently a number of species specific primers for the identification of

Meloidogyne species have been developed based on species specific RAPD fragments

(Williamson et al., 1997; Zijlistra 2000; Dong et al., 2001). The species specific

sequence characterized amplified regions (SCAR) primers work at higher annealing

temperatures as they are longer than RAPD primers (Zijlistra, 2000). The PCR

reaction is also less dependent on the amount of DNA and source of template thus the

PCR results are more reproducible and reliable (Williamson et al., 1997). SCAR-PCR

methods have the potential to be used in routine diagnostic applications using DNA

extracts from single juveniles, soil samples or even infected plant materials (Zijlistra,

2000).

Although molecular techniques are not free of error, it significantly reduces the

chances of bias and human error since the species identification is based on genetic

information rather than morphological characteristics which require more experience

and specialized skills for identification (Coomans, 2002). Molecular identification has

confirmed the validity of a number of classical nematode species and where necessary

revisions have been made to classifications based on molecular evidence. Molecular

systematics combined with related work in genetics has moved to the forefront lately

and has contributed a lot to taxonomy (Barker, 2004). Most significantly, attempts are

being made to incorporate morphological and molecular analyses of nematodes, an

approach which should provide a more effective means of characterizing nematodes

(Evans 1995; Thomas et al., 1997). For this investigation, both molecular and

morphological techniques will be used to determine the species identity of root-knot

nematodes.

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 CHAPTER 2

METHODS

2.1 SAMPLE COLLECTION AND SCREENING.

2.1.1 SAMPLE COLLECTION

Mainly vegetable farms and gardens from 10 different areas (Figure 2.1) on Viti Levu

were sampled over a one year period from April, 2007 till April, 2008, for the presence

of root-knot nematodes. A total of 675 randomly chosen farms were visited and

composite soil samples were taken from the root rhizosphere region of plants on each

of the farms. Soil sample collection for characterization of nematode distribution

should usually be done when nematode populations occur in maximum numbers

(Barker, 1985). The samples for this study were collected from a range of farms with

established crops, complete fallow, fallow with weeds and farms ready for planting.

The samples were taken from farms when the soil was not too wet or too dry as both

the extreme conditions make it difficult to collect and prepare samples for analysis

(Shurtleff and Averre, 2000).

The soil samples were taken from depths of approximately 0-15 or 30 cm using a hand

shovel from 10 different points on the farm while moving in a zigzag pattern (Figure

2.2) as outlined by Barker (1985). The hand shovel and footwear were cleaned after

sampling each farm to avoid contamination of soil samples and spread of nematodes

between farms. Approximately 2 kg of composite soil samples consisting of 10 sub

samples were taken from each of the farm. In cases where the soil was not easily

penetrable, a soil core (Figure 2.3) was used and samples were taken from a depth of

30cm. The soil samples were placed in clean polythene bags and the date, specific

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17
location, crop type, and sample number were written using a permanent ink pen on

adhesive paper labels stuck to the bag. Simultaneously the record was logged into the

field notebook with the sample number, farmers name, GPS coordinates, and the crop

type.

Figure 2.1: The 10 areas sampled from Viti Levu.

2 5 8

3 6 9

1 4 7 10

Figure 2.2: Sketch of sampling route on a rectangular farm.

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 c
a

b
Hollow Pipe with holes indicating Handle with weight for hammering
depth up to 45 cm pipe into the ground.

Figure 2.3: Soil core (Locally made).

a- Handle for hammering core tube into ground (weighs 3 kg)

b- Core tube for extracting soil samples made of steel pipe of diameter 3 cm

c- Core and handle assembled together. Handle is used to hammer the tube

into ground up to required depth (indicated by holes on side of core tube at 15

cm intervals). Once the tube is into the ground the handle is locked with the

core tube by inserting a pin and then pulled up while rotating the handle.

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 2.1.2 SCREENING SOIL SAMPLES FOR MELOIDOGYNE SPP.

Two different methods were used to determine the presence of root-knot nematodes on

the farms sampled. The first method involved direct examination of both crop and

weed roots for the presence of root galls on the farm. Where weeds were present, 5-10

specimens of the different species of weeds was dugout from soil and their roots were

examined for the presence of root galls. The root galls were distinguished from root

nodules by rubbing the galls between fingers. The infected plants were brought to the

laboratory and the root galls were dissected open to examine for presence of female

root-knot nematodes while viewing under a stereoscopic microscope (Olympus SZ61).

The results after direct examination of plant roots were recorded in a table with the

plant name, sample number and location. The samples found infected with root-knot

nematodes were denoted by a + sign while those that were not infected were denoted

by a – sign.

The second method involved bioassay of the collected soil samples by growing

susceptible tomato plant cv. Moneymaker. All composite soil samples collected from

the farms even those found infected after direct examination were screened using the

bioassay method. The bioassay method was used to detect low numbers of root-knot

nematodes in soil samples where no root-knot infections were seen by direct

examination. The root-knot nematode population propagated in the bioassay pot

provided nematode specimen and egg masses for identification via morphological and

molecular analysis respectively. The soil samples were placed in 2kg plastic pots and

two tomato seedlings (3 weeks old) were planted in the pots. Bioassay pots were

labeled with the date of planting, sample number and the area from which the soil

sample was collected. The tomato plants were allowed to grow for 8-10 weeks in the

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20
bioassay pots. Any other weeds germinating in the pots were also allowed to grow and

thinned where necessary to ensure that both tomato and weeds could grow. The weeds

were allowed to grow in the bioassay pots in order to determine their susceptibility to

the root-knot nematode population already present in the field soil sample. The weeds

in the bioassay pots germinated from the seeds already present in the field soil sample.

The weeds germinating in the pot soil samples were not necessarily the weeds seen or

collected from the same field.

The pots were kept in a screen house on raised benches and separated from each other

by a distance of at least 30 cm to avoid cross contamination between samples. The

plants were watered regularly taking care that the water draining out of the pots did not

contaminate other samples. After completion of eight weeks, the tomato plants plus

any weeds growing in the pots were carefully uprooted, labeled with sample number

and the roots were gently washed in tap water to remove adhering soil. The weed and

tomato roots were suspended in water kept in a Petri dish while viewing for presence

of root galls under a stereoscopic microscope.

The presence or absence of root galls on the tomato plant from the bioassay pot was

considered as the final indicator for the presence or absence of root-knot nematodes in

all the soil samples. The severity of the infection was determined by estimating the

number of root galls. The results after examination of the plant roots were recorded in

a table with the plant name, sample number and location. The infected samples were

denoted by a + sign while those not infected were denoted by a – sign. The percentage

incidence for each of the ten areas after bioassay was calculated using the following

formula: Percentage incidence per locality = Number of farms infected x 100

Number of farms sampled

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21
Similarly the total incidence was calculated using the formula:
Total incidence = Total number of farms infected x 100
Total number of farms sampled
The data recorded from the direct examination and the bioassay methods were also

compared by determining the difference between the percentage incidences as follows:

Difference in incidence = Total percentage incidence – percentage incidence

after bioassay after direct examination

The weeds found infected with root-knot nematodes were photographed and identified

with the help of reference books (Seemann and Fitch, 1865; Parham, 1958; Whistler,

1995) and verified in consultations with an expert botanist (Professor Arthur Whistler,

2007, USP). They were also tentatively categorized into poor and good hosts based on

gall count and egg mass count however there was no systematic comparative studies

on weed host status. Weeds were classified as non hosts of root-knot nematodes in

cases where the tomato plant was infected but the weeds remained uninfected. Poor

weed hosts of root-knot nematodes had only few galls (< 20) and egg masses (<10)

even in presence of extensive galling on the tomato plant from the same pot. Weeds

which had extensive galling (>20) and egg masses (>10) were considered as good

hosts of root-knot nematodes.

During the examination of plant roots, egg masses present were separated and placed

in Eppendorf tubes for later DNA extraction. Female nematodes were also dissected

out from the root galls and processed for later slide preparation. Males, where present,

were removed and processed and 1-2 egg masses were placed in distilled water to

obtain juveniles for later slide preparation. The nematode specimens (males, females

and juveniles) for morphological and molecular characterization were obtained from

populations maintained in the bioassay pots.

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2.2 IDENTIFICATION OF MELOIDOGYNE SPP.

2.2.1 MOLECULAR CHARACTERIZATION

2.2.1.1 SELECTION OF MOLECULAR MARKERS

Previously, restriction endonuclease analysis of the mitochondrial encoded gene

Cytochrome Oxidase subunit II had been used successfully by Powers and Harris

(1993) to characterise five common Meloidogyne species. Similarly, species specific

nuclear encoded Sequence Characterized Amplified Regions (SCAR) have been used

successfully for the rapid identification of root-knot nematodes (Zijlistra, 2000). There

are a number of SCAR molecular markers available for identification of the common

root-knot nematode species. In the present study, the populations found at 10 different

localities were screened using mitochondrial and nuclear encoded species specific

primers (Table 2.1).

2.2.1.2 EXTRACTION OF DNA

DNA was extracted from 5-10 egg masses collected from RKN infected tomato plants

maintained in the bioassay pots. The female nematodes from which the egg mass was

collected was also removed and processed for microscopic examination. The egg

masses were removed using dissecting needles and rinsed in distilled water to remove

as much of the adhering soil particles or debris. The egg masses were then placed in

pre sterilised DNA-free, 2 ml Eppendorf tubes and immediately placed in the

refrigerator at - 4 oC until DNA extraction.

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23
 Table 2.1: List of primers.

List of primers Reference sources

C2F3/1108 5’-GGTACAATGTTCAGAAATTTGTGG-3’ Powers and Harris, 1993

(mitochondrial) 5’-TACCTTTGACCAATCAGGCT-3’

C2F3/Mel450R 5’-GGTACAATGTTCAGAAATTTGTGG-3’ Reverse primer was

(mitochondrial) 5’-ATCCTAATAATAAATAAAAATG-3’ synthesized by Invitrogen

Far/Rar 5’-TCGGCGATAGAGGTAAATGAC-3’ Zijlistra et al., 2000

(M. arenaria) 5’-TCGGCGATAGACACTACAAACT-3’
SCAR
Fjav/Rjav 5’-GGTGCGCGATTGAACTGAGC-3’ Zijlistra et al., 2000
(M. javanica)
5’-CAGGCCCTTCAGTGGAACTATAC-3’
SCAR
MI-F/MI-R 5’-GTGAGGATTCAGCTCCCCAG-3’ Meng et al., 2004
(M. incognita)
5’-ACGAGGAACATACTTCTCCGTCC-3’
SCAR

The salting out procedure by Miller et al. (1988) was used with modifications to

extract DNA from egg masses. The steps used in the procedure for DNA extraction are

as follows:

1. Tissue digestion: involved the crushing of egg masses under liquid nitrogen in

Eppendorf tubes till a fine white powder was formed. The crushing was done using

polypropylene pestle, shaped to fit closely into the base of Eppendorf tubes. After

crushing, 600 µL of TNES solution (50mM Tris HCl, 400mM NaCl, 20mM EDTA,

0.5% SDS) and 10 µL of Proteinase K was added. The crushed egg masses were

suspended in the solution by vortexing and the tube was incubated for 3 hours at 55 oC

with occasional vortexing.

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24
2. Precipitation of proteins and cell debris: To precipitate out the proteins, the tubes

were removed after 3 hours and 170 µL of 5M NaCl was added. The suspension in the

tube was mixed thoroughly by shaking the tubes up and down for 15 seconds. The tube

was centrifuged at 14000 rpm for 5 minutes. While placing the tubes into the

centrifuge the lid hinge of the tube was pointed away from the centre to ensure that the

pellet was formed on one side. The supernatant containing the DNA was pipetted into

a clean new labeled tube ensuring that the cell and protein debris was not disturbed.

3. Precipitation of nucleic acids: After transferring the supernatant containing DNA

into a new tube, 750 µL of cold absolute ethanol was added and mixed by inverting the

tube gently for 30 seconds to precipitate out the DNA. The tube was kept in the freezer

at -20 oC for 15 minutes to allow for better precipitation of DNA. After precipitation of

DNA, the tube was centrifuged at 14000 rpm with the lid hinge pointed away from the

centre for 5 minutes. The ethanol and the salts were carefully poured off without

disturbing the DNA pellet formed after centrifugation. The DNA pellet was rinsed by

adding 400 µL of 70% ethanol and centrifuging at 14000 rpm for 5 minutes. The

ethanol was carefully poured off and the remaining ethanol was allowed to evaporate

by leaving the tube inverted for 20-30 minutes on top of clean paper towel. The DNA

pellet was suspended in 30 µL of TE buffer (10mM Tris HCl, 1mM EDTA) and stored

at - 20 oC for later use in PCR.

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 2.2.1.3 POLYMERASE CHAIN REACTION (PCR) PROTOCOL

The polymerase chain reactions (PCR) were carried out using the extracted root-knot

nematode DNA as the template. The master mix for 30 µL PCR reactions was

prepared using the following ingredients:

Sterilized distilled water (17.8 μl),

10X reaction buffer (3.0 μl),

MgCl2 50mM (3.0 μl),

dNTPs 2mM (3.0 μl),

Primer forward (1.0 μl),

Primer reverse (1.0 μl),

Taq polymerase (0.2 μl) and

Template (1.0 μl).

For all the reactions, a negative control was kept and when differentiating between the

mixed species, positive controls were also kept. To detect mixed populations, the DNA

samples were amplified using primers for all three common species in separate PCR

reactions. All PCR reactions were carried out in Eppendorf mastercycler gradient

thermocycler machine. First the primers were optimized using a temperature gradient

to select the most appropriate temperature. The optimized PCR programs for the

different primer combinations are represented in Table 2.2. PCR programs similar to

the ones given in Table 2.2 have been used to identify Meloidogyne spp. (Powers and

Harris, 1993; Zijlistra, 2000; Meng et al., 2004; Adam et al., 2007).

The products after PCR reactions using the general mitochondrial and the SCAR

primers were observed on 1.5 % agarose gels and their sizes were determined by

comparing against DNA Hyper ladder I (Appendix 3, p109).

26
26
Table 2.2: PCR programs

Step 1 Step 2 Step 3 Step 4 Step5 Step 6

Initial Denaturation Annealing at Extension Final Hold at
denaturation at 94oC for at 72 oC extension at 4 oC
at 94oC for 30 secs 61oC Far/Rar for 1 min 72 oC for 7
2 min min
64oC Fjav/Rjav

62oC MI-F/MI-R

55oC C2F3/1108

48oC C2F3/Mel

450R

For 30 secs
Repeated for 34 cycles

2.2.1.3 GEL ELECTROPHORESIS

The Gel used for electrophoresis was prepared by weighing out 0.5g of agarose

powder and dissolving in 50ml of 1X TAE (40mM Tris, 20mM NaOAc, 1mM EDTA,

pH 8 HOAc) buffer. The buffer was heated for 45 seconds in a microwave oven to

dissolve the agarose powder. The mixture was swirled to dissolve all agarose and

allowed to cool slightly before adding 5 μl of syber safe dye. The mixture was poured

into the electrophoresis chamber and allowed to cool. Combs with 10 teeth per row,

were placed in the liquid agarose to make 10x10 μl wells. Once the gel had solidified,

the electrophoresis chamber was filled with sufficient 10X TAE buffer to cover the

gel. Once the gel had set, the combs were gently removed to avoid damaging the wells.

The wells were loaded with 5 μl of the PCR products mixed with 2 μl of 10 X loading

buffer on a paraffin film and 5 μl of DNA hyper ladder I was loaded into the left corner

well. The electrophoresis was carried out at 110 volts for 20-30 minutes to allow for

the separation of the fragments.

27
27
The gel was placed on a UV transilluminator and digital pictures of the gel were taken

using a geldoc system. The gel pictures were digitally recorded and named with the

sample number(s). DNA marker ladder, hyper ladder I (10,000bp-200bp) was used to

approximate the size of the fragments. The size of bands on the gel was compared to

the expected band size of Meloidogyne spp. reported in literature for the particular

molecular marker to determine the root-knot nematode species.

The populations were categorized into five groups M. incognita, M. arenaria,

M. javanica, mixed populations and unidentified populations based on gel

visualization of the PCR results. Populations that showed amplification of a PCR

product of the appropriate diagnostic size with only one species specific primer

combination, and not with other combinations, were assigned to one of the appropriate

three groups. For instance, all populations that produced an amplification product of

1000bp with the primers MIF/MIR were assigned to M. incognita. Populations that

consistently showed amplification with more than one species specific primer were

considered as mixed populations and identification of mixed populations was

confirmed after morphological examination. The populations that did not amplify with

any of the species specific primers but did amplify with the mitochondrial primers

were categorized as unidentified Meloidogyne populations. Due to time limitations of

the current project, members of this latter group were not investigated further.

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 2.2.2 MORPHOLOGICAL CHARACTERIZATION

The root-knot nematode populations from various different geographical locations

were selected out of the five groups identified through molecular characterization and

their morphological features were studied to look for morphological variations and

confirm their species identity. For morphological characterization, adult females,

juveniles and males when available were studied. The adult female nematode and the

male specimen were obtained from field populations maintained on tomato plants in

the bioassay pots. Male specimens were not available from all the populations studied.

The egg masses from the females used for morphological characterization were used

for DNA extraction as outlined earlier under molecular identification. Juveniles (J2)

were obtained by incubating egg mass (obtained from the infected tomato roots prior to

dissecting adult female) kept in distilled water at room temperature (28-30 oC) in a

cavity block for 24 to 48 hours. The infected tomato roots were gently washed and

placed in a Petri dish and covered with water 2-3 hours prior to the dissection of

nematode specimen. All the dissections and sectioning for slide preparation were

carried out under stereoscopic microscope (Olympus SZ61) using mounted fine

pointed steel needles and number 11 surgical blades respectively. Picking and

manipulation of the nematode specimens were done using a fine pointed quill pick.

The morphological features studied from the female specimens included the perineal

pattern, body length, body width, body length/body width, stylet length, dorsal gland

orifice (DGO), vulva length and vulva-anus distance. In addition the juvenile body

length, stylet length, tail length and hayline tail terminus and male body length, stylet

length, distance to DGO, spicule length and gubernaculum length were measured to

assist with the species identification.

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 2.2.2.1 FEMALE

The whole females were placed in 45% lactic acid in glass cavity blocks for 48 hr.

After completion of 48 hr the females were removed from the lactic acid and placed in

a small drop of anhydrous glycerol. The slide with the whole female, without the

addition of a coverslip was examined under a compound light microscope at

magnification 400x and the body length and body width measurements were taken.

PERINEAL PATTERN

The procedure for preparing perineal patterns of root-knot nematodes as outlined by

Shurtleff and Averre (2000) was used with slight modifications. After measurements

were taken, the slide was placed under a stereoscopic microscope and while viewing,

an incision was made using a scalpel in the middle of the female to cut the cuticle into

half equatorially. The posterior region consisting of the perineal pattern was carefully

cut off and trimmed. The cut perineal pattern section was brushed gently using a fine

pointed quill pick to remove any attached debris. The cut perineal pattern was

manipulated using a quill pick and placed on another clean slide in a drop of anhydrous

glycerol. Three to four perineal patterns from a single population were positioned on

the slide with the outer side uppermost and a glass cover slip was applied. Excess

glycerol was soaked using a small piece of tissue and the slide was sealed using clear

nail polish.

The anterior half of female consisting of the stylet and neck region remaining after the

equatorial excision of the whole female was trimmed and mounted on the slide in

anhydrous glycerol. The measurements of the female stylet and dorsal gland orifice

(DGO) were taken. The stylet morphology and distance of the DGO from the stylet

30
30
base have often been used as morphometric characteristics for species descriptions

(Gerber and Taylor 1988).

2.2.2.2 JUVENILE AND MALE

The juveniles and males were heat killed by adding hot (≈ 70-75 oC) FA fixative (10

parts formalin (40% formaldehyde), 1 part glacial acetic acid, 2 part glycerol and 87

parts distilled water). After 48 hrs, most of the fixative was removed and the cavity

block was filled with slow clearing solution (8 parts 95% ethanol, 2 parts anhydrous

glycerol, and 90 parts water) and covered with a glass lid (Hooper et al., 2005). The

clearing solution was allowed to evaporate at room temperature (28-30 oC) for a week.

After evaporation of the slow clearing solution, the nematodes were left in glycerol.

The nematode specimens were transferred to a clean drop of anhydrous glycerol on a

glass slide using a quill pick. Broken pieces of cover slip arranged in a triangle shape

were used as supports to avoid crushing of the specimens under the cover slip.

All specimens mounted on the glass slide were covered with a cover slip and sealed

using clear nail polish. The specimens in prepared slides were marked on the cover slip

for easier location while viewing under the compound light microscope. All slides

were labeled with the sample number, number of specimen and the date of preparation

using a fine tip permanent marker.

The prepared slides were viewed under a compound light microscope (Olympus

BX51) attached with a digital camera (Q-imaging micropublisher 5.0 RTV) connected

to a computer with Image-Pro Plus software (Version 6.1) for processing and storing

the images (Figure 2.4). The Image-Pro Plus software was used to take measurements

31
31
from the image and snap digital pictures from the slides of the nematode specimen at

400x or 1000x magnification. The measure distances function of the image analysis

software was first calibrated using a stage micrometer for each of the objectives before

taking measurements of the specimen. All measurements were recorded and for each

of the species, the data was summarised by calculating the averages, range and

standard error using Sigma plot Version 11. The measurement values are summarised

and presented in Tables 3.1 - 3.4 under the results section. The molecular data,

morphological observations and measurements were compared with the species

descriptions given in the literature to confirm the species identity.

Figure 2.4: Microscope (Olympus model BX 51) with imaging system.

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 CHAPTER 3

RESULTS

3.1 DISTRIBUTION AND HOSTS.

3.1.1 MELOIDOGYNE SPP. DISTRIBUTION ON VITI LEVU.

Root-knot nematodes were found to be widely distributed in agricultural areas

throughout Viti Levu, Fiji with infections recorded from a total of 277 farm soil

samples out of the 675 farms sampled. Direct examination of plant roots revealed the

presence of root-knot nematodes on 189 (28%) of the farms while bioassay of the soil

samples revealed presence of root-knot nematodes in another 13% (88) of the soil

samples (Table 3.1). The total percentage incidence of root-knot nematodes was

determined to be 41% taking into account both the incidence recorded via direct

examination and bioassay of soil samples from all the 10 different localities.

Table 3.1: Incidence of root-knot nematodes on Viti Levu.

Locality Number Direct Total Difference %

of farms examination of incidence in incidence
sampled plant roots after incidence (C/A)x
(A) (B) Bioassay (C) (C-B) 100
1. Nausori 48 18 27 9 56
2. Suva-Nasinu 39 16 21 5 54
3. Navua 46 7 11 4 24
4. Sigatoka 92 43 62 19 67
5. Nadi 85 27 41 14 48
6. Lautoka 79 19 26 7 33
7. Ba 84 24 33 9 39
8. Tavua 73 11 20 9 27
9. Rakiraki 76 16 24 8 32
10. Korovou 53 8 12 4 23
Total 675 189 277 88 41

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The localities sampled had varying percentage incidence of root-knot nematodes with

Sigatoka having the highest incidence closely followed by Nausori, Suva-Nasinu and

Nadi. The levels of infestations observed during the survey ranged from severe on 8%,

moderate on 20% and low on 13% of the farms sampled. The farms or gardens

severely infected with root-knot nematodes were easily detected through direct

examination of crop or weed roots on the farm site. The severely infected plants had

extensive galling on the root systems and the plants also showed symptoms such as

stunted growth, yellow leaves and wilting (Figure 3.1). The plants on farms with

moderate level of infestation did not always show clear above ground symptoms but

root galls were present and identifiable after direct examination. Farms with low level

of infestation often had plants which did not show any above ground symptoms and

plant roots had few or no root galls after direct examination. Low levels of root-knot

infection were detected mostly after bioassay of soil samples. The bioassay tomato

plants (cv. Moneymaker) grown in soil samples with low initial population density of

root-knot nematodes had lower number of galls than the tomato plants grown in soil

samples with moderate to heavy root-knot population density.

The morphology of root galls induced by root-knot nematodes varied depending on the

host plant and the level of infestation. Most infected plants had clear and distinct root

galls (Figure 3.2) while some had large coalescing galls (Figure 3.3) and few plants

had really small and hard to determine root galls (Figure 3.4). The small root-galls

were observed mostly on grasses and were diagnosed after microscopic examination.

34
34
 Root Galls

Damage to
rhizome
(Disfigured

Figure 3.1: Ginger farm heavily infested with M. incognita in Nausori, Fiji.
(The area enclosed in the oval shape shows plants with stunted growth and wilt
symptoms and insert picture shows a ginger plant from the field.

Egg plant roots

Figure 3.2: Eggplant (Solanum melongena) roots with distinct root galls
(indicated by arrows) infected by M. incognita from a field in Sigatoka, Fiji.

35
35
 Okra root
Weed root

Weed root

Figure 3.3: Weed (Vernonia cinerea) and okra (Abelmoschus esculentus) roots
with coalescing root galls (indicated by arrows) infected by M. javanica and M.
arenaria collected from fields in Nadi and Sigatoka respectively.

Grass root

Figure 3.4: Grass (Eleusine indica) roots with small root galls (indicated by
arrows) infected by M. incognita collected from bioassay pot sample.

36
36
 3.1.2 CROP HOSTS OF MELOIDOGYNE SPP.

The hosts of root-knot nematodes included 33 species of crop plants including most

of the commonly grown crops (Table 3.2). The distribution of the root knot

infections and the parasitizing Meloidogyne spp. is also indicated in Table 3.2.

Although the host status was not studied in great detail, preliminary observations

indicate that crops such as tomatoes, okra, beans, egg plant, cucumber, pumpkin,

bele, pawpaw and ginger acted as good hosts of root-knot nematodes with greater

frequency of severe infections recorded on these hosts via direct examination. For

example, on a farm with maize and beans intercropped within the same row, the

maize plant had very few galls while the bean plant growing close by had extensive

galling of the root system caused by M. javanica.

The life cycle of root-knot nematodes is short in the tropical summer months

(November to April in Fiji) and is complete with the next generation produced within

6-8 weeks. The susceptible crops are easily infected and become severely infected

within one growing season. The susceptible tomato plants allowed to grow in infected

soil had fully developed egg masses protruding from the root galls (Figure 3.5) when

uprooted after 8 weeks from the bioassay pots.

Protruding
egg mass

0.6 cm Root galls 0.5 cm

.

Figure 3.5: Root gall with protruding egg mass produced by M. incognita on
tomato root collected from bioassay pot.

37
37
Root crops such as cassava, yam, dalo and sweet potato when infected had signs of

necrosis on the storage part and usually the secondary roots had the root galls. Direct

examination of the storage roots can be a bit confusing as there may not be distinct

galls present and necrosis of the outer tissue may be the only visible injury. The root

galls were best detected when the root crop plants were young (8-12 weeks old).

These root crops did not show many of the above ground symptoms such as wilting,

yellowing of leaves or stunted growth and appeared very similar to the uninfected

plants. Due to the varying nature of root-knot infection symptoms on root crops, it is

relatively more difficult to determine the presence or absence of root-knot nematodes

on root crops via direct examination of plant roots. Root crops were mostly infected

by M. incognita and M. javanica while infection by M. arenaria was less common

and noted from soil samples collected from dalo and cassava farms.

Apart from vegetables and root crops, sugarcane which is a major crop grown on the

western side of Viti Levu (Sigatoka, Nadi, Lautoka, Ba, Tavua and Rakiraki) was

also found to act as host to root-knot nematodes. The root galls on sugarcane roots

were smaller compared to the root galls observed on vegetable crop plants.

Sugarcane is usually grown as a mono crop and after yearly harvests, the ratoon

crops are allowed to grow for 5 years or more at times. M. incognita and M. javanica

were the common species found on sugarcane farms and in a few cases other crops

such as beans and tomatoes intercropped in-between the rows of sugarcane were also

found infected.

38
38
Table 3.2: Crop hosts of root-knot nematodes.

Scientific name Common Localities * Meloidogyne

name(s) spp. **
1. Abelmoschus Okra 1,2,3,4,5,6,7,9,10 Ma, Mj, Mi, U
esculentus
2. Amaranthus viridis Amaranthus*** 2, 4, 6, 7, 8 Ma, Mj, Mi
(Churaiya)
3. Ananas comosus Pineapple 1.4, 8 Mj, Mx

4. Arachis hypogaea Peanuts 1,5 Ma, U

5. Brassica campestris Cabbage 1, 2, 4, 5, 6, 7, 8, Mj, Mi

10
6. Cajanus cajan Pigeon pea 6 Mj
(Toor)
7. Capsicum annuum Chillies 1, 2, 4, 6, 7, 10 Mj, Mi, Mx

8. Carica papaya Pawpaw 1, 2, 4, 6, 8, 9, 10 Ma, Mi, Mx

9. Citrullus lanatus Watermelon 4, 8, 9 Mi

10. Colocasia esculenta Dalo 1, 7, 8 Mi, Mx

11. Coriandrum sativum Corriander 2, 4, 6, 7, 8, 9 Mi, Mx

(Dhania)
12. Cucumis sativus Cucumber 1, 4, 6, 9 Ma, Mj, Mi

13. Cucurbita maxima Pumpkin 1, 4, 5, 6, 7, 8, 9, Ma, Mj, Mi

10
14. Dioscorea esculenta Yam 5, 8, 9 Mi

15. Hibiscus manihot Bele 1, 2, 3, 4, 5, 6, 7, Ma, Mj, Mi

8, 9
16. Ipomoea batatas Sweet potato 1, 3, 4, 10 Mj, Mi

17. Lactuca sativa Lettuce 2, 6 Mi

18. Lagenaria siceraria Gourd (Laoki) 6 Mi

19. Luffa acutangula Toroi 5 Mj

20. Manihot esculenta Cassava 4, 5, 7, 8, 9 Mj, Mi

39
39
Table 3.2: Crop hosts of root-knot nematodes (continued).

21. Momordica charantia Bitter Gourd 5, 7 Mj

(Karela)
22. Musa musa Bananas 4, 8, 9 Mi

23. Nicotiana tabacum Tobbaco 4 Ma, Mi

24. Phaseolus vulgaris Bean (long green 1, 2, 4, 5, 6, 7, Ma, Mj, Mi,

beans) 8, 9, 10
25. Piper methysticum Kava 9 Mi

26. Raphanus sativus Raddish 4, 7 Ma

27. Saccharum officinarum Sugarcane 4, 5, 6, 7, 8, 9 Mj, Mi, Mx

28. Solanum melongena Eggplant 1, 2, 3, 4, 5, 6, Ma, Mj, Mi, U,

(Baigan) 7, 8, 9
29. Solanum lycopersicum Tomato 1, 2, 3, 4, 5, 6, Ma, Mj, Mi
7, 8, 9, 10
30. Vigna radiata Mung 5, 8 Mj

31. Vigna unguiculata Cow peas 2, 3, 4, 5, 6, 7, Mj, Mi, Mx

9
32. Zea mays Maize 4, 5, 8 Mj, Mi

33. Zingiber officinale Ginger 1, 3, 10 Mi

Key: *Localities: The localities are represented by numbers 1-10 as assigned in

Table 3.1, 1 = Nausori, 2 = Suva-Nasinu, 3 = Navua, 4 = Sigatoka, 5 = Nadi,
6 = Lautoka, 7 = Ba, 8 = Tavua, 9 = Rakiraki, 10 = Korovou.

** Meloidogyne spp. Ma = M. arenaria, Mi = M. incognita, Mj = M. javanica

U = unidentified, Mx = Mixed (Mi + Mj)

*** Amaranthus is commonly found on farms along with other crops and is
allowed to grow because it can also be used as a vegetable.

The same key for locality and Meloidogyne spp. applies to Table 3.3, Table 3.4 and
Table 3.5.

40
40
 3.1.3 WEED HOSTS OF MELOIDOGYNE SPP.

Weeds hosts of root-knot nematodes were found on farms after direct examination of

the weeds present and also after examination of weeds growing in the bioassay pots. A

total of 45 different species of weed hosts were identified out of which 25 different

species were categorized as good hosts (Table 3.3). Some of the commonly occurring

weeds (Amaranthus viridis, Ageratum conyzoides, Cassia obtusifolia, Coccinia

grandis, Hedyotis corymbosa, Ludwigia hyssopifolia, and Vernonia cinerea) were

found infected after direct examination on fields and are good hosts of root-knot

nematodes. The egg mass count was taken as an indicator of the reproduction potential

of the root-knot nematodes on the infected weed host. The remaining 20 species of the

weed hosts had lower number of root galls and fewer egg masses and were tentatively

categorized as poor hosts (Table 3.4). Another 11 weed species were identified as non

hosts of the prevalent Meloidogyne species as they remained uninfected even in soil

samples with high population density of root-knot nematodes (Table 3.5).

The host status categorization given is tentative because the host status of a plant

depends on other factors such as the parasitizing root-knot nematode species, initial

population density and other factors which need to be more systematically studied to

determine the pathogenicity of the weed species. For instance M. javanica was able to

infect the weed Phyllanthus amarus but M. arenaria could NOT as observed from two

different fields. In addition to such clear differences in host status with respect to the

Meloidogyne species, there were also differences in the degree of pathogenicity on

weeds by different Meloidogyne spp. For example it was observed that

Ageratum conzoides acted as a good host for M. incognita populations but was a poor

host for M. arenaria populations.

41
41
Table 3.3: Good weed hosts of root-knot nematodes.

Weed species Localities* Meloidogyne

spp.**
1. Ageratum conyzoides 1, 4, 5, 7 + bioassay pot Ma, Mi, Mj
2. Alternanthera sessilis Bioassay pot Mi
3. Amaranthus viridis 2, 4, 5, 7 + bioassay pot Mi, Mj, Mx
4. Cassia obtusifolia 5, 6, 9 + bioassay pot Mi, Mj, Ma
5. Coccinia grandis 2, 7, 10 + bioassay pot Mi, Mj
6. Cuphea carthagenensis Bioassay pot Mi
7. Digitaria setigera Bioassay pot Mi
8. Fimbristylis dichotoma Bioassay pot Mi
9. Hedyotis corymbosa 1, 2, 4 + bioassay pot Mi, Mj, Mx
10. Hyptis pectinata Bioassay pot Mi
11. Impatiens walleriana 4 + bioassay pot Ma, Mi
12. Ludwigia hyssopifolia 1, 3 + bioassay pot Mi, Mj, Mx
13. Macroptilium atropurpureum Bioassay pot Ma, Mi
14. Momordica charantia 5 Mj
15. Mullugo pentaphylla Bioassay pot Mi
16. Oxalis corniculata Bioassay pot Mi
17. Oxalis debilis Bioassay pot Mi
18. Peperomia pellucida Bioassay pot Mi
19. Phyllanthus amarus Bioassay pot Mj, Mx
20. Phyllanthus urinaria Bioassay pot Mj
21. Physalis angulata Bioassay pot Mi, Mj
22. Portulaca oleracea Bioassay pot Mi, Mx
23. Solanum torvum 1, 4 Mi
24. Triumfetta rhomboidea 6 + bioassay pot Mi
25. Vernonia cinerea 1, 3, 4, 7 + bioassay pot Mi, Mj

* and ** refer to key on p 39.

42
42
 Table 3.4: Poor weed hosts of root-knot nematodes.

Weed species Localities* Meloidogyne

spp.**
1.Clitoria ternatea Bioassay pots Mi
2.Commelina benghalensis 1 + bioassay pots Mi, Ma
3.Cyperus compressus 1, 4, 7+ bioassay pots Mi, Mj
4.Digitaria ciliaris Bioassay pots Mi
5.Echinochloa colona 4, 6 + bioassay pots Mi,
6.Echinochloa crus-galli Bioassay pots Mi, Mj
7.Eleusine indica 4, 5, 7, 8, 9 + bioassay pots Mi Ma
8.Eleutheranthera ruderalis Bioassay pots Mi
9.Ipomoea obscura 2, 3, 8 Mj
10. Ipomoea quamoclit 4 Mi, Mj
11. Ipomoea trifolia 1, 5 Mi
12. Lindernia nummulariifolia Bioassay pots Mi
13. Malvastrum coromandelianum 1, 4, 7 + bioassay pots Mi, Ma
14. Mikania micrantha 1, 4 + bioassay pots Mi,
15. Piper aduncum 1, 10 + bioassay pots Mj
16. Senna occidentalis 4 Mi
17. Setaria palmifolia 2 + bioassay pots Mj, Ma
18. Sporobolus indicus Bioassay pots Mi
19. Stachytarpheta urticifolia 4, 6 + bioassay pots Mi, Mj
20. Synedrella nodiflora 1, 2, 4 + bioassay pots Mi

* and ** refer to key on p39.

43
43
 Table 3.5: Weed non hosts of root-knot nematodes.

Weed species Locality* Meloidogyne

spp.**
1.Chamaesyce hypericifolia 5, 7 + bioassay pots Mi, Mj
2.Chamaesyce hyssopifolia 4 + bioassay pots Mi, Mj
3.Commelina diffusa 1, 3 + bioassay pots Mj
4.Euphorbia heterophylla 1, 3, 4 + bioassay pots Mi, Mj
5.Euphorbia hirta Bioassay pots Mi
6.Kyllinga polyphylla Bioassay pots Mj
7.Mimosa pudica Bioassay pots Mj
8.Passiflora foetida 4, 5, 9 + bioassay pots Mj
9.Pycreus polystachyos Bioassay pots Mj
10. Rottboellia cochinchinensis 7 + bioassay pots Mi, Mj
11. Spermacoce assurgens Bioassay pots Mi, Mj

* and ** refer to key on p39.

For Tables 3.3 -3.5, some of the weed specimens were found infected on the field as

well as in the bioassay pot while the other weed specimens along with the tomato plant

were found infected only after bioassay of soil samples.

44
44
3.2 MELOIDOGYNE SPP. CHARACTERIZATION.

A total of 277 populations were studied. First the populations were screened using

mitochondrial and SCAR markers and categorized into five different groups which

included M. incognita, M. javanica, M. arenaria, mixed populations and unidentified

populations. The molecular method, being more rapid than morphological

characterization, was chosen to categorize the populations into groups followed by

morphological examination of selected populations from the groups to verify the

species identifications.

M. incognita and M. javanica were the predominant species in all of the localities

while M. arenaria was less common (Figure 3.6). Mixed populations consisting of

M. incognita and M. javanica were found on 5% of the farms. There was no incidence

of mixed populations between M. incognita and M. arenaria or M. javanica and

M. arenaria. A small percentage of populations (1%) from 3 different localities could

not be identified at the species level as they did not amplify with any of the species

specific primers, and they were also morphologically different from the identified

species.

5% 1% M. incognita

10% M. javanica

56% M. arenaria

M. incognita +
M. javanica
28%
Unidentified
populations

Figure 3.6: Meloidogyne species composition on Viti Levu.

45
45
The unidentified populations were distinguished from one another on the basis of

morphological features and were classified as Meloidogyne spp. 1, 2 and 3.

Unfortunately, species designation of these populations could not be ascertained from

the molecular analyses undertaken and scanning electron microscopy was unavailable

within the time frame of this study. Thus inferences were limited to features

observable under light microscope. The Meloidogyne species description is given in

the following section and further studies are required to ascertain the identities of

Meloidogyne spp. 1, 2 and 3.

46
46
3.2.1 DESCRIPTION OF MELOIDOGYNE SPP.

MELOIDOGYNE INCOGNITA (Kofoid & White, 1919) Chitwood, 1949

Syn.: Oxyuris incognita Kofoid & White, 1919

Heterodera incognita (Kofoid & White, 1919) Sandground, 1923
M. incognita incognita (Kofoid & White, 1919) Chitwood, 1949
M. incognita acrita Chitwood, 1949
M. acrita (Chitwood, 1949) Esser, Perry & Taylor, 1976
M. incognita inornata Lordello, 1956
M. inornata Lordello, 1956
M. kirjanovae Terenteva, 1965
M. elegans da Ponte, 1977
M. grahami Golden & Slana, 1978
M. incognita wartellei Golden & Birchfield, 1978

(Cited from Karssen and Moens, 2006).

Hosts: (N= 26 crop hosts; 22 good weed hosts; 17 poor weed hosts).

Localities: All (1-10).

Refer to Tables 3.2, 3.3 and 3.4 for list of hosts and localities.

MOLECULAR CHARACTERIZATION

A total of 154 populations amplified with the SCAR primers MIF/MIR (Meng et al.,

2004) yielding 1.0 kb PCR product (Figure 3.7) and were categorized in the M.

incognita group. These populations did not amplify with the other common species

specific primers (Far/Rar and Fjav/Rjav).

1.0 kb fragments
characteristic of
M. incognita

10000 bp

1000 bp
800 bp Lane with
negative control

Figure 3.7: Gel photograph showing bands obtained from M. incognita

populations using species specific primers MIF/MIR.

47
47
 MORPHOLOGICAL CHARACTERIZATION

Following the molecular characterization, morphological examinations were carried

out on 30 populations from different localities to confirm the identification and to look

for morphological variations between the populations.

FEMALE

Measurements (µm) N= 10

L = 719.9 ± 8.1; Body width = 439.9 ± 8.3; a = 1.6; Stylet length = 14.8 ± 0.4,

DGO = 3.5 ± 0.1; Vulva length = 25.8 ± 0.7; Vulva- anus distance = 17.9 ± 0.3.

Description

M. incognita female body pear shaped; short projecting neck (Figure 3.8). No distinct

posterior protuberance in the perineal region. Stylet slightly curved, stylet knob

anteriorly indented. Perineal pattern with squarish dorsal arch, smooth, wavy or

zigzagged striations; no distinct lateral lines, although some striations appear to form

lines but upon closer examination at higher magnification (1000x) revealed no

distinctive lateral line. The perineal patterns observed (Figure 3.9 A, 1-3) very closely

resembled to the descriptions of M. incognita. (Figure 3.9 B). The morphological

measurements taken from the female specimens from the M. incognita populations

also closely resembled to the values given in literature for M. incognita (Table 3.6).

263um A B 78 um

Figure 3.8: M. incognita LM photograph of (A) Female, (B) Eggs with juveniles
ready to hatch from a population found infecting eggplant (Solanum melongena)
in Nausori.

48
48
 A1 A2

27 um 25 um
Farm 34 Nausori, host: Eggplant. Farm 7 Lautoka, host: Bele.

A3
B
25 um
Farm 4 Sigatoka, host: Pumpkin.

Figure 3.9 M. incognita (A, 1-3) LM photograph of perineal patterns,

(B) Drawing of perineal pattern (Source: Shurtleff and Averre, 2000).

The cuticle patterns for most of the M. incognita populations looked similar to the

patterns shown above however there were also slight variations recorded in the

M. incognita perineal patterns between the populations from Fiji (Figure 3.10 A, 1-4).

49
49
 A1 A2

Farm 19 Ba, host: Okra. 24 um Farm 34 Rakiraki, host: Cabbage. 23 um

A3 A4

Farm 24 Nadi, host: Tomato. 24 um Farm 18 Korovou, host: Pawpaw. 24 um

Figure 3.10 LM photographs of M. incognita perineal patterns showing variations

from the typical.

50
50
 MALE

Measurements (µm) N= 5

L = 1749.5 ± 18.9; Stylet length = 24.1 ± 0.3; DGO = 2.9 ± 0.1;

Spicule length = 28.5 ± 0.4; Gubernaculum length = 9.3 ± 0.2.

Description

M. incognita male body vermiform, head region not offset from body; stylet tip blunt,

shaft cylindrical, stylet knobs rounded to ovoid slightly indented anteriorly, short DGO

distance, four lateral incisures, tail region slightly curved (Figure 3.11). The

description of M. incognita matched the description given in literature (Eisenback,

1985; Karssen and Moens 2006).

A B
27 um 27 um

27 um
Farm 7 Lautoka, host: Bele.

Figure 3.11 M. incognita male LM photograph of (A) Head region, (B) Mid body
region, (C) Tail region.

51
51
 JUVENILE

Measurements (µm) N= 10

L = 359.3 ± 3.0; Stylet length = 10.6 ± 0.2; Tail length = 48.8 ± 0.7;

Hyaline tail terminus 10.7 ± 0.3.

Description

M. incognita juvenile body slender, tapering towards tail, (Figure 3.12) head cap

anteriorly flattened, head region rounded posteriorly, stylet cone and shaft gradually

increases in width posteriorly, stylet knobs prominent, set off from shaft, distinctive

hayline tail terminus, tail tip rounded.

A B C
Mag = 400X Mag = 1000X
45 um 11 um 18 um

Farm 7 Lautoka, host: Bele

Figure 3.12: M. incognita LM photograph of (A) Juvenile, (B) Juvenile head,

(C) Tail region.

52
52
 MELOIDOGYNE JAVANICA (Treub, 1885) Chitwood, 1949

Syn.: Heterodera javanica Treub, 1885

Anguillula javanica (Treub, 1885) Lavergne, 1901
M. javanica javanica (Treub, 1885) Chitwood, 1949
M. javanica bauruensis Lordello, 1956
M. bauruensis (Lordello, 1956) Esser, Perry & Taylor, 1976
M. lucknowica Singh, 1969
M. lordelloi da Ponte, 1969

(Cited from Karssen and Moens, 2006)

Hosts: (N= 20 crop hosts; 11 good weed hosts; 7 poor weed hosts).

Localities: All 10 localities sampled.

Refer to Tables 3.2, 3.3 and 3.4 for list of hosts and localities.

MOLECULAR CHARACTERIZATION

Root-knot nematode populations which produced a PCR amplification of size 670 bp

(Figure 3.13) using SCAR primers Fjav/Rjav (Zijlistra et al., 2000), were categorized

as M. javanica. These populations did not amplify with the other two species specific

primers (MIF/MIR and Far/ Rar). The PCR products from 78 populations were

categorized as M. javanica.

Lane with negative

control

10000 bp 670 bp fragment

characteristic of
1500 bp
M. javanica
600 bp
200 bp

Figure 3.13: Gel photograph showing bands obtained from M. javanica

populations using the primers Fjav/Rjav.

53
53
 MORPHOLOGICAL CHARACTERIZATION

The molecular identification of M. javanica was verified by morphological

examination of females and juveniles from 20 populations out of the M. javanica

group.

FEMALE

Measurements (µm) N= 10

L = 727.5 ± 8.2; Body width = 514.5 ± 9.6; a = 1.4; Stylet length = 16.4 ± 0.1;

DGO = 3.4 ± 0.1; Vulva length = 23.8 ± 0.2; Vulva- anus distance = 16.7 ± 0.2.

Description

M. javanica female body pear shaped with no distinct posterior protuberance, stylet

shaft cylindrical, stylet knobs not deeply indented; Dorsal striations low and rounded,

to slightly squarish, perineal pattern quite unique with distinct lateral ridges that divide

the dorsal and the ventral striae, lateral lines extend on both sides of tail terminus

cutting across perineal pattern (Figure 3.14 B, 1-3).

The perineal patterns observed for majority of the females matched closely with the

descriptions of M. javanica from the literature (Figure 3.14 C). In addition,

measurements taken from female and juvenile specimens from these populations

(Table 3.6) closely resembled to the measurements from M. javanica given in

literature. Some populations identified as M. javanica based on molecular

characterization had variations from the typical M. javanica perineal patterns which

included slightly higher than the typical dorsal arch with the dorsal striations more

squarish than rounded (Figure 3.15 A, 1-2).

54
54
 A B1

Farm 27 Nadi, host: Bean. 145 um Farm 27 Nadi, host: Bean. 27 um

B2
B3

Farm 15 Rakiraki, host: Bele. 25 um Farm 8 Suva-Nasinu, host: Chillies. 23 um

Figure 3.14: M. javanica LM photograph of (A) Female (B, 1-3) perineal patterns
(C) Drawing of perineal pattern (Source: Shurtleff and Averre, 2000).

55
55
A1 A2

24 um 25 um
Farm 9 Nausori, host: Cucumber. Farm 14 Lautoka, host: Churaiya.

Figure 3.15: (A, 1-2) LM photographs of M. javanica perineal patterns showing

variation from typical.

MALE

Specimen not found in this study but reported in literature (Rammah and Hirschmann,

1990).

JUVENILE

Measurements (µm) N= 10

L = 534.5 ± 6.4; Stylet length = 11.5 ± 0.2; Tail length = 55.6 ± 0.7;

Hyaline tail terminus 13.6 ± 0.3.

Description

M. javanica, juvenile body, long and slender, head cap anteriorly flattened, head region

posteriorly rounded, stylet similar to M. incognita, stylet knobs transversely elongate

and off set from stylet shaft, hayline tail terminus distinctive, long narrow tapering

tail, finely rounded tail tip.

56
56
M. INCOGNITA AND M. JAVANICA MIXED POPULATIONS

Hosts: (N= 7 crop hosts; 5 good weed hosts).

Localities: (1, 2, 4, 5, 6, 7 and 9).

Refer to Tables 3.2, 3.3 and 3.4 for list of hosts and localities.

MOLECULAR CHARACTERIZATION

A total of 14 populations from 6 different localities amplified with SCAR primers for

both M. incognita and M. javanica in separate PCR reactions hence were categorized

as mixed population consisting of both the species. The PCR products obtained with

MIF/MIR was of size 1.0 kb while the products obtained with Fjav/Rjav were of size

670 bp characteristic of M. incognita and M. javanica respectively (Figure 3.16). The

DNA used for the PCR reactions was extracted from pooled egg masses collected from

the infecting population which had both M. incognita and M. javanica egg masses.

Pure Mi Mix Mi+Mj Pure Mi Mix Mi+Mj

1.0 kb fragment
characteristic of
M. incognita

PCR using 670 bp fragment PCR using

Fjav/Rjav characteristic of MIF/MIR
M. javanica

Figure 3.16: Gel photograph showing bands obtained from a pure M. incognita
population and two mixed population of M. incognita and M. javanica.

(The lane furthest from the ladder in the gel above is the negative control for both
reactions).

57
57
There were no PCR products obtained with M. arenaria SCAR primers for the

populations represented in Figure 3.16. Also note that the band obtained with the

M. javanica SCAR primers is weak for one of the populations while the band for M.

incognita is strong for both the populations indicating that M. incognita is the

predominant species in both of the populations while M. javanica is present in lower

numbers in the population which has the weaker band with M. javanica primers as

represented in Figure 3.16.

MORPHOLOGICAL CHARACTERIZATION

The morphological features of 15-20 female specimens from the mixed populations

were also studied to verify that the morphological features also reflected the presence

of mixed population. The perineal patterns (Figure 3.17) for both M. incognita and

M. javanica were observed indicating coexistence of the two species with distinct

morphology in mixed populations. The measurements taken from the females were

within the range of values recorded for the pure populations of M. incognita and

M. javanica.

B
A

25 um 24 um

Mixed population, farm 14 Rakiraki, host: watermelon.

Figure 3.17: LM photograph of perineal pattern from mixed population
(A) M. javanica, (B) M. incognita.

58
58
MELOIDOGYNE ARENARIA (Neal, 1889) Chitwood, 1949

Syn.: Anguillula arenaria Neal, 1889

Heterodera arenaria (Neal, 1889) Marcinowski, 1909
M. arenaria arenaria (Neal, 1989) Chitwood, 1949
M. arenaria thamesi Chitwood, 1952
M. thamesi (Chitwood, 1952) Goodey, 1963

(Cited from Karssen and Moens, 2006)

Hosts: (N = 12, crop hosts, 4 good weed hosts, 4 poor weed hosts).

Localities: 9 out of 10 localities sampled.

Refer to Tables 3.2, 3.3 and 3.4 for list of hosts and localities.

MOLECULAR CHARACTERIZATION

A total of 28 populations of M. arenaria were recorded from 9 different localities after

molecular analysis using SCAR primers Far/Rar (Zijlistra et al., 2000) yielded PCR

product of 420 bp characteristic of M. arenaria (Figure 3.18). The M. arenaria

populations did not amplify with MIF/MIR or Fjav/Rjav primers.

Lane with
negative control

10000 bp
420 bp fragment
characteristic of
1000 bp M. arenaria
600 bp
400 bp
200 bp

Figure 3.18: Gel photograph showing the bands characteristic of M. arenaria with
primers Far/Rar.

59
59
 MORPHOLOGICAL CHARACTERIZATION

The molecular identification of M. arenaria was verified by morphological

examination of females and juveniles from 16 populations out of the M. arenaria

group.

FEMALE

Measurements (µm) N= 10

L = 782 ± 6.6; Body width = 602.8 ± 6.2; a = 1.3; Stylet length 15.2 ± 0.3,

DGO = 4.2 ± 0.1; Vulva length = 24.4 ± 0.4; Vulva-anus distance = 16.9 ± 0.2.

Description

M. arenaria, female body pear shaped, short projecting neck (Figure 3.19 A), stylet

broad and, robust with rounded stylet knobs gradually sloping backwards. Perineal

patterns with low dorsal arch slightly indented near lateral fields to form rounded

shoulders. No distinct lateral lines, striations short, irregular and wavy.

The perineal patterns observed matched the descriptions and LM photographs of

M. arenaria perineal patterns given in the literature (Figure 3.19 B, 1-3 and C). The

measurements taken from the female, juvenile and male specimens from the

M. arenaria populations were within the range of values given for M. arenaria in the

literature (Table 3.6 and Table 3.7).

Variations were also observed in the perineal pattern morphology of the M. arenaria

populations. The shoulders characteristic of M. arenaria was not clearly observable

and the striations were more coarse than the typical. In some perineal patterns, there is

slight wing formation. (Figure 3.20 A, 1-3).

60
60
 A B1

173 um 24 um
Farm 21 Sigatoka, host: Tomato. Farm 21 Sigatoka, host: Tomato.

B2 B3

25 um 23 um
Farm 48 Rakiraki, host: Cucumber. Farm 8 Navua, host: Okra.

Figure 3.19: M. arenaria LM photograph of (A) Female, (B, 1-3) Perineal

patterns, (C) Drawing of perineal pattern (Source: Shurtleff and Averre, 2000).

61
61
 A1 A2

20 um 22 um

Farm 14 Korovou, host: Okra. Farm 24 Nadi, host: Peanut.

A3

22 um
Farm 35 Nadi, host: Eggplant.

Figure 3.20: (A, 1-3) LM photographs of M. arenaria perineal patterns showing

variations from typical.

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62
 MALE

Measurements (µm) N= 5

L = 1065.5 ± 6.3; Stylet length = 22.3 ± 0.2; DGO = 4.9 ± 0.2,

Spicule length = 27.6 ± 0.4; Gubernaculum length = 8.0 ± 0.1.

Description

M. arenaria, male body long and vermiform with distinctive lateral lines

(Figure 3.21 A-D). Head cap low sloping posteriorly, head region not set off from the

rest of the body, stylet broad and straight, posterior part of stylet cone wider than the

stylet shaft, stylet shaft also increases in width posteriorly, stylet knobs rounded, DGO

distance relatively long, slightly smaller spicule and gubernaculum length than in M.

incognita males.

A1
21 um

23 um
B A2

15 um

C 17 um D
19 um
Figure 3.21: LM photograph of M. arenaria male (A 1-2) Head region with stylet,
(B) Mid-body region with lateral lines, (C) Posterior end, (D) Tail region with
spicules. Population from farm 21 Sigatoka, host: Tomato.

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63
 JUVENILE

Measurements (µm) N= 10

L = 461.8 ± 1.0; Stylet length = 11.0 ± 0.3; Tail length = 53.9 ± 0.4;

Hyaline tail terminus 9.5 ± 0.3.

Description

M. arenaria juvenile body long and slender, head cap anteriorly flattened but

posteriorly rounded, stylet cone and shaft wide and robust, stylet knobs broad, rounded

and posteriorly sloping, tail relatively long and slender, hyaline tail terminus not

clearly defined with pointed tail tip (Figure 3.22 A, B)..

A B

77 um 12 um

Farm 21 Sigatoka, host: Tomato.

Figure 3.22: LM photograph of M. arenaria (A) Juvenile, (B) Juvenile tail.

64
64
MELOIDOGYNE SP. 1

Hosts: Peanut (field) and tomato (bioassay).

Locality: Nadi.

MOLECULAR CHARACTERIZATION

The population Meloidogyne sp. 1 did not amplify with any of the three species

specific primers. The population did amplify with the mitochondrial primer set

C2F3/Mel 450R producing a band of size of 400bp (Figure 3.23). The DNA from this

species failed to amplify with the other species specific primers under standardised

conditions. The species was therefore placed in a separate group since it did not belong

to the three known Meloidogyne species.

Meloidogyne sp 1

Negative control
400 bp

Figure 3.23: Gel photograph showing Meloidogyne sp. 1, 400bp fragments with
primer C2F3/Mel450R

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65
 MORPHOLOGICAL CHARACTERIZATION

FEMALE

Measurements (µm) N= 10

L = 827 ± 7.0; Body width = 493 ± 5.4; a = 1.7; Stylet length 14.2 ± 0.5;

DGO 2.4 ± 0.2; Vulva length = 23.5 ± 0.5; Vulva-anus distance 19 ± 0.7

Description

Meloidogyne sp. 1, females pear shaped, relatively short neck and large body

(Figure 3.24 A). Stylet cone slightly curved, stylet shaft cylindrical, stylet knobs

rounded, backward sloping, and slightly indented. DGO value relatively smaller (2.4

µm) than in M. incognita (3.0 µm), M. javanica (3.9 µm) and M. arenaria (4.8 µm).

Perineal pattern with coarse wavy striations, dorsal striae forms a squarish arch, slight

wing formation on one side, phasmids are present, tail tip area well defined, perivulval

region not striated (Figure 3.24 B 1-3).

The morphological features of Meloidogyne sp. 1 were compared with M. incognita,

M. javanica and M. arenaria (Table 3.6) and four other Meloidogyne spp.

(M. enterolobii Yang and Eisenback, 1983; M. hispanica Hirschmann, 1986;

M. mayaguensis Rammah and Hirschmann, 1988 and M. brasilensis Charchar and

Eisenback, 2002) known to occur in warm and tropical climates (Table 3.9).

M. hispanica had the closest morphological resemblance to Meloidogyne sp. 1 out of

species compared from the literature (Table 3.8). Further molecular and morphological

investigation is required to ascertain the species identity.

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66
 A B1

28 um
223 um

B3
B2
29 um 23 um

Figure 3.24: LM photograph of Meloidogyne sp. 1 (A) Female, (B, 1-3) Perineal
patterns.

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67
 MALE

Specimen was not available in the current investigation.

JUVENILE

Measurements (µm) N= 10

L = 480 ± 7.0; Stylet length = 12.2 ± 0.6; Tail length = 42.6 ± 0.8;

Hyaline tail terminus 4.7 ± 0.3.

Description

Meloidogyne sp. 1, juvenile body moderately long, tapering towards posterior. Head

cap not set off from the body and posteriorly sloping. Stylet cone slightly curved, stylet

shaft cylindrical, stylet knobs rounded and posteriorly sloping. Hyaline tail terminus

relatively small, with rounded tail tip.

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68
MELOIDOGYNE SP. 2

Hosts: Eggplant (field) and tomato (bioassay).

Locality: Tavua.

MOLECULAR CHARACTERIZATION

The population Meloidogyne sp. 2 amplified with the mitochondrial primers

C2F3/Mel450R producing a band of size 400 kb (Figure 3.25) but did not amplify

with the primers C2F3/1108 under standard conditions. When amplified with species

specific primers, it failed to amplify with M. arenaria and M. javanica specific

primers.

No amplification Negative 400 bp amplification

with C2F3/1108 control with C2F3/Mel450R

400 bp

Figure 3.25: Gel photograph showing Meloidogyne sp. 2 PCR results with primers
C2F3/1108 (left half lanes 2-5 from hyper ladder) and C2F3/Mel450R
(lanes 7-10).

Meloidogyne sp. 2 also amplified with the M. incognita specific primers producing a

slightly larger PCR product of size 1.2 kb rather than the 1.0 kb fragment expected

from M. incognita population (Figure 3.26).

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69
 Negative 1.2 kb
control Meloidogyne sp. 2

1.0 kb
1.0 kb
M. incognita

L2 L3 L4 L5

Figure 3.26: Gel photograph showing PCR results with SCAR primers MIF/MIR
for Meloidogyne sp. 2 (L4 from ladder) compared with M. incognita (L3 and L5
from ladder).

MORPHOLOGICAL CHARACTRIZATION

FEMALE

Measurements (μm) N= 10

L = 684 ± 9.0; Body width = 478 ± 7.5; a = 1.4; Stylet length 14.3 ± 0.4;

DGO = 4.4 ± 0.2; Vulva length = 24.9 ± 0.8; Vulva-anus distance 18.8 ± 0.8.

Description

Meloidogyne sp. 2 female body typical pear shape, neck relatively long, neck straight

to bent at an angle to the longitudinal axis of female body (Figure 3.27 A). Perineal

pattern oval shaped, wider in the middle; dorsal arch moderately high, dorsal striations

more rounded rather than squarish, striations coarse, long and wavy, no distinctive

lateral lines. Phasmids present and distinctive, perivulval region oval shaped, not

striated, with sunken vulva and anus (Figure 3.27 B, 1-2).

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70
 The females from Meloidogyne sp. 2 as compared to M. incognita, M. javanica and

M. arenaria females, were smaller in size, had smaller stylet size, slightly larger DGO

distance, similar vulva length and slightly larger vulva-anus distance. The values

recorded for Meloidogyne sp. 2 resembled more closely to the range of values reported

for Meloidogyne floridensis (Table 3.8).

The perineal pattern morphology of Meloidogyne sp. 2 also has similarities to perineal

pattern of M. floridensis which has rounded or ovoid arch, with coarse striae in and

above anal area and smooth wavy lines in the outer field; prevulval region, without

striae; vulva and anus sunken, phasmids large and distinct (Handoo et al., 2004),.

However more detailed morphological and molecular analysis is required to confirm

whether the species is Meloidogyne floridensis.

B1
A B2
17 um 26 um 25 um

Figure 3.27: LM photograph of Meloidogyne sp. 2 (A) Female, (B, 1-2) Perineal

patterns.

MALE

Specimen was not available in the current investigation.

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71
 JUVENILE

Measurements (μm) N=10

L = 350 ± 2.0; Stylet length = 10.0 ± 0.4; Tail length 36.7 ± 0.9;

Hyaline tail terminus = 9.2 ± 0.3.

Description

Meloidogyne sp. 2 juveniles relatively short, anterior slightly tapering, heap cap

sloping posteriorly and slightly set off from body, stylet relatively short, stylet cone

straight and pointed, stylet knobs rounded and gradually sloping,. Posterior tail end

more slender than anterior, hayline tail terminus distinctive, tail tip rounded, slight

indentations around hyaline tail region, (Figure 3.28 A, B).

The Meloidogyne sp. 2 juveniles compared to the juveniles of M. floridensis had

similar features such as small vermiform body, tapering at both extremities but more

so posteriorly; truncate head, slightly offset from the body; short tail, tapering to a

bluntly rounded terminus (Handoo et al., 2004).

A B

62 um 9 um

Figure 3.28: LM photograph of Meloidogyne sp. 2 (A) Juvenile, (B) Juvenile tail.

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72
 MELOIDOGYNE SP. 3

Hosts: Okra (field) and tomato (bioassay)

Locality: Lautoka

MOLECULAR CHARACTERIZATION

The molecular analysis of Meloidogyne sp. 3 using the mitochondrial primer

C2F3/Mel450R produced amplification product of approximately 400 kb

(Figure 3.29). The population however did not amplify with species specific primers

for M. incognita, M. javanica and M. arenaria under standard conditions indicating

that it did not belong to any of the three species.

No amplification Negative 400bp amplification Negative
with C2F3/1108 control with C2F3/Mel450R control

Figure 3.29: Gel photograph showing Meloidogyne sp. 3 PCR results with primers
C2F3/1108 (left half lanes 2-4 from hyper ladder) and C2F3/Mel450R (lanes 7-9).

MORPHOLOGICAL CHARACTERIZATION

FEMALE

Measurements (µm) N= 10

L = 675 ± 8.5; Body width 428 ± 6.8; a = 1.6; Stylet length 15.7 ± 0.7;

DGO = 4.6 ± 0.3; Vulva length = 23.9 ± 0.5; Vulva-anus distance = 21.0 ± 0.3

73
73
Description

Meloidogyne sp. 3, female body globose to pear shape, neck relatively short, without

posterior protuberance. Stylet cone pointed, slightly curved dorsally, stylet knobs

posteriorly sloping. Perineal patterns oval shaped, high squarish to rounded dorsal arch

or no distinct arch, fine striations, smooth to wavy, no distinctive lateral lines,

formation of small lateral wings on one side (Figure 3.30 A, 1-4).

The perineal pattern morphology was distinctive from M. arenaria and M. javanica but

had some resemblance to the perineal pattern observed for M. incognita. The

morphological features of Meloidogyne sp. 3 female most closely resembled to the

features described for M. paranaensis (Table 3.8). M. paranaensis perineal pattern is

slightly different as compared to Meloidogyne sp. 3 and has rectangular to oval shape,

high squarish dorsal arch, fine to coarse dorsal striae varying from smooth to wavy

(Carneiro et al., 1996). Further morphological and molecular investigation is required

to determine Meloidogyne sp. 3, identity.

A1 A2
20 um 21 um

Figure 3.30: LM photograph of Meloidogyne sp. 3, perineal patterns (A, 1-2)

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 A3 A4
21um 22 um

Figure 3.30: LM photograph of Meloidogyne sp. 3, perineal patterns (A, 3-4)

MALE

Specimen was not available in the current investigation.

JUVENILE

Measurements (µm) N= 10

L = 450 ± 4.0; Stylet length = 13.0 ± 0.4; Tail length 49.0 ± 0.6;

Hyaline tail terminus 9.8 ± 0.4.

Description

Meloidogyne sp. 3, juvenile body vermiform, moderately long; head not off set from

body. Stylet slender with a sharp pointed stylet cone, cylindrical stylet shaft and

rounded stylet knobs set off from the shaft. Tail region narrow, distinct hayline tail

terminus, with rounded tail tip (Figure 3.31 A, B).

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Morphological features of Meloidogyne sp. 3, juveniles as compared to M. paranaensis

juveniles (Table 3.8) also had similar stylet and tail morphology. M. paranaensis

juvenile, stylet shaft is cylindrical, stylet cone increases in width gradually, and stylet

knobs set off from shaft; hayline tail terminus distinctive and narrowing tail (Carneiro

et al., 1996).

Based on the current morphological and molecular data for Meloidogyne sp. 3, the

species identity could not be resolved and more detailed morphological and molecular

analysis is required to determine the actual species identity.

A B

67 um 8 um

Figure 3.31: LM photograph of Meloidogyne sp. 3 (A) Juvenile, (B) Tail region.

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Table 3.6: Measurements from juveniles and females of M. arenaria, M. incognita and M. javanica.

Literature Literature Literature

Juvenile M. arenaria N= 10 Values M. incognita N=10 Values M. javanica N=10 Values
L 461.8 ± 1.0 503.6 ± 4.26 a 359.3 ± 3.0 534.5 ± 6.4
(456.5-467.4) (392-605)a (350.1-383.3) (350-450) b (510.4-570.5) (400-560) b
Stylet length 11 ± 0.3 11.1 ± 0.03a 10.6 ± 0.2 11.5 ± 0.2
(10-12.3) (10-12)a (9.7-11.5) (10-12)d (10-12.7) (10-12)d
Tail length 53.9 ± 0.4 56.0 ± 0.53a 48.8 ± 0.7 55.6 ± 0.7
(52-55.4) (44-69)a (45.6-51.2) (43-65)b (52.7-59.2) (47-60)b
Hayline tail terminus 9.5 ± 0.3 10.7 ± 0.3 13.6 ± 0.3
(8-10.7) (6-15)b (9.6-12.2) (6-14)b (11.9-14.6) (9-18)b
Female N= 10 N=10 N= 10
c
L 782 ± 6.6 741 ± 115 719.9 ± 8.1 727.5 ± 8.2 738.4 ± 8.07f
e
(734.5-801.7) (601-985)c (687.4-755) (600-1100) (688.9-757.7) (510.3-1012.5) f
Body width 602.8 ± 6.2 448 ± 89c 439.9 ± 8.3 514.5 ± 9.6 513.3 ± 6.37 f
(573.9-632.4) (334-626)c (396.1-473.5) (270-870)e (484.2-574.8) (356.4 -729.0) f
a 1.3 1.7 1.6 1.4 1.5 ± 0.02 f
(1.3-1.4) (1.5-1.8) (1.5-1.7) (1.2-2.2) (1.3-1.5) (1.0-2.1) f
Stylet length 15.2 ± 0.3 15.1 ± 0.05a 14.8 ± 0.4 16 (15-17)b 16.4 ± 0.1 16.1 ± 0.07 f
(14.3-17.1) (13-17)a (12.1-16.2) (15.8-17.1) (13.8-17.9) f
DGO 4.2 ± 0.1 4.8 ± 0.06a 3.5 ± 0.1 3.0d 3.4 ± 0.1 3.9 ± 0.05 f
(3.8-4.8) (3-7)a (2.8-4.3) (2-4)d (3-3.9) (1.9-5.0) f
Vulva length 24.4 ± 0.4 29.3 ± 4.1c 25.8 ± 0.7 n/a 23.8 ± 0.2 23.9 ± 0.33 f
(22.7-26.4) (24-37)c (21.4-28.3) (23-24.6) (16.7-59.2) f
Vulva-anus distance 16.9 ± 0.2 20.7 ± 2.6c 17.9 ± 0.3 n/a 16.7 ± 0.2 16.4 ± 0.20 f
(15.8-17.9) (18-21)c (16.4-19.3) (16-17.4) (4.1-23.3) f

All values are stated in µm and in the form mean ± se, (range) for Tables 3.6, 3.7 and 3.8.

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Table 3.7: Measurements from males of M. arenaria and M. incognita.

Literature Literature Literature

Male M. arenaria N = 5 Values M. incognita N = 5 Values M. javanica N = 0 Values
L 1065.5 ± 6.3 1720 ± 287a 1749.5 ± 18.9 (1000-2000)e Specimen not available 1347.1 ± 17.01 f
(1045.3-1079.2) (979-2279)a (1695.3-1795.2) (826.2-2008.8) f
Stylet length 22.3 ± 0.2 23.1 ± 1.46a 24.1 ± 0.3 24.5b 21.4 ± 0.09 f
(21.4-22.7) (22-28)a (23.3-24.8) (23-26)b (18.7-23.7) f
DGO 4.9 ± 0.2 6±1a 2.9 ± 0.1 3.0 ± 0.05 f
(4.4-5.3) (4-8)a (2.7-3.1) (2-4)b (1.1-4.2) f
Spicule length 27.6 ± 0.2 32 ± 2a 28.5 ± 0.4 29.5 ± 0.21 f
(27-28.3) (27-39)a (27.4-29.5) (25-33)e (22.9-35.3) f
Gubernaculum length 8.0 ± 0.1 9 ± 0.8a 9.3 ± 0.2 8.0 ± 0.06 f
(7.7-8.4) (7-10)a (8.7-9.8) (7-11)e (5.7-10.1) f

a
Values from (Cliff and Hirschmann, 1985).
b
Values from (Karssen and Moens 2006).
c
Values from (Jepson, 1987 cited in Skantar et al., 2008).
d
Values from (Eisenback and Triantaphyllou,1991).
e
Values from (Siddiqi 2000).
f
Values from (Rammah and Hirschmann, 1990).

Average, standard error and range values indicated in this table were calculated using SigmaPlot version 11.

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Table 3.8: Measurements from juveniles and females of Meloidogyne sp. 1, 2 and 3 compared with closest sp. from literature.

Meloidogyne M. hispanicaa Meloidogyne M. floridensisb Meloidogyne M. paranaensisc

Juvenile sp. 1 (N= 10) (Closest to sp.1) sp. 2 (N=10) (Closest to sp.2) sp. 3 (N=10) (Closest to sp.3)
L 480 ± 7.0 um 392 350 ± 2.0 um 355 450 ± 4.0 um 458 ± 5.1
(358.2-493) (356.0-441.4) (315.2-375.0) (310-390) (370.8-498.3) (389-513)
Stylet length 12.2 ± 0.6 11.1 10.0 ± 0.4 10.1 13.0 ± 0.4 13.5 ± 0.2
(11.6-12.7) (10.4-11.9) (9.1-11.3) (10-11) (11-14.1) (13-14)
Tail length 42.6 ± 0.8 46.4 36.7 ± 0.9 39.4 49.0 ± 0.6 49.0 ± 0.15
(40.1-44.8) (41.1-53.4) (34.1-43) (35-42.5) (46.2-50.8) (48-51)
Hayline tail terminus 4.7 ± 0.3 Not reported 9.2 ± 0.3 9.7 9.8 ± 0.4 10.1 ± 0.1
(4.2-6.1) Not reported (8.5-12.7) (8-12) (9.3-11.1) (9-10)
Female
L 827 ± 7.0 830 684 ± 9.0 697 675 ± 8.5 681 ± 12.5
(697.5-892.1) (570-1180) (530.4-810) (525-890) (542-750.3) (512-780)
Body width 493 ± 5.4 503 478 ± 7.5 491 428 ± 6.8 428 ± 11.7
(475.8-513.9) (330-740) (355-635.8) (356-648) (319-510.3) (320-532)
a 1.7 1.7 1.4 1.4 1.6 1.6 ± 0.1
(1.4-1.9) (0.9-2.2) (1.2-1.7) (1.2-1.7) (1.3-1.8) (1.1-2.1)
Stylet length 14.2 ± 0.5 14.1 14.3 ± 0.4 14.7 15.7 ± 0.7 16.1 ± 0.1
(11.6-14.7) (13.6-14.6) (13.7-15.8) (13-16) (15.2-17.5) (15.0-17.5)
DGO 2.4 ± 0.2 3.2 4.4 ± 0.2 4.6 4.6 ± 0.3 5.0 ± 0.1
(2.1-3.5) (2,8-4.0) (3.7-5.2) (3.5-6) (4-5.3) (4.2-5.5)
Vulva length 23.5 ± 0.5 23.5 24.9 ± 0.8 26 23.9 ± 0.5 25.9 ± 0.6
(20.1-25) (20.0-25.4) (20.5-26.2) (21-30) (21.3-25) (20-37)
Vulva-anus distance 19 ± 0.7 19.0 18.8 ± 0.8 19 21.0 ± 0.3 20.8 ± 0.5
(17-21.5) (17.2-22.6) (15.2-22.8) (15-25) (16.5-22) (15-25)
Male Not found Reported in Not found Reported in Not found Reported in literature
literature literature
a
Values from Hirschmann, 1985. b Values from Handoo et al., 2004. c Values from Carneiro et al., 1996.

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79
Table 3.9: Literature values from M. enterolobii, M. mayaguensis, and M. brasilensis for comparison.

Juvenile M. enterolobii a M. mayaguensisb M. brasilensisc

L 436.6 453.6 434 ± 34.5
(405.0-472.9) (390.4-528.0) (322-473)
Stylet length 11.7 11.6 11.0 ± 0.7
(10.8-13.0) (11.1-12.2) (9.7-12.2)
Tail length 56.4 54.4 53 ± 3.9
(41.5-63.4) (49.2-62.9) (43-58)
Hayline tail terminus Not reported Not reported 13.1 ± 1.6
(10-16)
Female
L 735.0 651.2 763 ± 94
(541.3-926.3) (518.4-769.5) (601-959)
Body width 606.8 501.0 508 ± 77
(375.7-809.7) (413.1-599.4) (387-728)
a 1.25 1.3 1.5 ± 0.1
(0.97-1.94) (1.1-1.6) (1.3-1.6)
Stylet length 15.1 15.8 14.3 ± 0.9
(13.2-18.0) (13.8-16.8) (12.6-16.4)
DGO 4.9 4.8 4.0 ± 0.6
(3.7-6.2) (3.5-6.7) (2.9-5.0)
Vulva length 28.7 26.1 23.9 ± 1.8
(25.3-32.4) (20.9-30.4) (19.3-27.1)
Vulva-anus distance 22.2 18.4 19.9 ± 1.9
(19.7-26.6) (12.7-21.1) (15.5-23.2)
Male Reoprted in literature Reported in literature Reported in literature

a
Values from Yang and Eisenback, 1983; b Values from Rammah and Hirschmann, 1988; c Values from Charchar and Eisenback, 2002.

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 CHAPTER 4

DISCUSSION

4.1 ROOT-KNOT NEMATODE DIVERSITY AND DISTRIBUTION ON VITI

LEVU, FIJI.

Meloidogyne spp. are one of the most economically important agricultural pests,

distributed worldwide (Sasser, 1977; Sasser and Carter, 1985; Abad et al., 2008). In

Fiji, root-knot nematodes are widespread in agricultural areas. The incidence of root-

knot nematodes from this investigation was determined to be 41% after a thorough and

systematic survey of 675 farms from Viti Levu, Fiji. This is the highest incidence of

root-knot nematodes recorded so far from Fiji when compared to previous reports on

root-knot nematode incidence of 31% (N = 700), (Orton Williams, 1980) and 28.7 %

(N = 185), (Khurma et al., 2008) from Fiji. In addition to the three Meloidogyne spp.

(M. arenaria, M. incognita and M. javanica), known to be present in Fiji (Orton

Williams, 1980; Khurma et al., 2008), three unidentified Meloidogyne spp. were also

recorded.

The wide range of crop (N =33 species) and weed hosts (N= 45 species) of root-knot

nematodes and the tropical weather conditions favour the widespread distribution of

root-knot nematodes. A combination of factors is likely to have contributed towards

the change in distribution of Meloidogyne spp. in Fiji since the initial survey by Orton

William. Over a period of time, movement of plant materials from root-knot infested

nurseries or farms and sharing of farming implements could have led to spread of

Meloidogyne spp. to new areas. Natural causes such as flooding and soil erosion also

assist in the spread of root-knot nematodes as soil and roots are washed over long

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distances to farms located down hill and downstream which are more prone to

receiving infected soil and root debris from infected uphill and upstream farms. The

farms located near the river banks such as the Sigatoka valley where flooding often

occurs, causes soil to be washed from farms across boundaries and could have

contributed to the high incidence of root-knot nematodes.

The sampling and diagnosis methods used in the current survey when compared to the

other two surveys included a more systematic sampling strategy and thorough analyses

using direct examination of plant roots and bioassay of soil samples. In the recent

survey by Khurma et al., (2008), bioassays were not used and the number of samples

was also smaller when compared to the current study. The survey by Orton Williams

(1980) sampled a greater geographical area (9 different islands) and used the extraction

procedure to determine presence of root-knot nematode juveniles in soil samples.

Direct examination of plant roots for the presence of characteristic root galls is a

relatively faster way of detecting root-knot nematodes and can be conducted by

farmers. When sampling, it is important that plants from various points on the farm

should be examined including weeds on the farm and along the boundaries as

nematodes can have varied distribution patterns (Barker 1985). However, examination

of plant roots may not totally reflect the presence or absence of root-knot nematodes

especially when the plants are tolerant to root-knot nematodes or when the population

density is very low. The results from this survey shows that the number of farms found

infected after direct examination (28%) was in fact lower than the actual incidence of

root-knot nematodes (41%) determined after bioassay of soil samples.

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Bioassay method involves the growth of a susceptible plant in composite soil sample

which gives enough time and suitable conditions for the juveniles present and those

hatching from eggs to infect the susceptible plant thus detecting presence of root-knot

nematode even when population density is low. The composite soil sample taken from

various different points on a farm provides better representation of the nematodes

present in the farm soil. Bioassay of soil samples has also been suggested as an

effective method for detecting low population density of root-knot nematodes by

Barker (1985). In addition to its sensitivity, bioassay also allows the development of

the nematode population on a susceptible host and was used to provide sufficient

number of specimens for species identification.

Another advantage of bioassay method which can be highlighted from this study is the

screening of weeds for their susceptibility to root-knot nematode populations. The

weeds germinating in the bioassay pots, allowed detection of 20 potential weed hosts

and 5 weed species as non hosts of root-knot nematodes (Tables 3.3-3.5). The

bioassay method also allowed confirmation of the host status of 25 other weed species

detected through direct examination. The three main advantages of bioassay procedure

used in this investigation include; detection of low population density of root-knot

nematodes; bioassay culture can be used as a source of nematode specimens for

identification studies; and confirmation of weed host and detection of additional weed

hosts and non hosts of root-knot nematodes. Bioassay method however, when

compared to direct examination requires much more time, bench space and resources.

Root-knot nematodes were found to infect a wide range of commonly grown crops and

weeds on farms sampled from Viti Levu, Fiji. The symptoms of root-knot nematode

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infections based on direct examination were variable depending on the levels of

infestation. The aboveground symptoms of infection such as stunted plant growth,

yellowish leaves, wilting on hot days were clearly observed on moderate to heavily

infected plants. However the symptoms caused by root-knot nematodes are very

similar to symptoms caused by nutrient deficiency (Hunt et al., 2005) thus farmers are

likely to think of nutrient deficiency as the cause of the symptoms rather than

nematodes. Plants heavily infected with root-knot nematodes have reduced nutrient

uptake capacity (Karssen and Moens, 2006) and are unable to effectively utilize

fertilisers or amendments added to the soil.

Currently, there are no systematic estimates available on the damage caused by root-

knot nematodes in Fiji but the fact that they were found to infect a wide range of

horticultural crops indicate their potential to cause crop loss. The damage from root-

knot nematodes is aggravated in presence of other microorganisms such as fungi

(species of Fusarium, Rhizoctonia, Phtytopathora, Pythium, Cruvularia etc.) and

bacteria (Pseudomonas, Agrobacterium etc.) which can cause further disease

complexes with Meloidogyne spp. (Siddiqi, 2000).

The presence of 25 good weed hosts of root-knot nematodes in current study is a clear

indication that weeds can act as alternative reservoir hosts of root-knot nematodes,

when left fallow or in-between the rows of crops and even along the boundaries. The

effectiveness of crop rotations and other cultural pest management techniques for

controlling root-knot populations is also limited due to the wide range of crop and

weed hosts of root-knot nematodes thus making root-knot nematode management a

challenging task.

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4.2 IDENTIFICATION OF ROOT-KNOT NEMATODE SPECIES.

Rapid and reliable identification of nematode species is required for a number of

reasons such as utilizing appropriate crop rotations, managing resistance effectively,

developing bio-control strategies, studying virulence, plant-nematode interactions, and

for quarantine purposes (Cenis, 1993; Zijlistra et al., 2000; Baicheva, 2002; Zijlistra

and Van Hoof, 2006, Abad et al., 2007). There has been increased pressure on

nematologists to accurately identify nematode species particularly after restrictions on

the use of broad spectrum chemical nematicides such as methyl bromide (Manzanilla-

lopez et al., 2004).

For this investigation, the root-knot nematode populations were first identified on the

basis of molecular characteristics. The use of species specific SCAR primers allowed

diagnosis of root-knot nematode populations into M. inocgnita, M. javanica, mixed

population of M. incognita and M. javanica, M. arenaria and unidentified

Meloidogyne spp. The diagnosis of the populations into groups based on PCR assays

using SCAR primers was adopted over the use of morphological characterization

because the former took much less time and provided more accuracy. PCR assays

using SCAR primers have also been recommended for routine molecular diagnostic of

root-knot nematodes as the procedure is straightforward, fast and reliable (Zijlistra et

al., 2000; Fourie et al., 2001; Randig et al., 2002; Meng et al., 2004). The molecular

technique using SCAR primers is able to determine species identity irrespective of the

developmental stage (juvenile, male, female or egg mass) and from small amounts of

tissue thus it can be used in nematode diagnostic labs.

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The SCAR-PCR technique was used to successfully detect the existence of mixed

populations of M. javanica and M incognita from 5% of the samples. Both of these

Meloidogyne species have a similar host range hence are able to coexist within the

same population. The SCAR-PCR technique can detect mixed populations even when

the proportion of one of the species is less than 1% (Fourie et al., 2001). The SCAR-

PCR technique however is not suitable for the identification of new species and the

specificity of SCAR primers should be tested on as many relevant species as possible

(Zijlistra, 2000).

The use of molecular methods for Meloidogyne spp. identification does not require a

great deal of nematological skills as compared to species identifications based mainly

on morphology. This is because molecular techniques rely on analyzing the genetic

information which is less influenced by environmental factors or the nematode’s

developmental stage (Hooper et al., 2005; Hyman, 1990) while Meloidogyne

morphological features exhibit variability, which makes species identification of root-

knot nematode based on morphology alone difficult and time consuming (Hooper et

al., 2005). The presence of physiological variability (good and poor weed hosts) as

observed for the Meloidogyne spp. in this study could be due to the existence of races

within the Meloidogyne spp. but races could not be determined through the SCAR-

PCR assays. More detailed investigation including differential host test, detailed

molecular and morphological analysis is required to distinguish between closely

related species and/or races within the species.

DNA sequencing now allows greater accuracy in distinguishing between closely

related species. In addition, isoenzyme profiles (Esbenshade and Triantaphyllou, 1990;

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Carneiro et al., 2000) can also be used to distinguish between Meloidogyne spp. The

inclusion of sequence information would increase the accuracy of species diagnosis but

the time required and cost of diagnosis also increases. DNA sequencing has an

additional advantage as the sequencing data can be used to carry out phylogenetic

analysis (Skantar et al., 2008) and is used frequently for new species descriptions and

genomic studies. There are a large number of Meloidogyne gene sequences already in

the Genbank database, and these data provide a phylogenetic framework for

investigating relationships of unknowns to annotated and referenced species.

Morphological characteristics of root-knot nematode female, male and juvenile have

been studied extensively and are still widely used to support species identifications.

Morphological features of root-knot nematodes are still valuable for species

identification as they can be used to distinguish between species and verify diagnosis

made on the basis of molecular information. Morphological descriptions are also

required when describing new species. Although relatively inexpensive, morphological

examinations require considerably more time and skill to determine species identity.

The accuracy of morphological examination depends on the number of morphological

characteristics studied and number of specimens as well. Some of the species are more

difficult to characterize based on morphological characteristics alone as there might be

differences in only a few morphological characteristics. The use of Scanning Electron

Microscopy (SEM) has helped a great deal in resolving subtle morphological

differences.

The perineal pattern is one of the most characteristic morphological features of the

genus Meloidogyne (Karssen and van Aelst, 2000) which has been widely used for

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species descriptions and is useful for distinguishing between some of the Meloidogyne

species. However, some of Meloidogyne species such as M. mayaguensis (Brito et al.,

2004); M. morocciensis (Rammah and Hirschmann, 1990); M. floridensis (Handoo et

al., 2004); M. hispanica (Hirschmann, 1986) have overlapping values for

morphological characters with commonly found species such as M. incognita,

M. arenaria and M. javanica hence morphology alone is also not sufficient to

accurately determine species identity. An integrated approach including examination

of molecular, morphological and physiological characteristics is therefore required to

determine the Meloidogyne species diversity.

For instance, Meloidogyne spp. 1 and 3 populations in the present study did not

amplify with species specific SCAR primers but did amplify with Meloidogyne

mitochondrial primers. These species were also found to be morphologically

distinctive from the three common species. Meanwhile Meloidogyne sp. 2 amplified

with the SCAR primers for M. incognita but had a slightly larger amplification product

than the amplification product expected for M. incognita. This population upon

morphological examination was found to differ from M. incognita and had closer

morphological resemblance to M. floridensis which also has morphological features

close to those of M. incognita (Handoo et al., 2004). The three unidentified

Meloidogyne spp. 1, 2 and 3 were also morphologically distinct from each other even

though they had the same size bands from the mitochondrial DNA fragment.

The M. incognita, M. arenaria and M. javanica populations had intraspecific variations

in their perineal pattern morphology but the measurements of other morphological

parameters such as the juvenile body length, tail length, stylet length and length of

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hayline tail terminus along with the values measured from the female such as the stylet

length and DGO distance were useful in verifying the species identity. The

measurements of the morphological features from the female, male and juvenile

specimens (Table 3.6) were distinctive enough to separate M. arenaria, M. incognita

and M. javanica from each other. In cases where the measurements had overlapping

values for some of the morphological features, other supporting data such as the

perineal pattern morphology, stylet morphology and tail morphology were found

useful in supporting the species identifications.

The variable nature of perineal pattern between individuals from the same population

makes it relatively difficult and at times creates confusion while determining the

species identity. The variability of perineal pattern could be sometimes due to mixture

of Meloidogyne spp. coexisting as one population. Mixed populations with distinctive

morphological features are more easily detected through the examination of a large

number of male, female and juvenile specimens. However, mixed populations where

the species are morphologically very similar or exist in different proportions, (i.e. one

species is more dominant than the other) are more difficult to diagnose on the basis of

morphological examination and molecular method as outlined above becomes useful.

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4.3 CURRENT AND FUTURE IMPLICATIONS FOR AGRICULTURE IN

FIJI.

This study can form the basis for further investigation on the development of rapid and

reliable diagnostic method for planning root-knot nematode management strategies,

extension advisory, and quarantine purposes in Fiji. The widespread presence of M.

incognita, M. javanica and M. arenaria in agricultural areas from Fiji is of concern and

if not managed, root-knot nematodes can cause significant loss in the quality and

quantity of common vegetable crop production in Fiji. The presence of other

Meloidogyne species is also of concern and their exact identity, pathogenicity and

damage potential needs to be determined. New species introductions can be avoided by

ensuring strict quarantine regulations while dealing with fresh agricultural produce.

There is also a need to create greater awareness amongst farmers in Fiji about root-

knot problems and nematode management practices so that crop loss and unintentional

spread of root-knot nematodes to new areas can be minimized.

The large number of local crop and weed hosts of root-knot nematodes makes

nematode management a difficult task. For instance the Sigatoka Valley has the most

intensive vegetable farming and majority of the farmers use pesticides to control pests

but the area still has the highest incidence of root-knot nematodes. In Fiji, the use of

integrated pest management strategies such as use of crop rotations in combination

with soil solarization, pesticides, weedicides and proper sanitation of farm equipment

seem to be the most practical approaches to nematode management. There is no single

method that can be completely effective (Barker 1985; Sikora and Fernandez 2005;

Karssen and Moens 2006) in managing root-knot nematodes.

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Nematode specific control measures are hardly used in Fiji except on commercial

ginger farming where hot water treatment, soil fumigation and soil solarization is used

(Gowen et al., 2005). The subsistence farmers in Fiji mostly rely on cultural pest

management techniques such as crop rotations, fallow, soil solarization, intercropping

and integrated pest management techniques. The effectiveness of non chemical pest

management techniques such as use of plant resistance, crop rotations, biological

control and other integrated pest management techniques can be improved if they are

carefully planned after considering the parasitizing species (Hussey, 1990). The

establishment of nematode advisory service in Fiji is recommended to help farmers to

effectively manage root-knot nematodes.

The damage caused by root-knot nematodes to agricultural crops in Fiji is not well

studied and given their high level of incidence, further studies on damage assessment

and control strategies need to be carried out on this important pest in Fiji.

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 APPENDIX 1

Some weed hosts of root-knot nematodes from Fiji.

Ageratum conyzoides Cassia obtusifolia Coccinia grandis

Digitaria setigera Eleusine indica Impatiens walleriana

Mullugo pentaphylla Peperomia pellucida Vernonia cinerea

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 APPENDIX 2
Meloidogyne incognita statistical analysis of measurements (using sigma plot version 11)

Data Juvenile Female Male

Hayline Vulva-
Tail tail Body Stylet Vulval anus Gubern
L Stylet length terminus L width a length DGO Slit dstance L Stylet DGO Spicule aculum
Mean 359.29 10.55 48.76 10.7 719.92 439.93 1.64018 14.77 3.47 25.75 17.87 1749.48 24.12 2.92 28.46 9.26
Medi
-an 356.35 10.4 49.25 10.65 716.95 443.55 1.62811 15.05 3.4 26.55 18 1749.3 24.1 2.9 28.3 9.3
Std 9.4756 0.5126 25.741 0.4037
Dev 4 2 2.11618 0.79861 8 26.2551 0.08522 1.16909 0.44485 2.24413 0.86673 42.2018 0.72938 0.14832 0.91815 3
Std 2.9964 8.1402 0.1805
Err 6 0.1621 0.6692 0.25254 8 8.30258 0.02695 0.3697 0.14067 0.70966 0.27408 18.8732 0.32619 0.06633 0.41061 5
95% 6.7786 0.3667 0.5012
conf 1 1 1.51386 0.5713 18.415 18.7822 0.06096 0.83634 0.31823 1.60539 0.62004 52.3992 0.90563 0.18416 1.14001 9
99% 9.7388 0.5268 0.8310
conf 9 6 2.17497 0.8208 26.457 26.9845 0.08759 1.20157 0.45721 2.30647 0.89081 86.8714 1.50142 0.30532 1.88999 7
Size 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5
Total 3592.9 105.5 487.6 107 7199.2 4399.3 16.4018 147.7 34.7 257.5 178.7 8747.4 120.6 14.6 142.3 46.3
Min 350.1 9.7 45.6 9.6 687.4 396.1 1.53266 12.1 2.8 21.4 16.4 1695.3 23.3 2.7 27.4 8.7
Max 383.3 11.5 51.2 12.2 755 473.5 1.75259 16.2 4.3 28.3 19.3 1795.2 24.9 3.1 29.5 9.8
Min
Poss 350.1 9.7 45.6 9.6 687.4 396.1 1.53266 12.1 2.8 21.4 16.4 1695.3 23.3 2.7 27.4 8.7

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 Meloidogyne arenaria statistical analysis of measurements (using Sigma Plot version 11)

Data Juvenile Female Male

Hayline Vulva-
Tail tail Body Stylet Vulva anus Gubern
L Stylet length terminus L width a length DGO length ditance L Stylet DGO Spicule aculum
Mean 461.87 10.98 53.88 9.48 781.95 602.76 1.29789 15.18 4.2 24.37 16.92 1065.54 22.2 4.88 27.66 8
Medi
-an 461.7 11 53.8 9.55 788.15 601.7 1.29009 14.95 4.1 24.45 16.9 1065.7 22.4 4.9 27.7 8
Std 3.0144 0.8547 20.804 0.2738
Dev 3 9 1.14095 0.94845 3 19.7062 0.03472 0.95778 0.33665 1.22751 0.66633 14.0689 0.51478 0.34928 0.49295 6
Std 0.9532 0.2703 6.5789 0.1224
Err 5 1 0.3608 0.29993 1 6.23164 0.01098 0.30288 0.10646 0.38817 0.21071 6.29179 0.23022 0.1562 0.22045 7
95% 2.1564 0.6114 14.882 0.3400
conf 4 9 0.81621 0.67849 9 14.0973 0.02484 0.68517 0.24083 0.87813 0.47668 17.4684 0.63917 0.43368 0.61206 4
99% 3.0981 0.8785 21.382 0.5637
conf 7 4 1.17265 0.9748 3 20.2537 0.03568 0.98438 0.346 1.26161 0.68485 28.9604 1.05967 0.719 1.01473 4
Size 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5
Total 4618.7 109.8 538.8 94.8 7819.5 6027.6 12.9789 151.8 42 243.7 169.2 5327.7 111 24.4 138.3 40
Min 456.5 10 52 8 734.5 573.9 1.25749 13.8 3.8 22.7 15.8 1045.3 21.4 4.4 27 7.7
Max 467.4 12.3 55.4 10.7 801.7 632.4 1.36679 17.1 4.8 26.4 17.9 1079.2 22.7 5.3 28.3 8.4
Min
Poss 456.5 10 52 8 734.5 573.9 1.25749 13.8 3.8 22.7 15.8 1045.3 21.4 4.4 27 7.7

All the measurements stated in table above are in µm.

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 Meloidogyne javanica statistical analysis of measurements (using Sigma Plot version 11)

Data Juvenile Female Male

tail Hayline Body Body Body Stylet Vulval Anus to Gubern
length stylet length tail length width L/W length DGO Slit Vulva Length Stylet DGO Spicule aculum
Mean 534.54 14.88 55.58 13.61 727.5 514.45 1.41766 16.38 3.42 23.81 16.73
Medi
-an 530.9 14.95 54.8 13.85 740.35 500.55 1.41296 16.3 3.45 23.8 16.65
Std 20.279 0.4939
Dev 2 6 2.18113 0.87108 25.976 30.4752 0.08239 0.44422 0.30478 0.59339 0.499
Std 6.4128
Err 6 0.1562 0.68973 0.27546 8.21434 9.6371 0.02605 0.14048 0.09638 0.18765 0.1578
95% 14.507 0.3533
conf 2 7 1.56032 0.62315 18.5825 21.8011 0.05894 0.31778 0.21803 0.42449 0.35697
99% 20.842 0.5076
conf 6 9 2.24173 0.89528 26.6977 31.3218 0.08468 0.45656 0.31324 0.60987 0.51286
Size 10 10 10 10 10 10 10 10 10 10 10
Total 5345.4 148.8 555.8 136.1 7275 5144.5 14.1766 163.8 34.2 238.1 167.3
Min 510.4 14 52.7 11.9 688.9 484.2 1.28913 15.8 3 23 16
Max 570.5 15.7 59.2 14.6 757.7 574.8 1.52393 17.1 3.9 24.6 17.4
Min
Poss 510.4 14 52.7 11.9 688.9 484.2 1.28913 15.8 3 23 16

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 APPENDIX 3

DNA HyperLadder I used for estimating band sizes of PCR product visualised on
agarose gels.

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