Abelmoschus Esculentus (L.) Moench.) in the Vegetable Growing Areas of the Punjab, Pakistan

STUDIES ON BIOLOGY, DISTRIBUTION AND MANAGEMENT OF Meloidogyne spp. ON OKRA

MUHAMMAD ARSHAD HUSSAIN 05-arid-1183

Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2011 STUDIES ON BIOLOGY, DISTRIBUTION AND MANAGEMENT OF Meloidogyne spp.ON OKRA

Muhammad Arshad Hussain (05–arid-1183)

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy In Plant Pathology

Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2011

In the name of Allah, The Compassionate, The Merciful

DEDICATION

To, my loving mother and my late father To, my wife, for her quiet understanding and patience To, my cute daughter Hareem Zahra and loving son Rumman Arshad

CERTIFICATION

I hereby undertake that this research is an original one and neither part of this thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences.

Student’s Name: Muhammad Arshad Hussain Signature: ______

Registration No. 05 - arid - 1183 Date: ______

Certified that the contents and form of thesis entitled “Studies on Biology,

Distribution and Management of Meloidogyne spp. on Okra” submitted by

“Muhammad Arshad Hussain” have been found satisfactory for the requirement of the degree.

Supervisor: ______(Dr. Tariq Mukhtar)

Member: ______(Prof. Dr. Irfan Ul-Haque)

Member: ______(Prof. Dr. Muhammad Aslam)

Chairperson: ______

Dean: ______

Director, Advanced Studies: ______TABLE OF CONTENTS TITLE PAGE TABLE OF CONTENTS …………………………………………………. i LIST OF TABLES ………………………………………………………… v LIST OF FIGURES ……………………………………………………….. vi LIST OF ACRONYMS ……………………………………………………. viii ACKNOWLEDGEMENT ………………………………………………… ix ABSTRACT ………………………………………………………………... 1 1 GENERAL INTRODUCTION …………………………………………… 3 2 REVIEW OF LITERATURE …………………………………………….. 6 2.1 HISTORICAL BACKGROUND …………………………………… 6 2.2 IDENTIFICATION …………………………………………………. 7 2.3 SCANNING ELECTRON MICROSCOPY ………………………... 8 2.4 DIFFERENTIAL HOST TEST …………………………………….. 8 2.5 GALL FORM ………………………………………………………. 8 2.6 ISOZYME PHENOTYPING ………………………………………. 9 2.7 MOLECULAR DIAGNOSTICS …………………………………… 9 2.8 BIOLOGY ………………………………………………………….. 11 2.9 DISEASE CYCLE OF ROOT KNOT NEMATODE ……………… 13

2.10 DISTRIBUTION …………………………………………………… 14 2.11 DAMAGE ASSESSMENT ………………………………………… 17 2.12 PLANT RESISTANCE …………………………………………….. 20 2.13 MANAGEMENT …………………………………………………… 24 2.13.1 Organic soil amendments ………………………………… 24 2.13.2 Biological control ………………………………………..... 29 2.13.2.1 Pasteuria penetrans …………………………… 29 2.13.2.2 Pochonia chlamydosporia (= Verticillium chlamydosporium ) ……………………………. 33

2.13.2.3 Paecilomyces lilacinus ………………………... 35 2.13.2.4 Trichoderma …………………………………... 37 3 GENERAL MATERIALS AND METHODS ……………………………. 40

i 3.1 i. EXPERIMENTAL SITE 40

3.2 MULTIPLICATION OF M. INCOGNITA …………………………. 40 3.3 EXTRACTION OF EGGS AND JUVENILES …………………….. 40 3.4 CONCENTRATION OF JUVENILES ……………………………... 41 3.5 STANDARDIZATION OF NEMATODES ………………………... 41 3.6 COLLECTION OF OKRA GERMPLASM ………………………... 41 3.7 COLLECTION OF PLANT MATERIAL FOR SOIL 42 AMENDMENTS …………………………………………………… 3.8 INOCULATION OF ROOT-KNOT NEMATODES ………………. 42 3.9 SOURCES OF ANTAGONISTS USED IN EXPERIMENTS …….. 42 3.10 MASS PRODUCTION OF PASTERIA PENETRANS ……………… 43 3.11 PRODUCTION OF INOCULUM OF POCHONIA 43 CHLAMYDOSPORIA ………………………………………………. 3.12 MASS MULTIPLICATION OF P. LILACINUS AND T. 44 HARZIANUM ………………………………………………………. 3.13 MEDIA USED ……………………………………………………… 45 3.15.1 Semi-selective medium for P. chlamydosporia …………... 45 3.15.2 Potato Dextrose Agar (PDA) ...... 45 3.14 THE SOIL USED FOR POT EXPERIMENTS …………………….. 46 3.15 DATA COLLECTION ……………………………………………… 47 3.16. EXPERIMENTAL DESIGNS AND STATISTICAL ANALYSES .. 48 4 DISTRIBUTION AND INFESTATION OF ROOT-KNOT 49 NEMATODES INFESTING OKRA (ABELMOSCHUS ESCULENTUS (L.) MOENCH.) IN THE VEGETABLE GROWING AREAS OF THE PUNJAB, PAKISTAN

4.1 INTRODUCTION …………………………………………………. 49 4.2 MATERIALS AND METHODS …………………………………. 50 4.2.1 PREVALENCE, INCIDENCE AND SEVERITY OF 50 ROOT-KNOT NEMATODES ……………………………. 4.2.2 IDENTIFICATION OF MELOIDOGYNE SPECIES …….. 51

ii 4.3 RESULTS ………………………………………………………….. 57 4.3.1 PREVALENCE OF ROOT-KNOT NEMATODES …….. 57 4.3.2 INCIDENCE OF ROOT-KNOT NEMATODES ………… 57 4.3.3 SEVERITY OF ROOT-KNOT NEMATODES …………. 58 4.3.4 OCCURRENCE OF ROOT-KNOT NEMATODES 58 4.4 DISCUSSISPECIESON ……………………………………………………… 65 5 EVALUATION OF DIFFERENT OKRA CULTIVARS FOR THEIR 68 RESISTANCE AND SUSCEPTIBILITY AGAINST ROOT-KNOT NEMATODE M. INCOGNITA 5.1 INTRODUCTION …………………………………………………. 68 5.2 MATERIAL AND METHODS …………………………………… 69 5.3 RESULTS ………………………………………………………….. 71 5.4 DISCUSSION ……………………………………………………… 77 6 ASSESSMENT OF THE DAMAGE CAUSED BY M. INCOGNITA ON 80 OKRA AT DIFFERENT INOCULUM LEVELS. 6.1 INTRODUCTION …………………………………………………. 80 6.2 MATERIAL AND METHODS …………………………………… 81 6.3 RESULTS ………………………………………………………….. 82 6.4 DISCUSSION ……………………………………………………… 88 7 EFFECT OF BIOCONTROL AGENTS ON M. INCOGNITA 90 INFECTING OKRA 7.1 INTRODUCTION …………………………………………………. 90 7.2 MATERIAL AND METHODS …………………………………… 91 7.3 RESULTS ………………………………………………………….. 92 7.3.1 SHOOT AND ROOT WEIGHT ………………………….. 92 7.3.2 SHOOT AND ROOT LENGTH ………………………….. 93 7.3.3 NEMATODE INFESTATION …………………………… 94 7.4 DISCUSSION ……………………………………………………… 103 8 EFFICACY OF ORGANIC AMENDMENTS IN 105 CONTROLLING M. INCOGNITA ON OKRA 8.1 INTRODUCTION …………………………………………………. 105

iii 8.2 MATERIAL AND METHODS …………………………………… 106 8.3 RESULTS …………………………………………………………... 107 8.3.1 ROOT AND SHOOT LENGTH ………………………….. 107 8.3.2 SHOOT AND ROOT WEIGHT ………………………….. 108 8.3.3 NUMBER OF GALLS, EGG MASSES AND 108 REPRODUCTION FACTORF …………………………… 8.4 DISCUSSION ……………………………………………………… 114 9 GENERAL DISCUSSION ………………………………………………… 116 CONCLUSIONS …………………………………………………………… 128 LITERATURE CITED ……………………………………………………. 129 APPENDICES ……………………………………………………………... 190

iv Table LIST OF TABLES Page No. 3.1 Physico-chemical characteristics of experimental soil……………….…. 46 4.1 The districts and localities surveyed for root-knot nematodes ……….… 53

4.2 Prevalence, incidence and galling index (severity) of root-knot 60 nematode (Meloidogyne species) on okra in different districts of Punjab. 4.3 Distribution of Meloidogyne species associated with okra in different 64 districts of Punjab ………………………………………………………

5.1 Response of different okra cultivars against M. incognita……………... 77

7.1 Effect of biocontrol agents on increase in fresh shoot weight at various 95 concentrations…………………………………………………………… 7.2 Effect of biocontrol agents on increase in dry shoot weight at various 96 concentrations……………………………………………………………. 7.3 Effect of biocontrol agents on decrease in root weight at various 97 concentrations…………………………………………………………… 7.4 Effect of biocontrol agents on increase in shoot length at various 98 concentrations……………………………………………………………. 7.5 Effect of biocontrol agents on increase in root length at various 99 concentrations……………………………………………………………. 7.6 Effect of biocontrol agents on decrease in root galls at various 100 concentrations…………………………………………………………… 7.7 Effect of biocontrol agents on decrease in egg masses at various 101 concentrations…………………………………………………………… 7.8 Effect of biocontrol agents on decrease in Rf at various concentrations. 102

v Fig. No. LIST OF FIGURES Page

4.1 Map of Punjab pointing districts ……………………………………….. 54

4.2 Sampling method used for survey ……………………………………… 55 4.3 Root galling rating scheme for evaluation of Meloidogyne infestation 56 (Bridge and Page, 1980)………………………………………………… 4.4 Prevalence, incidence and severity of root-knot nematodes 59 (Meloidogyne species) in different districts of Punjab………………….. 5.1 Effect of M. incognita on shoot weight of different okra cultivars ……. 73 5.2 Effect of M. incognita on dry shoot weight of different okra cultivars .. 73 5.3 Effect of M. incognita on root weight of different okra cultivars ……... 74 5.4 Effect of M. incognita on shoot length of different okra cultivars ……. 74 5.5 Effect of M. incognita on root length of different okra cultivars …….. 75 5.6 Effect of M. incognita on Number of galls on different okra cultivars ... 75 5.7 Effect of M. incognita on Number of egg masses on different okra 76 cultivars ………………………………………………………………… 5.8 Effect of M. incognita on Reproduction factor (Rf) on different okra 76 cultivars ………………………………………………………………… 6.1 Effect of inoculum levels on the reduction (%) of root length of okra…. 84 6.2 Effect of inoculum levels on the reduction (%) of shoot length of okra... 84 6.3 Effect of inoculum levels on the (%) increase in root weight of okra …. 85 6.4 Effect of inoculum levels on the (%) decrease in shoot weight of okra… 85 6.5 Effect of inoculum levels on the (%) decrease in dry shoot weight of 86 okra ……………………………………………………………………. 6.6 Effect of inoculum levels on number of galls caused by M. incognita … 86 6.7 Effect of inoculum levels on the egg masses of M. incognita …………… 87 6.8 Effect of inoculum levels on the Reproduction factor (Rf) of M. 87 incognita ……………………………………………………………….. 8.1 Effect of organic amendments at various concentrations on % increase in root length. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura 110 stromonium) and T.E ( Tagetes erecta) ......

8.2 Effect of organic amendments at various concentrations on % increase 110 in shoot length.

vi A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta) ...... 8.3 Effect of organic amendments at various concentrations on % increase in shoot weight. 111 A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)...... 8.4: Effect of organic amendments at various concentrations on % increase in dry shoot weight. 111 A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E ( Tagetes erecta)...... 8.5 Effect of organic amendments at various concentrations on % reduction in root weight. 112 A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E ( Tagetes erecta)...... 8.6 Effect of organic amendments at various concentrations on % reductions in galls. 112 A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)...... 8.7 Effect of organic amendments at various concentrations on % reductions in egg masses. 113 A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)...... 8.8 Effect of organic amendments at various concentrations on % reductions in reproduction factor (Rf). 113 A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)......

vii LIST OF ACRONYMS

ANOVA Analysis of Variance cfu Colony forming units cv Cultivar oC Centigrade cm Centimeter

DMRT Duncan’s Multiple Range Test et al And others

FAO Food and Agriculture Organization

GOP Government of Pakistan g Grams

J2s Second stage juveniles

Kg Kilogram ml Milliliter

NaOCl Sodium hypochlorite Pf Final nematode population

PDA Potato Dextrose Agar Pi Initial nematode population

RKN Root-knot nematodes Rf Reproduction factor

SOD Superoxide dismutase µm micrometer v/v Volume by volume W/W Weight by weight

viii ACKNOWLEDGEMENTS

I feel pleasure to extend my deep sense of gratitude to my kind Supervisor

Dr. Tariq Mukhtar, Associate Professor, Department of Plant Pathology, PMAS

Arid Agriculture University, Pakistan, for his guidance, suggestions, significant insight and unswerving encouragement throughout the course of study. I have greatly been benefited through his deep knowledge, experience and skillful learning.

I have no words to express thanks to Supervisory Committee Members, Dr.

Prof. Dr. Irfan Ul-Haque and Prof. Dr. Muhammad Aslam for extending their keen interest, sincere guidance, continuous encouragement and sympathetic attitude that enabled me to complete these studies.

With deep sense of honor I wish to extend my sincere thanks to Mr.

Muhammad Zameer Kayani and Dr. Nasir Mukhtar Assistant Professor,

Faculty of Veterinary and Animal Sciences, PMA Arid Agriculture University

Rawalpindi, for their useful suggestions, moral support and kind hospitality during writing this dissertation.

The author feels pleasure to place on record his profound obligations to his friends for their excellent cooperation that enabled me to complete the dessertation.

Sincere thanks are also due for my wife (Jawaria), son (Rumman Arshad “

Rumma”) and daughter (Hareem Zahra “Haaney”) for their continuous cooperation and patience. I pay cordial thanks to my colleagues, my brothers and other family members for their prayers, moral support and encouragement.

(Muhammad Arshad Hussain)

ix ABSTRACT

The survey of 17 districts of the Punjab province of the country revealed

that root-knot nematodes prevailed in 85.25% of okra fields with an average

incidence of 38.89%. Hundred percent prevalence was recorded in Multan, Okara,

Dera Ghazi Khan, Bahawalnagar, Vehari, Rahim Yar Khan and Rawalpindi

districts and a minimum prevalence of 22.4% was found in Lodhran district. The

incidence was above 60% in Bahawalnagar, Rahim Yar Khan, Dera Ghazi Khan

and Vehari and was only 4.44% in Lodhran. The severity of infection of the

nematodes was highest in Bahawalnagar and Vehari, while it was lowest in

Lodhran. Of the four most common root-knot species, M. incognita contributed

74.74%, M. javanica 24.02%, M. arenaria 2% and M. hapla 0.78%.

Of the twelve cultivars of okra screened for resistance against M. incognita,

none was found tolerant, highly resistant or moderately resistant. Two cultivars

viz. Selection-31 and Okra Sindha were susceptible and the cultivar Punjab

Selection was found highly susceptible. The rest of the cultivars showed moderate

susceptibility towards the nematode. All the cultivars caused reduction in various

growth parameters to varying levels over their respective controls.

When the effect of different inoculum levels of M. incognita was

investigated on the highly susceptible okra cultivar ‘Punjab Selection’, all the

densities of nematode behaved differently. The reduction in growth parameters and

increases in number of galls and egg masses were found directly proportional to

the inoculum level as against, the nematodes build up which was found to be

inversely proportional. 2

All the tested antagonists proved effective in controlling M. incognita and significantly increased the root and shoot lengths and weights and caused reductions in number of galls and egg masses. Pochonia chlamydosporia and

Pasteuria penetrans were found equally effective at a concentration of 8 ¯ 103 chlamydospores / endospores per gram of soil.

Incorporation of leaves of Azadirachta indica, Calotropis procera, Tagetes erecta and Datura stramonium in the soil @ 25, 50 and 75 g / kg of soil controlled

M. incognita to varying degree. A. indica and C. procera caused maximum reductions in number of galls, egg masses and reproduction factor (Rf) of the nematode resulting into an increases in various growth parameters.

Chapter 1

GENERAL INTRODUCTION

Okra (Abelmoschus esculentus (L.) Moench.), also known vernacularly as bhindi, is usually a hot weather crop and mostly grown in sub-tropical, tropical and temperate regions where the areas are relatively warm. The vegetable grows well in hot humid climate with an average temperature of 25 oC and relative humidity

between 65-85%. It is very sensitive to drought, low temperature, frost and water-

logged conditions.

The fresh fruits are utilized as cooked vegetable. Dry seeds contain edible

oil (13-22%) and edible protein (20-24%). The stems and dry fruit shells of okra

are used in the manufacturing of cardboard and paper. Okra has high nutritive

value and a good cheap source of energy, vitamin C, riboflavin, niacin, thiamin,

carotene, iron, calcium and protein (Grubben, 1977).

There are contradictory reports as to its origin. The genus Abelmoschus is considered to be of Asiatic origin, particularly the region now comprising Burma,

India and Pakistan (Zeven and Zukovsky, 1975). It is also thought to be originated

in the African region (Vavilov, 1936). A late nineteenth century record reveals the

wild occurrence of Abelmoschus on the Nile in Sudan (Singh et al., 1975). It is an

important vegetable crop of India and Pakistan and its distinguished cultivars are

commercially grown in many parts of the world for its unique taste and nutritional

value.

The area under okra cultivation in Pakistan is about 14.78 thousand

hectares and total production of 0.112 million tones with average yield of about

7.55 tones/ha. In Pakistan per hectare yield of okra is very low as compared to

Cyprus (17.8), Jordan (17.7), Egypt (14.1) and Barbados (11.1) (Anonymous,

2006). The low yield can be attributed to many biotic and abiotic factors. Among biotic factors, nematodes particularly root knot nematodes are of significant importance. In Pakistan, 23 nematodes belonging to different genera have been reported to be associated with okra (Maqbool and Shahina, 2001) and the root knot nematodes (Meloidogyne spp.), are the most pathogenic among them.

Meloidogyne incognita being ubiquitous in distribution is considered the most important constraint. The nematode has a very wide host range and has been reported to attack more than 2000 cultivated plant species (Agrious, 2005). There are four races of M. incognita known so far. This pathogen reproduces and causes infections more readily at a temperature, ranging between 25 to 30°C preferably in light-textured sandy soils having a pH range of 4.0 to 8.0. Almost all the vegetables in tropical and warm areas of temperate regions are severely attacked by this nematode (Sikora and Fernández, 2005).

As the nematode completes it life cycle at a suitable temperature in a short period of 5-6 weeks which enables them to build up their populations to the maximum resulting into severe galling in the presence of a suitable host. In such situations if proper control measures are not adopted, nematodes cause colossal yield losses and in some cases crop plants die before attaining their physical maturity. (Shurtleff and Averre 2000). It has been estimated that worldwide crop losses due to Meloidogyne and other nematodes an average are about 12.3%

(Sasser, 1989). Losses up to 22 % have been reported in okra. (Lamberti, 1979).

The greater part of the losses is borne by the least affording growers in

undeveloped countries as their losses reached as much as 25% to 50%

(Sasser1989; Taylor and Sasser. 1978). As there is meager information regarding root-knot nematodes on okra in the country, so the studies reported in the dissertation was planned with the following objectives.

1. Determination of prevalence, incidence and intensity of root-knot

nematodes in okra growing areas and identification of major root-knot

nematode species associated with the crop.

2. Evaluation of different okra cultivars for their resistance/susceptibility

against Meloidogyne incognita.

3. Assessment of the damage caused by M. incognita on okra at different

inoculum levels.

4. Management of M. incognita with Biological control agents and Organic

amendments.

Chapter 2

REVIEW OF LITERATURE

2.1. HISTORICAL BACKGROUND

Although root knot nematodes might have existed on the earth since the time immemorial, they were first observed by Berkeley in 1855 when he noticed galls on roots of cucumber plants grown in greenhouse in England and described them as “Vibrios”. For about a century, these nematodes have been described from various countries and named differently by many workers. In Germany, Greef

(1872) named the nematode as Heterodera radicicola and Licopoli (1875) and

Jobert (1878) in Italy called them “small worms” and “cysts” respectively. Cornu

(1897) after observing root galls on Onobrychis sp. (sainfoin), named root-knot nematodes as Anguillula marioni which Muller in 1884, changed to Heterodera

Radicicola. Treub (1885) in Java called these nematodes as Heterodera javanica while Goeldi (1887) described these organisms as Meloidogyne exigua in Brazil.

Neal (1889) called this nematode as Anguillula arenaria, Atkinson (1889) as

Heterodera radicicola, Cobb (1890) as Tylenchus arenaria, Lavergne (1901) as

Anguillula vialae and Kofoid and White (1919) as Oxyuris incognita. Goodey

(1932) assigned the name of Heterodera marioni to root-knot nematodes.

Chitwood (1949) reestablished the genus Meloidogyne proposed by Goeldi in

1887. He retained four species viz. javanica, arenaria, exigua and incognita and described a new species M. hapla and a variety M. incognita acrita from the United

States. Chitwood in 1952 also described a sub species M. arenaria thamesi. A year later M. brevicauda was described by Loos (1953) from Sri Lanka. There were

only six species of Meloidogyne by 1953. In the subsequent years new species of

Meloidogyne have been discovered and their number kept on increasing. To date,

more than 100 Meloidogyne species have been described (Karssen and Moens,

2006). Of all the root knot nematode species, four viz. Meloidogyne incognita, M.

javanica, M. hapla and M. arenaria have been frequently found in various

agricultural crops (Sasser, 1980). M. incognita and M. javanica among

Meloidogyne spp., are economically important, attacking several vegetables and

field crops in Pakistan, in sandy and warm soils which favors nematode infestation,

especially in irrigated areas where susceptible crops are grown continuously

(Anwar et al., 2006).

2.2. IDENTIFICATION

The accurate identification of root-knot nematode species and races is essential for their management and is a prerequisite for a purposeful research study.

Morphology of the perineal pattern, located in the posterior body region of

adult females is the most frequently used character for identification of

Meloidogyne species. The area comprises the vulva–anus area (perineum), tail

terminus, phasmids, lateral lines and surrounding cuticular striae. Preparation of

perineal patterns for observation and identification of Meloidogyne species has

been described by Taylor et al. (1955), Eisenback (1985), Franklin (1965b), Sasser and Carter (1982), Hartman and Sasser (1985), Hirschmann (1985), Jepson (1987),

Riggs (1990) and Charchar and Eisenback (2000). A more detailed account on root-knot perineal pattern development was given by Karssen (2002).

2.3. SCANNING ELECTRON MICROSCOPY

Meloidogyne species have also been identified by using scanning electron microscopy by Eisenback (1991) and Charchar and Eisenback (2000).

2.4. DIFFERENTIAL HOST TEST

There are variations in the host range of some Meloidogyne species which have been characterized on the basis of differential host range. Sasser (1954) proposed a simple method for the identification of 4 out of 5 Meloidogyne species recognized by Chitwood (1949) on the basis of responses to a series of differential hosts and the amount of galling induced. This test, known as the ‘North Carolina differential host test, has been used to detect host races within the ‘Chitwood species’ (Sasser and Carter, 1982). The responses of about 1000 populations of the four most common species of Meloidogyne and their races to differential hosts have been summarized in ‘International Meloidogyne project’ (IMP) and details are given by Taylor and Sasser (1978); Eisenback et al, (1981); Sasser and Carter

(1982) and Hartman and Sasser (1985).

2.5. GALL FORM

Species of Meloidogyne form characteristic galls on plant roots and other underground organs. However, stems, leaves or flowers are also parasitized and galls are formed in these tissues in several genera of plants (Lehman, 1985).

Meloidogyne spp. are the most common and best-known nematodes that cause extensive root galls or ‘root-knots’, although a few species do not produce galls at all, e.g. M. sasseri (Handoo et al., 1994). The physical appearance and position of galls on roots can be of some assistance in diagnostics. For example, galls

produced by M. javanica and M. incognita are large and irregular which are at

some distance from the root tip, whereas galls of M. exigua on coffee are small, more or less spherical and located at the root tip. Galls of M. graminicola on rice are elongate and usually located just behind the root tip and affected roots assume a characteristic hook shape. The galls incited by M. hapla are relatively small and irregular.

2.6 ISOZYME PHENOTYPING

Isozyme electrophoretic profiles, often using esterase and malate dehydrogenase, have also been used, for identification of M. incognita and M. javanica (Esbenshade and Triantaphyllou, 1985a, b, 1987; Karssen et al., 1995;

Carneiro and Almeida, 2001; Carneiro et al., 1998, 2000; Hernandez et al., 2004 ;).

2.7 MOLECULAR DIAGNOSTICS

There is no denying that PCR-based methodologies are of ever-increasing importance in species diagnostics and phylogeny within the genus Meloidogyne.

Techniques include RFLP (restriction fragment length polymorphism) profiles of the ITS (internal transcribed spacer) region of rDNA, RAPD (random amplified polymorphic DNA) fragments, 18S rDNA sequences, satellite DNA probes and species-specific primers (De Ley et al. (2002) and Tigano et al. (2005). Carta et al.

(2006) recommended molecular protocols for identification of root-knot nematodes on potato. Mitkowski et al. (2002) identified species and races of root knot nematodes by perineal pattern morphology of adult female, scanning electron microscopy of lip region of juveniles and males. Santos (2002) proposed an improved scanning electron microscopy technique for preparing perineal pattern of

root knot nematodes. Many Meloidogyne species are easily identified on the basis of distinct morphological characters and restricted host ranges. Several species are difficult to identify due to their similarity to other species and poor taxonomic descriptions. M. incognita, M. javanica, M. arenaria, and M. hapla are the four most common root-knot nematode species, comprising 98% of all worldwide populations, (Hussey, 1985). Difficulty in identifying root-knot nematodes may result from morphological variations within and between populations from a same species. Since the reevaluation of Meloidogyne spp. by Chitwood in 1949, female perineal patterns became the dominant diagnostic character of the four most common Meloidogyne species. Morphological features of the perineal patterns of

M. incognita, M. javanica, M. arenaria, and M. mayaguensis are describes as follows: M. incognita. Striae are smooth, wavy, sometimes in a zigzag pattern.

Lateral lines are absent. A squarish, high dorsal arch containing a distinct whorl around the tail terminus is the most conspicuous diagnostic character of this perineal pattern. M. javanica. Striae are smooth and somewhat wavy. The dorsal arch is often low and rounded but may be high and squarish, frequently possessing a whorl in the tail terminus area. Unique to this species are distinct lateral ridges that run across the pattern, fading away around the tail terminus. M. arenaria.

Striae are smooth and slightly wavy, often extended laterally, forming wings on one or both lateral sides of the pattern. Distinctive lateral ridges are absent, but are marked by forked, irregular lateral fields. The dorsal arch is low and indented near the lateral fields, forming rounded shoulders (Eisenback, 1985). However, the identification of root-knot nematode species solely on the basis of the differential host test is unreliable due to the possibility of mixed populations, and should be

used in conjunction with morphological, morphometric, and biochemical

evaluations to determine root-knot nematode species (Hartman and Sasser, 1985).

Dickson et al. (1970) first studied the root-knot nematode protein profile stability and its utilization in the identification of root-knot nematodes. Morphological characters, particularly female perineal patterns, are the useful method for root- knot nematode identification. (Hussey, 1985).

2.8 BIOLOGY

Christie (1959) described the life cycle of root-knot nematode as it is largely indifferent with respect to individual species, host-parasite relationships and physiological characteristics. According to Maggenti (1987) the root-knot nematode eggs are protected within a gelatinous egg mass produced by the female.

Hussey (1985b) revealed that inside the egg, a first-stage root-knot nematode juvenile (J1) molts prior to hatching into a J2 while egg hatching is usually spontaneous and does not correlate with plant-root stimuli or root diffusates. Once hatched, the J2 move through the soil in search of a suitable feeding site (Christie,

1959). According to Hussey (1987) the root penetration by the pathogen involves the mechanical disruption of host tissues. However, cellulose and pectin-dissolving enzymes may also aid in the penetration process. The penetration may occur anywhere in the root system, J2 are often observed aggregating and penetrating behind the root cap, near the meristematic zone. Other penetration sites include cracks and lesions of mature roots and areas of secondary root formation (Lewis,

1987). Once root-knot nematode establishes within the plant tissue, it becomes sedentary endoparasite, and stopped further movement (Christie, 1959). Upon establishment of a suitable feeding site, sexually-undifferentiated J2 begin to

modify the host’s physiology by transforming healthy, undifferentiated cells into

specialized feeding sites referred to as giant cells. Modified cells exhibit nuclear,

nucleolar, and surface hypertrophy, an increase in cytoplasmic density, organelle

hyperplasia, and disappearance of the central vacuoles (Lewis, 1987). Late in the

J2 stage, following feeding initiation and giant cell formation, an increase in J2

width is observed, the third-stage juvenile (J3) of both sexes is emerged by the J2

cuticle surrounding the J3, the loss of the stylet and the median esophageal bulb

valve, and the loss of the tail spike, which becomes rounded. The J3 stage passes in a few hours, at time a third molt giving rise to the fourth-stage juvenile (J4). In this

stage, the median esophageal bulb valve is reformed and the excretory pore opens.

The female rectal glands, uterus and vagina, and male vas deferens differentiate

and enlarge, and the male undergoes metamorphosis, attaining an elongated,

cylindrical shape. At this stage the stylet reappears in both sexes, the perineal

pattern is observed in females, and sperm production is initiated in males prior to

the disappearance of the previously-molted cuticles (Triantaphyllou and

Hirschmann, 1960). As male production occurs in most root-knot nematode

species, M. hapla, which reproduces through facultative meiotic parthenogenesis,

produce relatively more males than M. incognita, M. javanica, and M. arenaria,

which reproduce through obligate mitotic parthenogenesis (Triantaphyllou, 1985).

Regardless of their reproductive behavior, Meloidogyne spp. produces fewer eggs

and more males in response to increasing populations within a root system (Lewis,

1987). Upon maturation, females produce a gelatinous matrix through their rectal

glands, into which they deposit eggs, the gelatinous matrix provides eggs with

physical protection and acts as a barrier to temperature fluctuations and water

evaporation from eggs (Maggenti, 1987; Van Gundy, 1985). The viability and development of Meloidogyne spp. is influenced by various environmental and physical stresses, including temperature, soil texture, moisture, aeration, osmotic potential, and host suitability. Temperature has the greatest influence on egg development and hatching, growth, reproduction, and survival. Optimal temperature for egg development of M. incognita, M. javanica and M. arenaria is

10 to 15 °C, and approximately 9 °C for M. hapla. While, the optimal temperatures for growth and development of juvenile and adult stages of M. incognita, M. javanica, and M. arenaria is 25 to 30 °C (Van Gundy, 1985). It is widely accepted that the activity of Meloidogyne spp. is optimum in sandy loam soils and reproduction is maximum in fine sand, primarily due to the low water holding capacity of such soils (Wallace, 1969; Benson and Barker, 1985).

2.9 DISEASE CYCLE OF ROOT-KNOT NEMATODE

Eggs and juveniles of the nematode survive from one crop season to next on plant debris or in soil, and the adults survive in perennial hosts. In soil, the egg stage is more capable of withstanding adverse conditions than the second stage juvenile. They are spread by surface run-off water, agricultural implements and contaminated soil through animals or man. Eggs hatch, infective J2s emerge out of egg and are attracted to the host roots. Second stage juveniles penetrate the epidermal cells of meristematic root tissue. The Juvenile feeds on surface, their sub-ventral pharyngeal gland is activated, and juveniles penetrate root cortex.

Juveniles enter into stele region, and dorsal pharyngeal gland is stimulated.

Hypertrophy of the cortex is started within 4 days of the penetration. Within fifth day, feeding site is established and the vascular cells near the head of the juvenile

begin to swell. Eight to nine days after inoculation, second stage juvenile becomes swollen and sedentary. Within 10-15 days, second and third moults occur. Giant cells gradually attain full size. Twenty days after inoculation, males leave root.

Giant cells become denser, syncytia around males are disintegrated. After twenty five days of inoculation, egg mass is formed inside female nematode. Giant cells are fully developed. Within 30 days, female lays eggs in a protective covering called gelatinous matrix. Giant cells start disintegration, galls rot inside soil. Eggs hatch and J2s become active whenever susceptible hosts or favorable conditions are available (Dasgupta, 1998).

2.10 DISTRIBUTION

Root knot nematodes are known to occur throughout the world. Their occurrence has been reported from all the continents including Asia, Europe, North and South America and Australia. The four commonly occurring root knot species are M. incognita, M. javanica, M. hapla and M. arenaria and have been reported from almost all the areas of tropical and temperate regions. However, M. hapla is mainly confined to cooler areas. The historical background of these nematodes has been given in section 2.1 of this chapter. Since its recognition, various surveys were conducted worldwide to ascertain its occurrence and incidence on various crops. Dutt and Saha (1973) examined the association of Meloidogyne species with jute in West Bengal and showed that M. incognita was 58%, M. javanica 21% and

M. hapla was 21%. Nono-Womdim et al. (2002) identified root knot nematode species occurring in tomatoes in Tanzania by surveying the country for two years and doing field evaluations for next three years to know resistant lines of tomato.

Meloidogyne hapla, Meloidogyne incognita and Meloidogyne javanica were found

in 1, 19 and 89% of samples out of 87, in tomato, respectively. Choi (1976) conducted a survey of root-knot nematodes and reported 48% infestation of M. hapla, 38% of M .incognita, 8% of M. arenaria and 5.7% of M. javanica from seven provinces of Korea. Ibrahim et al. (1976) found that 79% samples of different crops were positively infected with root-knot nematodes (Meloidogyne spp.) collected from Egypt. Curi and Silveira (1978) reported widespread distribution of M. incognita and M. exigua in coffee plantations from the State of

Sao Paulo in sandy soil. Carneiro and Carneiro (1982) showed that M. incognita was predominant on coffee in Parana State of Brazil. M. exigua was found in restricted areas associated with M. incognita. M. javanica was not found in coffee roots although it was present in inter-crops and weeds. M. coffeicola was absent in this region. Lordello et al. (1984) reported that M. incognita was the most common species (56 %) on cotton in the Brazilian state of Sao Paulo. Cho et al. (1987) investigated the distribution and density of root-knot nematodes in fruit, vegetable and field crops in Korea Republic and reported that M. hapla was dominant in fields in central parts of the Korean peninsula while M. incognita was more prevalent in the southern parts. M. javanica was limited to Jeju Island and southern seaside areas. Gul and Saeed (1987) found that M. javanica was the most prevalent species with respect to geographical distribution and natural host-range, followed by M. incognita, M. arenaria and M. hapla. Egg plant, okra and tomato were hosts of all the four Meloidogyne species. Berney and Bird (1992) in surveys conducted during 1986 and 1988 detected M. hapla from 69.8% of the fields from all the major carrot-growing counties of Michigan. Sorribas and Verdejolucas (1994) found that Meloidogyne spp. occurred in 49% of the tomato sites sampled in Baix

Llobregat County, Barcelona, Spain. Corrales et al. (1999) found Meloidogyne spp. in 90% of soil samples and in 83% of root samples of lulo (Solanum quitoense) crop in 8 counties of the Cauca Valley, Colombia. Carrillo-Fasio et al. (2000) reported that the frequency of M. incognita was 82.5% and those of M. arenaria and M. javanica were 2.5% and 5.0% respectively in Sinaloa, Mexico on tomato, bell pepper, cucumber and egg plant. Wheeler et al. (2000) found M. incognita in

39% and 43% of the fields in 1995 and 1996, respectively in the High Plains of

Texas. Dautova and Gommers (2000) surveyed the occurrence of Meloidogyne in several areas of the Republic of Macedonia. M. incognita (47.9%) and M. javanica

(35.6%) were the predominant species followed by M. arenaria (13.7%). M. hapla

(2.7%) was found sporadically. del Prado-Vera et al. (2001) sampled 47 localities from 18 states of Mexico. A total of 56 populations were obtained from which

60.7% belonged to M. incognita, 21.4% to M. arenaria, 12.5% to M. javanica and

5.3% to M. hapla. Khanzada et al. (2002) conducted a survey of different eggplant fields in order to observe the incidence of root-knot nematodes and recorded the maximum disease incidence in Mission (62%) followed by Bhawal Zanoor (56%) and minimum in Kamil Jamali (26%). Barbosa et al. (2004) reported 70% of the plantations infected with M. exigua from the State of Rio de Janeiro of Brazil.

Olowe (2004) revealed the occurrence of M. incognita, M. javanica and M. arenaria singly or in combination in all the cowpea farms sampled in Nigeria. The overall distribution of M. incognita was 51.8%, M. javanica 44.1% and that of M. arenaria was 4.1%. Bhosle et al. 2004 conducted a survey in the Parbhani district of Maharashtra, India, in the summer season, to determine the prevalence of plant parasitic nematodes in okra fields and found that M. incognita was the most

predominant nematode species associated with okra. Khan et al. (2005) conducted a survey on the occurrence and geographical distribution of the nematode in the province of Punjab and reported that (40.92%) samples were infested by

Meloidogyne spp., maximum infection (67%) was recorded from the Faisalabad district, while the minimum (9%) was observed from the Attock district. Root-knot infection was above 50% in four districts (50-76%) and 40% in 3 districts (40-

48%), above 30% in 14 districts (30-38%), and above 20% in 9 districts (20-29%), while other 4 districts showed 9-15% infection. Out of 47 hosts of Meloidogyne spp., maximum infection was recorded on tomato (81%) followed by okra (78%), egg plant (70%) and cucumber (52%). Anwar et al. (2007) during a survey found

85.10 % occurrence of Meloidogyne species associated with vegetable crops in the

Punjab.

2.11. DAMAGE ASSESSMENT

The pathogenecity tests conducted on various crops revealed that nematode infection causes reduction in various plant growth parameters, formation of conspicuous galls on roots, stunted growth of the plant and chlorotic effect on leaves. The pathogenic effects of different levels of different species of root knot nematodes studied by various researchers are reviewed as followed.

Ogunfowora (1977) observed in an experiment that the yield of 7 tomato cultivars was considerably reduced at all tested inoculum levels of 222, 741, 6666 and 20000 larvae of M. incognita / kg of soil. Das and Sukul (1984) inoculated tomato plants in pots with five levels of M. incognita (250, 500, 750, 1000 and

1250 larvae/pot) and found that the number of root galls were directly proportional

to inoculum density up to 1000 larvae / pot. Mani and Sethi (1984) made

inoculations of M. incognita at 1.0, 2.0, 4.0 and 8.0 larvae / gm of soil and found a

direct relationship between plant growth reduction and inoculum levels; 2

larvae/gm of soil being the damaging threshold level. Chindo and Khan (1988)

evaluated the effect of seven different initial population densities (0, 500, 1000,

2000, 4000, 8000 and 16000 larvae) of M. incognita Race 1 on growth and yield of tomato and observed progressive decline in the growth and yield with increasing nematode population. Wonang and Akueshi (1990) made inoculations with 500,

1000, 2000 and 4000 eggs of M. incognita in pot experiment on tomato seedlings and found that stem height, vitamin C content of tomato fruit and percentage fruit yield were decreased at 3 highest inoculum levels. Govindaiah et al. (1991) inoculated mulberry plants with M. incognita @ 0, 10, 100, 1000 and 10000 second stage larvae/plant in a pot experiment. Plant growth, leaf yield, moisture %, shoot and root weight showed a progressive decrease with increasing inoculum level. Khan and Saxena (1992) reported significant reductions in plant growth when okra plants cv. Red Wonder were inoculated with 0, 10, 100, 1000 or 10000

J2s of M. incognita race 2 in pot trials. Devi and Das (1994) noticed significant reductions in shoot length, fresh and dry weight of shoot and tap root at or above

100 nematodes/plant when carrots plants cv. Early Nentes were inoculated with 0,

10, 100, 1000, 5000 or 10000 M. incognita J2s / pot Sarmah and Sinha (1995) observed a progressive decrease in plant growth of cowpea and rate of reproduction of M. incognita with increased initial inoculum levels (10, 100, 1000 and 10,000 J2 / plant). Mohanty and Das (1996) tested different inoculum levels of

M. incognita (0, 10, 100, 1000, 5000, 10,000 and 20,000 J2s / pot) on tuberose and

found a progressive reduction in plant height and root length with the increased

inoculum levels over the control. Aparajita et al. (1998) studied the pathogenicity of M. incognita on papaya with 5 inoculum levels of 100, 1000, 3000, 5000 and

10,000 juveniles per 3 kg of soil and found a progressive decrease in growth of the plant with the increase in inoculum level. Khan et al. (2004a) studied the pathogenic affect of M. javanica on bitter gourd (Momordica charantia), bottle

gourd (Lagenaria siceraria), red gourd (Cucurbita maxima) and sponge gourd

(Luffa cylindrical) by inoculation of different inoculum levels of second stage

juvenile viz. 250, 500, 1000, 2000, and 8000 J2s / kg of sterilized soil in earthen

pots and found significant reduction in growth of bottle gourd and red gourd at

initial inoculum level of 1000 J2s of M. javanica / kg of soil which was the

damaging threshold level. Similarly, a level of 500 and 2000 J2s / kg of soil was

found damaging threshold in case of sponge gourd and bitter gourd respectively.

Yousuf and El-Nagdi (2004) observed under green house conditions that faba bean

(Vicia faba L.) plants inoculated with 0, 10, 100, 1000 and 10,000 larvae of M.

incognita per pot reduced plant growth significantly, the maximum being at the

highest inoculum levels. Bora and Neog (2006) after inoculation of 2-week-old

seedlings of tea cv. TS-378 with 10, 100, 1000 or 10 000 J2s of M. incognita

observed a reduction in seedling height, root length, and number of lateral roots as

the inoculum level increased. The number of galls, egg masses, and final nematode

population in the soil increased as the inoculum level increased from 10 to 1000

J2s per seedling, and then declined at 10,000 J2s per seedling. Khan et al. (2006)

tested different inoculum levels (0, 250, 500, 1000, 2000, 4000 and 8000 second-

stage juveniles or J2s per plant) of M. javanica on balsam (Ipomea). They found

that a minimum inoculum level of 500 J2s per plant caused significant reduction in plant height and dry shoot weight of plants. The number of galls and nematode population both in soil and roots increased as the initial nematode density increased from 250 to 8000 J2s per plant. The rate of nematode build up declined with an increase in the inoculum level and was greatest at 250 J2s per plant and lowest at 8000 J2s per plant. Senthamarai et al. (2006) studied the pathogenecity of

M. incognita @ 10, 100, 1000, 10000 and 100000 J2s / pot containing 5 kg soil on

Coleus forskohlii and observed significant reductions in root-shoot length and shoot weight. Mishra and Usha (2007) evaluated the effect of different inoculum levels of M. incognita (10, 100, 1000 and 10000 J2s / pot) and found significant reductions in the growth parameters of okra cv. Pusa Sawani when the nematode infestation was high. Nasiruddin and Azam (2005) reported that all inoculum densities of M. incognita @ 0, 500, 1000, 2000 or 4000 J2s / pot) caused reduction in all the growth characteristics of green gram, irrespective of initial inoculum level, with maximum damage being at 4000 juveniles per pot.

2.12. PLANT RESISTANCE

Direct control of nematodes, especially with the application of nematicides is dangerous and pollutes the soil and environment. However, use of resistant cultivars is economical and durable. There is plethora of literature available on the screening of different varieties/cultivars of various crops and vegetables for their resistance against nematodes. It is difficult to encompass all of them and some of it is being reviewed briefly.

Alam et al. (1974) discovered that when tested in the greenhouse, all the

okra varieties were found susceptible to M. incognita. Rao and Singh (1977) tested

34 varieties and selections of A. esculentus for their susceptibility to M. incognita and showed that all were susceptible to the nematode to varying degrees. Mahajan and Sharma (1979) screened numerous okra cultivars and lines for resistance to M. incognita in field studies between 1975 and 1979. Only cv. Abtalia (from Iraq) was found to be slightly susceptible. The remaining lines and cultivars were either susceptible or highly susceptible. Montasser and Ai-Sayed. (1985) evaluated the reaction of twelve okra cultivars to M. javanica under greenhouse conditions and

found that all tested cultivars were susceptible to the nematode infection. Resende

and Ferraz (1987) tested 112 cultivars and 128 introductions of okra against M.

incognita race 3 and M. javanica respectively. None of these cultivars or

introductions showed promise as a source of resistance against these nematodes.

Darekar and Sharma (1990) found that 3 cultivars of okra viz. 92/82-2, 118/82-74

and IC-52314 showed resistance to race-3 of M. incognita while the reaction of the

rest of 142 cultivars was either highly susceptible or susceptible. Sharma and

Trivedi (1990) tested 26 okra cultivars and found all of them susceptible to M.

incognita to varying degrees. Singh et al. (1993) reported that out of 24 okra

varieties/cultivars screened for resistance against M. incognita, KS-381, KS-114

and KSL-380 were resistant. Five cultivars were moderately resistant and the rest

were susceptible or highly susceptible. Hazarika et al. (1995) screened 45 brinjal

cultivars in pot experiments and found none of them resistant to root knot

nematodes. Sharma and Singh (1996) tested 10 okra varieties against M. incognita

and found all of them to be susceptible. Das et al. (1997) screened 16 coriander

(Coriandrum sativum) genotypes for reaction to M. incognita and classed CO2 and

UD21 as resistant, whilst RCr41, UD20, Co1 and CO3 were moderately resistant and 10 other varieties were classified as susceptible. Han and Kim (1997) bioassayed 175 red pepper varieties for selecting resistance to M. hapla. Fifteen native varieties (IT 102794, 104806, 105516, etc. and two imported varieties were proved to be resistant while the varieties such as Hongtap, Kangsan, Hongsil, and

Bookang were moderately resistant to the nematodes. Pinochet et al. (1998) tested

15 accessions and cultivars of banana for their resistance towards Pratylenchus goodeyi, M. incognita and M. javanica and found that most of the tested material was highly susceptible to all the three nematodes. Chavda et al. (1999) observed that of 25 green gram (Vigna radiata) lines, variety IC 10488 was highly resistant to M. incognita but highly susceptible to M. javanica. IC 8955 and IC 11438 showed moderate susceptibility to M. javanica. The remaining lines were either susceptible or highly susceptible to both the species of root-knot nematodes.

Debanand (1999) noticed that out of 25 rice cultivars screened against M. graminicola, only MTC 23/A showed any resistance. Rekha and Gowda (2000) screened 18 okra germplasm and 3 cultivars for their resistance to M. incognita under greenhouse conditions. No cultivar or germplasm was resistant. AROH-10,

HOE-202, VLC-1, AROH-9, VB 9101, IIHR-91, and Arka Anamika were susceptible, whereas the rest were highly susceptible. Martinello et al. (2001) evaluated 22 okra genotypes for resistance to M. incognita race 2 and M. javanica.

No resistance was observed among okra genotypes to infection by M. javanica.

The line, CGO 8180A7, presented the highest resistance level to M. incognita race

2. Fazal et al. (2001) evaluated 34 soybean cultivars for their resistance to M.

incognita and found that 18 were resistant, 5 were moderately resistant, 5 were

susceptible and 7 were highly susceptible to M. incognita. Aparajita et al. (2004) evaluated a total of 282 genotypes of green gram for resistance to M. incognita.

Seventy-four genotypes were found to be susceptible while the rest of the

genotypes were highly susceptible. Pathan et al. (2004) studied the response of

seven tomato cultivars against M. incognita. None of the variety was found

immune to M. incognita. Bibha and Bora (2005) tested 20 jute cultivars for

resistance to M. incognita and found them either susceptible or highly susceptible.

Choudhury et al. (2005) screened 149 cultivars of cowpea for their resistance

against M. incognita and found that 19 were resistant, 42 were moderately resistant, 61 were susceptible and 27 were highly susceptible to the nematode.

Nabanita and Sinha (2005) tested twenty cultivars of okra for their susceptibility / resistance to M. incognita, under screen house conditions and found that IC-8991

and IC-27878 were susceptible and the other cultivars were highly susceptible to

the nematode. Sheela et al. (2006) screened 293 cultivars of okra for resistance to

M. incognita. None of the cultivars / accessions was highly resistant, 3 were

resistant, and 123 were moderately resistant while the rest were susceptible and

highly susceptible. Bansa et al. (2006) tested 300 accessions of cultivated

germplasm of urdbean (Vigna mungo) for resistance to M. javanica under field and

reported that 11 genotypes showed resistant and 42 gave moderately resistant

reaction. Adegbite (2007) found that of the 34 varieties, TGM 344, TGM 1784 and

TGX 1448-2E were the most resistant to M. incognita. Ten varieties exhibited

tolerance; four were hyper-susceptible while the rest were susceptible to root-knot

nematode. Simon (2009) reported that among various rice cultivars tested for

resistance against M. graminicola, 13 were highly resistant, whereas 24 cultivars were found to be resistant. Six cultivars exhibited moderate resistance. Seven cultivars were susceptible and 3 cultivars were highly susceptible.

2.13. MANAGEMENT

2.13.1 Organic soil amendments

The use of organic amendments in the soil has been greatly emphasized as an alternative easy, cheap and satisfactory method of nematode control. The application of organic amendments as a mean of biological control of plant diseases has been demonstrated to cause better plant growth, reduction in inoculum density and capacity and reduction in host susceptibility. The application of organic amendments results in the alteration of biotic environment thus increasing the population of natural enemies in the soil. Various oil-cakes and meals like neem, mahua, castor, peanut, soybean, linseed, mustard, sesamum and safflower have been used as source of organic amendments. Linford (1937) and Linford et al.

(1938) applied chopped pineapple leaves @ 125-500 t/ha to control root knot nematodes. O’Brien and Prentice (1932) earlier suggested that potato cyst nematode infection would be delayed by organic manuring. Lear (1959) noticed a significant decline in the population of three plant parasitic nematodes due to castor bean pomace added to soil. Mankau and Minteer (1962) observed complete control of citrus nematode by the addition of castor paomace in the soil. Efficacy of karanj cake (Pongamia glabra) was reported by Singh (1965b) to reduce root- knot by about 50% at low field dosages and by about 100% at high field dosages.

Singh and Sitaramaiah (1966, 1969) found that oil cakes of margosa, castor,

groundnut, linseed, mustard and mahua were capable of reducing root knot galls when incorporated into infested soil in field plots three weeks before planting okra or tomatoes. They also reported that margosa and groundnut cakes applied at the rate of 1600 lbs per acre gave significant control of the disease. Suatmadji (1969) reported that the marigold significantly suppressed lesion nematodes (Pratylenchus spp.) and root-knot nematodes (Meloidogyne spp.). Singh and Sitaramaiah (1971) reported effective control of M. javanica by dressing of seven oil cakes with or without the addition of sawdust. Srivastava et al. (1971) tested some oil cakes against M. javanica on tomato and brinjal. Neem @ 486 Kg per acre proved most effective. Goswami and Swarup (1971) tested linseed, margosa, groundnut and karanj oil cakes against M. incognita on tomato seedlings. Karanj and groundnut cakes showed considerable decrease in the population of nematodes with improved growth of crop. Gowda (1972) with the application of six oil cakes could reduce nematode population and improve plant growth, the best being neem cake.

Mukhopadhyaya et al. (1972) controlled Heterodera avenae by using mustard and castor cakes with significant improvement in the yields of wheat and barley. Eight locally available oil cakes mixed with soil @ 25 q per ha reduced root galling on okra, mahua and groundnut cake being the most effective (Mammen, 1972).

Mishra (1972) reported the effect of eight cakes (karanj, neem, mahua, mustard, groundnut, cotton, linseed and sesamum) on various nematodes attacking various crops kike wheat, mung bean and tomato. All cakes excepting linseed and cotton cakes were effective in reducing nematode population, neem cake being the best.

Theses cakes were also found to possess residual effect and they increased the plant growth and reduced nematode in the nest crop (Mishra, 1972; Mishra and

Prasad, 1974). Prasad et al. (1972) reported significant reduction in the population

of plant parasitic nematodes with wheat straw + neem seed + NPK and wheat straw

+ ½ NPK. They also reported that the effectiveness of these soil amendments could

be better. Khan et al. (1973), Singh and Sitaramaiha (1973) and Alam et al. (1975)

have observed that margosa cake treatment was the cheapest and most effective for

root-knot control. Sitaramaiha et al. (1978) found salwood (Shorea robusta)

sawdust very effective against nematodes when applied @ 2.5 t / ha. Hussain et al.

(1984) studied the effect of eggplant seedlings with plant extracts, nematicides, oil-

cake extracts and anthelmintic drugs on plant growth and nematode development.

They observed that root-dip treatment of eggplant seedlings with margosa and

marigold leaf extracts, aldicarb, decries, mustard cake and carbofuran reduced root

knot development quite considerably. Masood and Husain (1986) have

demonstrated that the application of oil-cake amendments besides acting as

fertilizer for better plant growth and a good control measure also increase the bio-

chemical resistance in tomato and brinjal against root-knot nematodes. Nandal and

Bhatti (1993) revealed that the application of leaves of Calotropis procera, Datura stramonium and Xanthium strumarium significantly reduced the penetration of root-knot nematodes of brinjal roots and gall formation in green house studies.

Owino and Waudo (1992) found that soil amendment with Datura metel, Ricinus

communis and Galium aparinoides significantly reduced infection by M. incognita

on okra. Ramakrishnan et al. (1997) used Neem (Azadirachta indica) leaves (80

g/pot) and recorded highest reduction in root-knot index and increase in yield of

okra infected with M. incognita. Asawalam and Adesiyan (2001) carried out

greenhouse study to compare the nematicidal potential of Carbofuran (Furadan)

and Azadirachta indica (neem) leaf extract against root knot nematode (M. incognita) on okra and found that nematicidal potential of A. indica and carbofuran, in terms of the reduction of root galls in okra, was similar. The use of

A. indica and carbofuran significantly reduced root galls, and increased mean fruit number and fruit weight. Wang et al. (2002) reported that various plant species such as asparagus, castor bean, marigold, neem, partridge pea, rape seed, sesame and sun hemp are effective in reducing nematode populations. Shah et al. (2003) observed that population of root-knot nematodes was significantly reduced in soil treated with neem oil followed by Furadan plus ammonia. Furadan, ammonia and

NPK fertilizers also suppressed the nematode population as compared with untreated plants of tomato. Zarina et al. (2003) observed that soil amendments with leaf extracts of AK, Datura and Neem significantly reduced root-knot nematodes infection and improved growth of eggplant plant. Bari et al. (2004) showed that incorporation of poultry refuse, mustard oil cake, Dholkalmi, neem leaf powder,

Furadan and Rugby significantly reduced the severity of root-knot nematode and augmented vegetative growth of lady's finger. Khan et al. (2004b) used Datura

(Datura stramonium L.), Ak (Calotropis procera L.), Neem (Azadirachta indica

L.) leaves and sawdust of Shisham (Dalbergia sisso) alone and in combination against M. incognita in pots on tomato and found significant increase in plant height and shoot weight and reduction in number of galls. Wani (2006) reported that soil amendments with oil cakes of castor and neem and leaves of castor,

Persian lilac (bakain) and neem, caused significant reductions in root galling and improved plant growth and chlorophyll content of okra and lentil. Sharma et al.

(2006) studied the efficacy of neem oil, D. stramonium and C. procera leaves,

alone and in combination with kalisena (a commercial formulation of Aspergillus niger) and Trichoderma harzianum against M. incognita in okra and found that C. procera and neem oil alone mitigated root galls by 62% resulting into improvement of plant growth paramenters. Zarina and Ghaffar et al. (2006) reported that the soil amendments with leaf extracts of calotropis, datura and neem significantly reduced root-knot infection caused by M. javanica and improved growth of okra plant as compared with unamended control. Pereira (2008) found that soil amendment with fresh neem (A. indica) leaves was efficient in the control of both Meloidogyne and Fusarium separately as well as in their interaction on okra. Wani (2008) evaluated the effect of soil amendment with dry crop residues of marigold, mustard and rocket-salad at different doses and found significant reductions in the populations of M. incognita and an improvement in plant growth.

Ismail et al. (2009) reported that waste residues from black seed (Nigella sativa L.) and jojoba (Simmondsia chinensis (Link.) Schneider.) when applied @ 1.25, 2.5 and 3.75% significantly reduced soil and root populations of M. incognita, resulting consequently into lower root galling and nematode build-up as compared to untreated plants. All the tested organic amendments caused significant increase in shoot, root, and flowers yield parameters of chamomile. Radwan et al. (2009) found that oil cakes of flax, olive, cotton, sesame and soybean incorporated in the soil @ 5, 10, 15, 20 or 50 g / kg soil, significantly reduced gall formation by M. incognita and number of juveniles in the soil. Sesame cake proved to be the most effective as against olive cake. Gitanjali (2010) determined that incorporation of

Tithonia leaf and stem significantly increased the plant growth and decreased the host infestation by M. incognita. Gomes et al. (2010) observed that the poultry

compost and the cow manure applied homogenously under the canopy of guava

provided the highest suppression of M. mayaguensis and productivity. Ogwulumba

et al. (2010) showed that soil amended with poultry droppings, grass ash and rice

husk ash @ 10 to 20 t / ha were good for optimum growth, performance and

control of M. javanica infecting tomato. Seenivasan (2010) revealed that among all organic amendments, neem cake, reduced M. incognita populations by 31.2% in soil on coleus plants and increased the root tuber yield by 42.4% under glasshouse condition. Wani (2010) found that dry crop residues of marigold (T. erecta), mustard (Brassica juncea) and rocket-salad (Eruca saliva) at different doses brought about significant reduction in root-knot development caused by M. incognita. The plant growth of okra and lentil was highest in plants treated with the higher doses of soil amendments as compared to the lower doses.

2.13.2 Biological control

2.13.2.1 Pasteuria penetrans

Pasteuria penetrans is a very promising biological control agent against root-knot nematodes. The role of P. penetrans in suppressing plant-parasitic nematodes has been tested by many workers on many crops, mostly in greenhouse pot tests. Prasad (1971) was perhaps the first to report that greenhouse tomatoes inoculated with M. incognita had fewer galls on roots when grown in soil containing P. penetrans than in bacterium free soil. Birchfield and Antonopoulos

(1976) reported that P. penetrans can reduce root-knot population by 99% within a short span of only three weeks. Mankau and Prasad (1977) found that P. penetrans reduced mobility of Meloidogyne spp. juveniles in soil and resulted in lower

number of galls. Mankau (1980) found that pot cultures of Meloidogyne

populations infested with Bacillus penetrans approached extinction in 4-5

generations. Stirling (1984) applied a dried root population laden with spores into

field soil at 600 mg/kg soil and found significant reduction in nematode and root-

galling. Page and Bridge (1985) reported almost complete destruction of

glasshouse M. acronea cultures by the bacterium. Brown and Smart (1985)

observed inhibition of tomato root penetration of M. incognita J2 by P. penetrans

in laboratory and greenhouse tests. Channer and Gowen (1988) reported that the

application of P. penetrans endospores to soil resulted in significant control of

root-knot nematodes on tomato. Jaya Raj and Mani (1988) obtained 48.4% and

94.42% reduction in M. javanica multiplication with application rates of 250 and

1100 mg spore powder preparation/kg soil respectively with an increase in tomato growth. Minton and Sayre (1989) observed reductions of tomato root penetration by J2 of M. arenaria, root galling and nematode reproduction using P. penetrans infested soil. Sekhar and Gill (1990) reported that P. penetrans reduced root penetration by M. incognita juveniles, gall formation and nematode multiplication.

Daudi et al. (1990) showed that P. penetrans suppressed galling and egg masses and increased the shoot weight of tomato. Ahmad and Gowen (1991) found 6% reduction in egg masses by applying 90,000 endospores / g of soil in pot treatment.

Davies et al. (1991a) demonstrated that P. penetrans reduced the motility of M. incognita and the number of females in tomato roots. Vargas et al. (1992) showed

that roots of tomato plants infected with M. incognita and P. penetrans had

significantly lower root-knot indices and egg masses than those infected with the

nematode only. Walia et al. (1992) reported that application of P. penetrans

aqueous spore suspension to nematode-infested soil resulted in improved growth of brinjal seedlings and suppressed root galling due to M. javanica to the extent of

36%. Sharma (1992) demonstrated that P. penetrans reduced root galling on chickpea due to M. javanica by 81 % and 58% in two greenhouse tests. Zaki and

Maqbool (1992) found that P. penetrans application into M. javanica infested soil reduced nematode infection and increased length and fresh shoot and root weights of okra. Kasumimoto et al. (1993) showed that incorporation of P. penetrans endospores in soil reduced M. incognita J2 in soil and root-gall indices. Adiko and

Gowen (1994) observed that P. penetrans endospores caused 81% and 97% suppression of root galls and reproduction of M. incognita respectively. Walia and

Dalal (1994) obtained a significant reduction in M. javanica population and an increase of 18-20% in tomato yield after treating the soil with P. pentrans.

Weibelzahl-Fulton et al. (1996) reported that P. penetrans caused reductions in M. incognita and M. javanica infections on tobacco in nematode-suppressive soil in

Florida. Duponnois et al. (1997) found that P. penetrans reduced root galls caused by M. graminicola and increased tomato root biomass. Chen et al. (1997b) reported that numbers of eggs per root system, J2 of M. arenaria per 100 cm3 soil at harvest, root galls and pod galls decreased with increasing P. penetrans infestation levels. Dube (2001) found that application P. penetrans suppressed populations of M. javanica, reduced root galling and increased bean yield by 40%.

Maximiniano et al. (2001) reported that P. penetrans caused reductions in the population density of M. exigua J2s in the soil in a 10 year old coffee plantation.

Sankaranarayanan, et al. (2001) observed in pot experiments that combined application of P. fluorescens and P. penetrans inhibited formation of galls and egg

masses by M. incognita on tomato than individual inoculations. Tateishi and Sano

(2001) examined the suppressive effects of P. penetrans on M. incognita infesting

two sweet potato cultivars by six seasons of consecutive cropping from 1994 to

1997. Application of P. penetrans significantly reduced population densities of the root-knot nematode in the 7th and 8th cropping and increased the marketable yields of fleshy storage root. Mukhtar et al. (2002) found that combined application of P. penetrans and V. chlamydosporium caused 48.74, 67.25 and

38.3% reductions in the number of galls and egg masses and in the population of the M. javanica resulting into 25.50 increases in yield of tomato. Gogoi and Neog

(2003) reported that the application of P. penetrans and nematicides significantly improved the plant growth characteristics of green gram compared with the control and decreased number of galls per plant and nematode population of M. incognita in soil. Anil et al. (2005) proved that P. penetrans controlled M. javanica infesting aubergine as effectively as carbofuran in microplot and field tests. Gomathi et al.

(2006) achieved 73.4% suppression in populations of M. incognita and 77.7% reduction in egg masses by the application of P. penetrans at 6 ¯106 spores / g of soil. Suloiman (2007) investigated the effect of P. penetrans in a greenhouse

experiment over a 3-year period. P. penetrans significantly reduced the densities of

J2s six months after inoculation and at the end of the third year; treatments with

higher rates had the greatest percentage of dead and infected J2s (90% and 100%,

respectively).

2.13.2.2 Pochonia chlamydosporia (=Verticillium

chlamydosporium)

Verticillium chlamydosporium is a wide spread fungus that parasitizes

females and eggs of cyst and root-knot nematodes. The fungus has potential as a

biocontrol agent for both the nematodes when thoroughly incorporated through the

soil. It caused the decline of cereal cyst nematode populations in monocultures of

susceptible crops (Kerry et al., 1982c). Thomas (1982) found that 80-90% of young cysts, eggs, larvae and females of Heterodera schachtii were infected with

V. chlamydosporium at 6 sites in GFR. Infection rates were similar at 0-20 and 30-

40 cm depth. Godoy et al. (1983) reported that application of V. chlamydosporium

resulted in 69% reduction in galls caused by M. arenaria in greenhouse studies.

Morgan-Jones et al. (1983) found that an isolate of V. chlamydosporium prevented

egg hatching of M. arenaria in vitro. Kerry et al. (1984) showed that isolates of V.

chlamydosporium added to soil on ground oat grain reduced the number of H.

avenae on wheat between 26 and 80%. Freire and Bridge (1985) found that V.

chlamydosporium infected about 12.1% eggs in egg masses from roots of black

pepper seedlings inoculated with the fungus. De Leij and Kerry (1991) achieved

significant population reductions of > 80% of M. arenaria after the first nematode

generation with isolate 10 of V. chlamydosporium. Clyde (1992) reported that V.

chlamydosporium reduced nematode populations of H. schachtii significantly in

pot experiments. Crump and Irving (1992) showed that the most effective isolate of

V. chlamydosporium gave 75 and 76% control of first generation eggs of H.

schachtii and Globodera pallida, respectively. De Leij et al. (1992b) found that V.

chlamydosporium in combination with Pasteuria penetrans reduced population

density of M. incognita in pots by 92% after 14 weeks. De Leij et al. (1993a)

obtained 90% control of M. hapla populations on tomato plants in microplot

experiment with V. chlamydosporium. Combining fungus with aldicarb gave 98% control. Mousa et al. (1995) found that inoculation of soil with V. chlamydosporium, colonised on rice medium @ 30 g / kg soil, reduced the number of galls and mature females of M. javanica by 50 and 84.3%, respectively. Zaki and Maqbool (1996) found that V. chlamydosporium significantly reduced gall formation on chickpea caused by M. javanica by 40%. Shahid (1999) showed that integration of V. chlamydosporium with P. penetrans suppressed significantly the

multiplication of M. javanica and ultimately resulted in reduction of egg masses,

root galling and final female population over three crop cycles. Siddiqui et al.

(1999) found that V. chlamydosporium when combined with Pseudomonas

aeruginosa significantly reduced egg mass production and number of juveniles in soil and enhanced plant growth. Gopinatha et al. (2002) used various combinations i.e V. chlamydosporium + carbofuran, marigold + carbofuran, and V. chlamydosporium + marigold against Meloidogyne incognita on tomato and found improved plant growth and lowest galls and egg masses number. Tariq and Ijaz

(2003) reported that different dilutions (0, 20, 40, 60, 80 and 100%) of the culture filtrates of V. chlamydosporium significantly inhibited the eggs hatching of M. javanica. Rao et al. (2003) applied P. chlamydosporia [V. chlamydosporium] to control M. incognita on aubergin and confirmed the efficacy and potential of the bio-agent against the nematode. El-Shanshoury et al. (2005) noticed that the nematophagous fungi P. chlamydosporia (Verticillium chlamydosporium), P. lilacinus and A. dactyloides reduced the density of M. incognita populations by

95.4-98.9% and were comparable to Furadan and Nameless resulting into an

improvement of the root and shoot growth of faba bean. Nyongesa et al. (2007) evaluated the V. chlamydosporium and A. oligospora against M. incognita on the rhizosphere of celery (Apium graveolus L.) and tomato and found that V. chlamydosporium was most virulent as compared to Arthrobotrytis oligospora against the nematodes population on both the crops. Tzortzakakis (2007) reported significant reductions of root galling and juvenile density when P. chlamydosporia applied @ 5,000 and 60,000/ chlamydospores/ g of soil on M. incognita (450 and

1,400 juveniles/plant) on tomato and pepper plants in pots after 14 weeks of inoculations. Dallemole-Giaretta et al. (2008) found that the application of P. chlamydosporia var. chlamydosporia chlamydospores caused 82 % reduction in number of eggs and 50 % in the number of galls per root system caused by M. javanica. Ebadi et al. (2009) reported that Strain 50 of P. chlamydosporia var. chlamydosporia infected 40 % eggs and caused 56% reduction in the population of

M. javanica on pistachio roots, while 15% parasitism of the eggs was observed where isolate 40 of the antagonist was applied causing 36% decreased in the population of the pathogen

2.13.2.3 Paecilomyces lilacinus

Paecilomyces lilacinus is a soil-inhabiting fungus, capable of parasitizing nematode eggs, juveniles and females, hence reducing soil populations of plant parasitic nematodes. Walia et al. (1991) in a pot experiment applied P. lilacinus

(cultured on wheat bran) as a soil treatment 10 days after sowing @ 1 g/kg soil (5

¯ 108 spores to sandy soil infested with M. javanica that resulted in better top

growth of okra. Root galling was also reduced in fungus treatments. Zaki (1994) showed that the fungal filtrates of P. lilacinus inhibited the hatching of M. javanica at all concentrations used when exposed for 72 hours. Shahzad et al. (1996) studied the efficacy of P. lilacinus as soil drench against M. incognita on mashbean and found effective in suppressing root-knot nematodes. Zaki and Maqbool (1996) found that V. chlamydosporium significantly reduced gall formation by 40% on chickpea caused by M. javanica. Dhawan et al. (2004) tested the efficacy of P. lilacinus against M. incognita on okra as seed treatment at 10, 15 and 20 g / kg seed and found significant improvement in plant growth as well as reduction in number of galls, egg masses and eggs per egg mass. Khan et al. (2004c) applied P. lilacinus @ 0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0 and 8.0 g / pot against M. incognita in okra and recorded an increase in plant growth parameters. No significant differences were found among 6.0, 7.0 and 8.0 g concentrations. Similarly, the nematode multiplication was significantly reduced. Sharma et al. (2007) conducted a green house experiment in earthen pots to manage M. incognita on okra with P. lilacinus alone and in combined application with carbofuran, phorate and neem cake and observed that P. lilacinus alone reduced number of galls, eggs per egg mass by 32% each and soil population by 77%. El-Shanshoury et al. (2005) determined that P. chlamydosporia, P. lilacinus and Arthrobotrytis dactyloides reduced population densities of M. incognita up to 98.9% and were as effective as carbofuran and Nameless and improved the root and shoot growth of faba bean.

Reduction in galls and egg masses per root system and soil population of M. incognita and consequent improvement in growth of tomato due to P. lilacinus were also recorded by Pathan et al. (2005). Xiao et al. (2006) observed that fungal

inoculation to potted plants and greenhouse plots controlled Meloidogyne spp. by

77.7 and 73.3%, respectively, resulting into a 19.7% increase in cucumber yield.

Kiewnick and Sikora (2006) reported that application of P. lilacinus to soil infested with M. incognita prior to planting reduced galls, egg masses and the final nematode population in the roots by 66, 74 and 71% respectively. Sharma et al.

(2007) observed that P. lilacinus along with addition of neem cake reduced number

of galls, eggs per egg mass by 64 % each and soil population by 77 %. Thakur and

Devi (2007) found that A. oligospora alone and in combination with P lilacinus significantly suppressed root galling and nematode population of M. incognita on okra thereby improving plant growth parameters. Kannan and Veeravel (2008) evaluated the effect of P. lilacinus against M. incognita in tomato and recorded maximum enhancement in shoot / root length and shoot weight and highest reduction in soil nematode population, galls/plant and egg mass / g root. Rao

(2008) found that application of 10 g P. lilacinus, 10 g Pseudomonas fluorescens and 250 g of neem seed cake per tree of acid lime (Citrus aurantifolia) once in six months, for a period of two years, reduced the population of M. javanica and increased the yield of the crop. Cadioli et al. (2009) reported that all of the isolates of P. lilacinus reduced the population of eggs and J2s of M. paranaensis in the root system as well as in the soil and favored the growth of the coffee plants.

2.13.2.4 Trichoderma

There have been several successful endeavors where Trichoderma spp. has been tried for the management of nematodes infecting various plant species.

Sharon et al. (2001) reported that T. harzianum caused significant reduction in galling caused by M. javanica on tomato and increased fresh weight of the tomato

plants. Hemlata and Gopal (2002) observed that the combination of organic

amendment with neem cake and T. harzianum increased the chickpea growth over the control and reduced population of M. incognita. Pathak and Kumar (2003) reported that the root dip of rice (cv. Rajshree) seedlings in the culture filtrate of T. harzianum and G. virens for different durations and concentrations had significant effects on the penetration ability of M. graminicola. Goswami et al. (2005) revealed that different isolates of T. harzianum showed variability in their potentiality against M. incognita and wilt fungus Fussarium oxysporum f. sp.

Lycopersici. Verma et al. (2006) evaluated the efficacy of Gliocladium virens and

T. viride against M. javanica and found significant increase in yield of okra by 48.4 and 29.1 %.Dababat et al. (2006) reported that treatment of the soil with the biocontrol agents T. harzianum and T. viride before transplanting tomato, improved control of M. incognita. Saifullah et al. (2007) showed that the culture filtrates of T. harzianum had strong nematicidal effect against Meloidogyne spp.

Sahebani and Hadavi (2008) showed that T. harzianum BI significantly declined

infection by M. javanica and egg hatchability after penetration into the egg masses.

Baharullah et al. (2008) reported that Th1 and Th9 Isolates of T. harzianum collected from Jabban and Shamozai (Pakistan), were aggressive against M. javanica. Parasitism of M. javanica eggs by T. harzianum ranged from 16.65% in control (T0) to 90.00% in isolates collected from Shamozai (Th9). Barua and Bora

(2008) observed that Pseudomonas fluorescens, T. harzianum, carbofuran 3G,

Streptocycline and Neemcake alone significantly reduced the final soil nematode population of M. incognita and wilt disease incidence in brinjal. Bokhari (2009) found that culture filtrates of T. koningii, T. hamatum and T. harazianum,

significantly reduced the egg-masses and females of Rotylenchulus reniformis and

Meloidogyne species. Ashoub et al. (2009) evaluated the ability of some fungi species as bio-agent against the root-knot nematode, M. incognita infecting some vegetable crops and found that Trichoderma viride was the most effective against

M. incognita. Sharon et al. (2010) reported that in pot experiments Trichoderma treatments reduced the populations of M. incognita and M. javanica. Loganathan et al. (2010) observed that in vitro studies the Tvc1, Tvc2 and The isolates of

Trichoderma effectively inhibited the growth of Sclerotinia sclerotiorum and egg hatchability of M. incognita.

Chapter 3

GENERAL MATERIALS AND METHODS

3.1 EXPERIMENTAL SITE

All the experiments reported in this dissertation were carried out in the

Plant Pathology Laboratory and greenhouse of the Regional Agricultural Research

Institute, Bahawalpur, Pakistan.

The survey for prevalence and incidence of root-knot nematodes was conducted in 17 vegetable growing districts of Punjab. Punjab is one of the five provinces of the country and constitutes more than 60% population of Pakistan.

Vegetables are widely grown in majority of the districts of the province.

3.2 MULTIPLICATION MELOIDOGYNE INCOGNITA

M. incognita, raised from a single egg mass, was used in all the experiments. For mass multiplication, the most susceptible variety of tomato

(money maker) was used as host plant. Three weeks old tomato plants were transplanted in pots containing 5 kg formalin sterilized sandy loam soil. One week after transplantation, the plants were inoculated with approximately 5000 J2s of M. incognita by making holes around the stem of plants (Campos and Campos, 2005).

The plants were watered as needed and kept in the green house at 25 ± 2 oC.

3.3 EXTRACTION OF EGGS AND JUVENILES

For extraction of eggs the infected roots were removed from the pots, washed with gentle stream of water to remove soil particles. The roots were chopped into pieces, put in a bottle containing 250 ml of 0.5% solution of NaOCl

and agitated vigorously for about 5 minutes. The agitation in sodium hypochlorite

solution dissolved the egg masses and eggs were released in the solution. The eggs

were then collected on 38 µm sieve after passing through 150 µm sieve to trap root

fragments. The eggs on 38 µm sieve were gently washed to remove any of the

excess bleach and collected in a beaker (Hussey and Barker, 1973). The eggs were

then processed on extraction trays for emergence of second stage juveniles (J2s).

The freshly hatched juveniles were used in pot experiments.

3.4 CONCENTRATION OF JUVENILES

For concentration of J2s, the nematode suspension was kept undisturbed for

about 4 hours, and then the excess water was decanted off without disturbing the

nematodes in the bottom.

3.5 STANDARDIZATION OF NEMATODES

To estimate the inoculum density the juvenile suspension was poured into a

beaker. The suspension was mixed vigorously by blowing with pipette. The

numbers of J2s were estimated in 10 aliquots of 1 ml each in a counting dish under

a dissecting microscope at 40 ¯ magnification. The total population was estimated

by multiplying the mean of 10 aliquots with the total volume. When the nematodes

were in higher concentrations then the suspension was diluted by adding the required amount of water.

3.6 COLLECTION OF OKRA GERMPLASM

Okra germplasm viz. Sanam, Dikshah, Sabz Pari, Arka Anomika, Ikra-1,

Ikra-2, Selection-31, Super Star, Punjab Selection, PMS-55, Okra Sindha, PMS

Beauty was collected from Vegetable Section, Ayub Agricultural Research

Institute, Faisalabad.

3.7 COLLECTION OF PLANT MATERIAL FOR SOIL AMENDMENTS

Datura stromonium (Dhatura) and Calotrois procera (Ak) were collected

from the Cholistan dessert of Bahawalpur, while Tagetes erecta (Marigold) and

Azadirachta indica (Neem) were collected from the Botanical Garden of

Agriculture Extension Department, Model Town-A, Bahawalpur.

3.8 INOCULATION OF ROOT-KNOT NEMATODES

For inoculation of nematodes, 4-6 holes were made around the stems of plants with the help of wooden rod. The required numbers of juveniles contained in a small volume of nematode suspension were pipetted in these holes. The holes were covered with soil to prevent drying.

3.9 SOURCES OF ANTAGONISTS USED IN EXPERIMENTS

An isolate of Pasteuria penetrans designated as Pp3, derived from M. incognita originating from South Africa, and the fungal isolate Pochonia chlamydosporia obtained from University of Reading, UK were used in the experiments. Paecilomyces lilacinus and Trichoderma harzianum were obtained from the National Nematological Research Centre (NNRC), University of Karachi,

Karachi, Pakistan and the Institute of Plant Pathology, Punjab University, Lahore,

Pakistan respectively.

3.10 MASS PRODUCTION OF PASTEURIA PENETRANS

The isolate of P. penetrans was multiplied on M. incognita on tomato cv.

Money maker in green house by the method of Stirling and Wachtel (1980).

Hundred mg of root powder containing spores was ground in a small amount of water with a pestle and mortar to slurry (Chen et al., 1996b). The slurry was diluted with tap water and filtered through a 38 µm aperture sieve to remove debris and collected in a beaker. Newly hatched second stage juveniles of M. incognita were added to this spore suspension and agitated by bubbling air through it. After 24 hours, juveniles were examined under inverted lens microscope for spore attachment. Majority of juveniles had 6-8 spores attached to their cuticles.

The spore-nematode suspension was poured through a 20 aperture µm sieve to separate encumbered J2s from excessive spores unused in the suspension. The spores encumbered J2s were washed into a 250 ml beaker and the required volume was made up using tap water. These spore encumbered J2s were inoculated around the stem of plants grown in sterilized pots by making holes. Pots were kept in the green house at 25 ±2 oC and watered regularly. After seven weeks, the root system

were carefully removed, gently washed, air dried and ground in pestle and mortar.

This root powder served as P. penetrans inoculum. The spore concentration of powdered roots was determined with the help of haemocytometer.

3.11 PPRODUCTION OF INOCULUM OF POCHONIA

CHLAMYDOSPORIA

Dried milled barley was washed over a 53 µm aperture sieve and mixed 1:1

(V/V) with coarse sand and left to dry until moist and easily friable. About 130-

150 ml of the medium, in a 250 ml flask was autoclaved (30 min, 15 psi), cooled,

shaken and inoculated with 5 plugs (7 mm) of P. chlamydosporia on corn meal

agar. Three weeks after incubation at 25 oC, the colonized sand bran was washed

through 250 µm, 53 µm and 10 µm aperture sieves with a fine water spray to

remove the sand and bran and the fungal propagules were collected on 10 µm

aperture sieve.

The deposit was further washed to remove conidia and small hyphal

fragments leaving mainly chlamydospores and blotted dry to remove extra

moisture. Chlamydospores were scraped off and thoroughly mixed in a 1:10 (w/w)

with fine sand (40-100 mesh) which acted as inert carrier. A 1 g sub-sample of

inoculum was shaken in 9 ml of water and the number of chlamydospores per gram

of sand was estimated using a haemocytometer.

3.12 MASS MULTIPLICATION OF P. LILACINUS AND T. HARZIANUM

For mass multiplication of the inoculum of P. lilacinus and T. harzianum,

the chopped wheat grains were immersed in water for about 10 to 12 hours,

surface dried using a paper towel and autoclaved (250 g / 500 flask) at 15 psi for about 50 minutes. The sterilized wheat grains in flasks were inoculated with pure cultures of both the fungi and incubated at 25 ± 1 oC for 15 days. The flasks were shaken at alternate days for uniform colonization of the fungus. The colony forming units per gram of the grains were counted by using haemocytometer after making spore suspension in suitable amount of distilled water.

3.13 MEDIA USED

The following media were used for culturing of fungi used in the experiments.

Semi-selective medium for P. chlamydosporia (De Leij and Kerry, 1991)

Corn meal agar 17 g

NaCl 17.5 g

Rose Bengal 75 mg

Water 1000 ml

After autoclaving add

Triton X-100 3 ml

Streptomycin sulphate 50 mg

Chlortetracycline 50 mg

Chloramphenicol 50 mg

Thiabendazol 37.5 mg

Carbendazim 37.5 mg

3.13.2 Potato Dextrose Agar (PDA)

Potato starch 20 g

Dextrose 20 g

Agar agar 20 g

Water 1000 ml

3.14 THE SOIL USED FOR POT EXPERIMENTS

The soil used in the pot experiments was collected from an experimental field of the institute. The soil was got analyzed from the Soil and Water Testing

Laboratory, Bahawalpur for various physical and bio-chemical properties which are given in Table 3.1. The soil was then sterilized with formalin. Diluted formalin

(1:320) was poured on a small heap of soil and covered with polythene sheet for one week. After one week plastic sheet was removed, the soil was thoroughly mixed, air dried by spreading in a thin layer on a polythene sheet to remove effects of formalin. The soil was then sieved through a 3.5 mm pore size sieve to remove large stones and plant residues and used in pots.

Table 3.1: Physico-chemical characteristics of experimental soil

CHARACTERISTICS UNIT SOIL PROPERTIES

Soil type - Sandy clay loam

Sand % 55.6

Silt % 19.4

Clay % 25 pH 7.6

TSS % 0.19

Organic matter % 0.98

Total nitrogen % 0.048

Available phosphorous ppm 5.9

Available potassium ppm 259

3.15 DATA COLLECTION

For recording data of pot experiments, the plants were gently removed from the pots after stipulated period (six weeks). The shoots were excised from the roots. The length of shoots and roots were measured with the help of a scale. The shoots and roots of individual plants were weighed in an electric balance. For dry shoot weight, the shoots were dried in an oven at 60°C 10 24 hours. The galls and egg masses of the whole root systems of plants were counted under Stereoscope at magnification of 40¯.

For estimation of total nematode population, eggs were extracted from the roots of individual plants by using the method described described in Section 3.3.

The juveniles were extracted from the soil of each individual plant from their respective pots following Whitehead and Hemming Tray Method (Whitehead and

Hmming, 1965). The total number of eggs and nematodes in soil constituted the total population. The reproduction factor (Rf) was calculated by dividing the final population (Pf) by the initial one (Pi).

The percent reduction/increase in growth parameters were calculated by the following formula.

A-B % reduction/ increase = ------X 100 A

Where A = value of the control plants

B = value of the inoculated plants

3.16 EXPERIMENTAL DESIGNS AND STATISTICAL ANALYSES

In pot experiments the Randomized Complete Block Design (RCBD) was

used. All the data were subjected to Analysis of Variance (ANOVA) using GenStat

package 2009, (12th edition) version 12.1.0.3278 (www.vsni.co.uk). The means were compared by Duncan’s Multiple Range Test (DMRT) at 5 %. Standard errors of means, trend lines, regression equations and R2 were calculated in Microsoft

Excel 2003.

Chapter 4

DISTRIBUTION AND INFESTATION OF ROOT-KNOT NEMATODES (MELOIDOGYNE SPP.) ON OKRA (ABELMOSCHUS ESCULENTUS (L.) MOENCH.) IN THE VEGETABLE GROWING AREAS OF THE PUNJAB PROVINCE.

4.1 INTRODUCTION

Among the nematode pathogens, root-knot nematodes (Meloidogyne spp.) pose a serious threat to crops throughout the world and are considered the most destructive and cause huge losses. Almost all the vegetables in tropical and warm areas of the temperate regions are severely attacked by this nematode (Sikora and

Fernández, 2005). Okra (Abelmoschus esculentus (L.) Moench.) is considered to be one of the world’s oldest crops and is cultivated in almost all the inter-tropical and

Mediterranean regions for its young fruits. The vegetable is an important source of vitamins and essential mineral salts including calcium, which lacks in the diet of poor people of most of the developing countries of the world. This vegetable, vernacularly known as “Bhindi” is one of the most important summer vegetable crops of Pakistan. In the Punjab province of Pakistan, okra is sown from mid

February to mid May. The total area under okra cultivation in Pakistan is about

5 6 2.21 ¯ 10 hectares with production of 2.86 ¯ 10 tons of green pods (GOP, 2005-

06). The yield obtained in Pakistan is relatively lower, for which there are many constraints including prevalence of diseases caused by different pathogens. Among various pathogens responsible for the low yield, the root-knot nematodes are of considerable economic importance and cause annual losses in tropics to an extent of 22 per cent (Sasser, 1979). In India the losses have been estimated up to 99 per

cent (Bhatti and Jain, 1977). In Pakistan damages to plants by nematodes are more serious and complex than in the developed countries due to many reasons. The country is situated in the tropical region where environmental conditions are conducive throughout the year for infection, development and reproduction of these nematodes. In the arid zone of the country, the sandy soils favour the activities of the nematodes. In irrigated areas perennial crops or susceptible crops grown in the same field year after year allow the nematodes to reproduce profusely and consequently cause severe infections and damage. Plant parasitic nematodes, in Pakistan, have received little attention and only a few surveys have been made in the past (Brown, 1962; Kafi, 1963; Saeed and Ashrafi, 1973; Ahmad and Khan,

1973; Khan et al., 2005). There is limited information regarding the association of root knot nematodes with okra in the country. Therefore, the objective of present studies was to conduct a survey to determine and document the occurrence, prevalence and intensity of root knot nematodes on okra cultivations in the vegetable growing areas of Punjab.

4.2 MATERIALS AND METHODS

4.2.1 PREVALENCE, INCIDENCE AND SEVERITY OF ROOT-

KNOT NEMATODES

A survey of okra fields located in different randomly selected localities of the major vegetable growing districts (Table 4.1 and Figure 4.1) of the Punjab province of Pakistan was conducted during the year 2007 for the determination of prevalence, incidence and severity of root-knot nematodes. Form each locality, three fields of okra were randomly selected. From each okra field, 25 plants were

selected after each 10 steps following zig zag pattern as shown in Figure 4.2. The selected plants were carefully uprooted upto 15-20 cm soil depth with the help of trowel. The soil adhering to the root system was gently removed and roots were observed for root-knot nematodes infection (presence or absence of galls).

The root systems of individual plants were rated following the galling index developed by Bridge and Page (1980) given in Figure 4.3 for the determination of severity of root-knot nematodes. The infected roots along with soil were put into polythene bags, labeled properly and brought to the laboratory of Plant Pathology

Section, Regional Agricultural Research Institute, Bahawalpur for identification of root knot nematode species.

The incidence of root-knot nematodes of individual okra fields was determined as followed.

Total number of infected plants Incidence (%) = ------x 100 Total number of observed plants

The prevalence of root-knot nematodes in each district was calculated as followed

Number of fields infected with RKN Prevalence (%) = ------x 100 Total number of fields surveyed

4.2.2 IDENTIFICATION OF MELOIDOGYNE SPECIES

Root-knot nematodes (Meloidogyne species) were identified on the basis of female perineal patterns described by Taylor and Netschler (1974). Mature females of root-knot nematodes were dissected out from the infected okra roots and placed in watch glass containing distilled water. The live mature females were then picked

up with fine bristle and were placed in plastic petri dish containing 45 percent

lactic acid and were left for two hours. The posterior end then was cut off with a

fine needle and the body tissues were removed by lightly brushing the inner surface of the cuticle with a flexible bristle. When all the tissues were removed, the cuticle was transferred to a drop of glycerin where it was carefully trimmed, the piece of cuticle containing vulval portion with the typical perineal pattern was then transferred to a drop of glycerin on a micro slide, a cover slip was applied and sealed with nail polish, and was observed under microscope. The perineal pattern was compared with standard diagrams and Meloidogoyne species was identified. In this way perineal patterns of 40 females were prepared from each infected okra field and the distribution of each Meloidogyne species in each district was calculated.

Table 4.1: The districts and localities surveyed for root-knot nematodes

S.No District Locality/Area

1. Multan Gulzar Pur, Jumaa Wali, Muzafarabad, Murlay Wala and Bohar Wala 2. Gujranwala Raitali Virkan, Muriala Waraich, Killa Deedar Singh

3. Jhang Rajoa Sadat, Bhowana, Ahmadpur Sial,

4. Faisalabad Samundri, Jaranwala,Tandlianwala, Chak Jhumra

5. Toba Tek Singh Chak 431 JB Walah, Saraba, Chak 377 JB Javan, Chak 307 JB Saroki 6. Okara Rajuwal, Hawailian, Gogaira

7. Kasur Bhai Pharoo, Attari Karampur, Terath

8. Dera Ghazi Adil peer, Kotla Sikhani,Kot Chutta, Wadoor,Choti Khan Zareen 9. Muzafar Garh Khan Garh, Roulhnawali , Jatoi, Gazi Ghat

10 Khanewal Chak 167/10R, Cahak 7/9R, Chak 168/10 R, Kabirwala

11 Bahawalnagar Haroonabad, Chishtian, Maroot

12 Rajanpur Hajipur, Kotla Mughlan, Fazilpur

13 Rahim Yar Dari Sanghi, Ranjhay Khan, Chandrami, Aman Garh Khan 14 Vehari 15/WB, 75/WB, 53/WB, 561/EB

15 Bahawalpur Bahawalpur suburb, Jhangiwali, Abbasnagar, Goth Mehro, Ahmadpur East, Chak 13 BC

16 Lodhran Qureshi Wala, Adamwahan, Dhanot, Lodhran, Kehror

17 Rawalpindi Sehala, Tarnoul, Pindi Ghaib

Figure 4.1: Map of Punjab pointing districts surveyed for root-knot nematodes

Figure 4.2: Sampling method used for recording incidence of root-knot nematodes.

Figure 4.3: Root galling rating scheme for evaluation of Meloidogyne infestation

(Bridge and Page, 1980).

4.3 RESULTS

4.3.1 PREVALENCE OF ROOT-KNOT NEMATODES

The prevalence of root-knot nematodes in different districts is given in

Figure 4.4. It is evident from the figure that maximum prevalence (100%) was

recorded in districts Multan, Okara, Dera Ghazi Khan, Bahawalnagar, Rahim Yar

Khan, Vehari and Rawalpindi, while the minimum (22.4%) was found in district

Lodhran. The prevalence in Gujranwala, Jhang, Faisalabad, Toba Tek Sing, Kasur,

Muzafar Garh and Bahawalpur ranged from 66.62 to 91.65%. The individual

prevalence of root-knot nematodes in each locality of each district is given in Table

4.2 column 3.

4.3.2 INCIDENCE OF ROOT-KNOT NEMATODES

The incidence of root-knot nematodes was recorded from all the okra

growing areas. The incidence varied in all the districts and ranged from 11.1-

69.95%. The maximum incidence (69.98%) of root-knot nematodes was found in

Bahawalnagar district followed by Rahim Yar Khan (69.95%), Dera Ghazi Khan

(66.18%) and Vehari (63.30%). The lowest incidence was recorded from Lodhran

(4.44%) followed by Jhang (11.1 %) and Rajan Pur (13.30%). The incidence

ranged from 13.30 to 63.30 % in Multan, Faisalabad, Toba Tek Singh, Okara, Dera

Ghazi Khan, Bahawalnagar, Rahim Yar Khan and Vehari districts. The mean

individual incidence in each district and in each locality of the district is given in

Figure 4.4 and Table 4.2 column 4 respectively.

4.3.3 SEVERITY OF ROOT-KNOT NEMATODES

The maximum severity (6.16) measured in terms of galling index suggested

by Bridge and Page (1980) was found in district Bahawalnagar followed by Vehari

(6.00) and Rahim Yar Khan (5.83) while the minimum was observed in district

Lodhran which was only 0.55. The individual mean galling index (Severity)

recorded in each district and in each locality of the district is given in Figure 4.4

and Table 4.2 column 5 respectively.

4.3.4 OCCURRENCE OF ROOT-KNOT NEMATODES SPECIES

The different species of Meloidogyne associated with okra in different

districts are given in Table 4.3. Of all the associated species of root-knot

nematodes, M. incognita constituted 73.74%, M. javanica 24.02%, M. arenaria 2.0

% and M. hapla 0.78%.

Both M. incognita and M. javanica were found in all the districts, M.

incognita being predominantly found. M. arenaria and M. hapla was found only in Rawalpindi district. The individual percentage of each Meloidogyne species in each district is given in Table 4.3.

Maximum mean occurrence of M. incognita (97.5%) was observed in Toba

Tek Singh and the lowest (33.3%) was recorded in Rawalpidi District. The highest occurrence of M. javanica (48%) was recorded in Dera Ghazi Khan and lowest

(2.5%) was found in Toba Tek Singh. The distribution of Meloidogyne species in individual districts is given in Table 4.3.

Prevalence Incidence Severity ) x

120 7 e d In

6 g 100 llin a 5

80 (G e g

4 erity ta

60 sev

3 e d ercen P to

40 a

2 m e t n

20 o

1 n t-k o

0 0 o R l r a r n h r n i r i n l g d h a a r n d a a r u a r u a a u t n g s a g a n l a b a wa p h p r i wa n a l u a i G e n e a h p h l Kh n Kh n J S Ka n l a d l a a Ok i r j r V w M a a o r s k z h a a j i a f wa L wa a e Ra h u h a K a Y F T z G h Ra G u m Ba a a i b a r M B h o e T D Ra Districts surveyed

Figure 4.4: Prevalence, incidence and severity of root-knot nematodes

(Meloidogyne species) in different districts of Punjab.

Table 4.2: Prevalence, incidence and galling index (severity) of root-knot nematode (Meloidogyne species) on okra in different districts of Punjab. Disease District/ Disease Incidence Locality Prevalence Galling index Tehsils (%) (%) 1 2 3 4 5 Range Mean Range Mean Gulzar Pur 100 33.3-40 39.96 2-3 2.33

Jumaa Wali, 100 40-60 48.86 3-4 3.33

Muzafarabad 100 33.3-53.3 44.4 2-4 3 Multan Murlay Wali, 100 26.6-46.6 35.5 3-5 4

Bohar Wala 100 40-53.3 46.63 5-6 5.33

Mean 100 26.6-53.3 43.07 2-6 3.6

Raitali Virkan 66.6 0-20 11.1 2-2 1.33

Muriala Waraich 100 13.3-20 17.76 2-3 2.33

Gujranwala Killa Deedar 100 20-33.3 26.63 3-5 3.66

Singh

Mean 88.86 0-33.3 18.5 2-5 2.44

Rajoa Sadat 100 13.3-20 15.53 2-4 3

Bhowana 33.3 0-20 6.66 0-7 2.33 Jhang Ahmadpur Sial 66.6 0-20 11.1 0-6 3

Mean 66.63 0-20 11.1 0-7 2.77

Samundri 100 80-86 82.2 7-8 7.33

Jaranwala 100 53.3-80 64.43 5-7 6.6

Faisalabad Tandlianwala 100 53.3-60 57.76 5-7 5.66

Chak Jhumra 33.3 0-20 6.60 0-5 1.66

Mean 83.32 0-86 52.76 0-8 5.33

Chak 431 JB 66.6 0-46.6 22.2 0-5 3

Walah

Saraba 100 66.6-80 75.53 6-7 6.66

Toba Tek Chak 377 JB 100 60-86.6 62.16 5-7 5.66

Singh Javan

Chak 307 JB 100 46.6-73.3 71.06 7-8 7.33

Saroki

Mean 91.65 0-86.6 57.74 0-8 5.66

Rajuwal 100 60-80 57.76 6-7 6.66

Hawailian 100 33.3-60 46.63 4-6 5 Okara Gogaira 100 53.3-86.6 73.3 5-6 5.66

Mean 100 33.3-86.6 59.23 4-7 5.77

Kasur Bhai Pharoo, 66.6 0-26.6 15.53 0-4 2.33

Attari Karampur 100 13.3-33.3 22.2 4-5 4.33

Terath 66.6 0-20 11.1 0-4 2.33

Mean 77.73 0-33.3 16.27 0-5 3

Adil peer 100 80-93.3 86.6 6-8 6.66

Kotla Sikhani 100 46.6-60 51.06 3-7 4.66

Dera Ghazi Kot Chutta 100 46.6-66.6 57.7 5-6 5.33

Khan Wadoor 100 80-93.3 79.96 6-8 7

Choti Zareen 100 46.6-60 55.53 4-5 4.33

Mean 100 46.6-93.3 66.18 3-8 5.6

Muzafar Khan Garh 100 13.3-20 15.53 3-4 3.66

Garh Roulhnawali 100 13.3-26.6 19.96 2-4 3

Jatoi 100 6.6-26.6 15.50 1-3 2

Gazi Ghat 66.6 0-20 11.10 0-4 2.33

Mean 91.65 0-26.6 15.52 0-4 2.75

Chak 167/10R 33.3 0-33.3 11.10 0-2 0.66

Cahak 7/9R 66.6 0-46.6 28.86 0-5 3

Khanewal Chak 168/10 R 100 33.3-53.3 42.2 4-5 4.33

Kabirwala 66.6 0-20 11.10 0-3 1.66

Mean 66.62 0-53.3 23.31 0-5 2.41

Haroonabad 100 73.3-80 77.76 6-7 6.66

Bahawal Chishtian 100 53.3-73.3 62.20 5-7 5.66 nagar Maroot - - - - -

Mean 100 53.3-80 69.98 5-7 6.16

Hajipur 100 6.6-20 11.06 2-4 3.33

Kotla Mughlan 66.6 0-33.3 19.96 0-4 2.33 Rajanpur Fazilpur 66.6 0-20 8.86 0-3 1.66

Mean 77.73 0-33.3 13.30 0-4 2.44

15/WB 100 53.3-73.3 62.2 6-7 6.33

75/WB. 100 40-66.6 51.06 5-6 5.33

Vehari 53/WB 100 46.6-80 64.4 5-7 6.33

561/EB 100 60-86.60 75.53 4-7 6

Mean 100 40-86.60 63.3 4-7 6

Rahim Yar Dari Sanghi 100 46.6-60 51.06 4-5 4.66

Khan Ranjhay Khan 100 66.6-80 73.30 5-7 6.33

Chandrami 100 73.3-86.6 82.16 6-7 6.33

Aman Garh 100 60-86.6 73.30 5-7 6

Mean 100 46.6-86.6 69.95 4-7 5.83

Bahawalpur 33.3 0-20 6.6 0-2 0.66

suburb

Jhangiwali 100 26.6-60 42.2 3-5 4.33

Abbasnagar 66.6 0-20 8.86 0-6 3.33 Bahawalpur Goth Mehro 100 20-40 31.10 4-6 5

Ahmadpur East 100 46.6-60 51.06 4-6 5

Chak 13 BC 100 80-93.3 88.86 7-8 7.66

Mean 83.31 0-93.3 38.12 0-8 4.33

Lodhran Qureshi Wala 33.6 0-20 6.66 0-2 0.66

Adamwahan 33.6 0-20 6.66 0-3 1

Dhanot 0 0 0 0 0

Mean 22.4 0-20 4.44 0-3 0.55

Sehala 100 20-46.6 33.3 4-6 5

Tarnoul 100 20-60 42.2 4-6 5 Rawalpindi Pindi ghaib 100 13.3-60 39.96 3-6 4.66

100 13.3-60 38.48 3-6 4.88

Mean 85.28 - 38.89 - 4.08

Table .4.3: Distribution of Meloidogyne species associated with okra in different

districts of Punjab

Number of Meloidogyne spp. (percent) District/locality Meloidogyne Species M. M. M. M. incognita javanica arenaria hapla Multan 2 86.0 14.0 0 0 Gujranwala 2 86.6 13.3 0 0

Jhang 2 70.0 30.0 0 0

Faisalabad 2 70.0 30.0 0 0

Toba Tek Singh 2 97.5 2.5 0 0

Okara 2 83.3 16.6 0 0

Kasur 2 90.0 10.0 0 0

Dera Ghazi Khan 2 52.0 48.0 0 0

Muzafar Garh 2 82.5 17.5 0 0

Khanewal 3 77.5 15.0 0 0

Bahawalnagar 2 80.0 20.0 0 0

Rajanpur 2 86.6 23.3 0 0

Rahim Yar Khan 2 70.0 30.0 0 0

Vehari 2 65.0 35.0 0 0

Bahawalpur 2 63.3 36.6 0 0

Lodhran 2 60.0 40.0 0 0

Rawalpindi 4 33.3 26.6 26.6 13.3

Average 73.74 24.02 1.57 0.78

4.4 DISCUSSION

The results of the present survey showed variations in the prevalence,

incidence and severity of root-knot nematodes in different districts of Punjab.

Similar results were also reported by Lamberti et al. (1975); Bhatti and Jain

(1977); Khan et al. (2005); Shahid et al. (2007). The results of these scientists confirmed the present findings regarding the prevalence of plant parasitic nematodes and occurrence of Meloidogyne specie on vegetables. It is clear from the results of these researchers that okra crop was the most susceptible host of

Meloidogyne species in vegetable growing areas. These variations in infestations are attributed to many environmental and edaphic factors, as differences in various climatic and edaphic factors of these districts have been found. There are reports which confirmed that distribution, prevalence, incidence and severity of root knot nematodes are affected by varying agro-climatic conditions of the areas, soil type, moisture, soil pH and particular cropping sequence (Taylor et al., 1982; Sasser and

Carter, 1985; Van-Gundy, 1985; David, 1985).

Root-knot nematodes are also influenced by the biological, chemical and physical characteristics of the soil environment (Upadhyay et al., 1972). In district

Lodhran, the prevalence of root-knot nematodes is quiet low. This is because of higher amount of organic matter in the soil of the district and also due to the fact that okra was sown in the soils which were fallow for the last few years. This fact is supported by the findings of other researchers who found that fallowing increased the organic matter contents of the soils and thereby reduced the number of nematodes (Ferris and Bernard, 1971; Netscher, 1985; Aung and Prot, 1990;

Floret and Serpantie, 1993).

The root-knot disease was found 100% in Multan, Okara, Bahawalnagar,

Rahim Yar Khan, Vehari and Rawalpindi. This high incidence is due to intense vegetable cropping pattern and the availability of suitable host throughout the year in these districts which allowed rapid multiplication of root-knot nematodes.

Earlier a number of researchers reported that abundance of root-knot nematodes is highly dependable upon the presence of the suitable host plants (Jacq and Fortuner,

1979; Yeates, 1976 and 1981; Ferris et al., 1985; Cuc and Prot, 1992).

The high incidence and severity of root-knot nematodes in these districts were also due to the cultivation of susceptible varieties, high temperature and comparatively less annual rainfall. These conditions favored the multiplication, development and infection of root-knot nematodes. There are reports which showed that nematode populations are influenced by soil type (Kincaid, 1946;

Wallace, 1969; Prot and van Gundy, 1981; Jain, 1992). In the present studies M. incognita and M. javanica were found in all the districts in varying proportions and

M. incognita was predominant in all the districts. Trudgill et al. (2000) reported that M. incognita and M. javanica were the most widespread root-knot nematode species in all the countries. Similar results have also been reported by many workers (Khan, et al., 1993; Campos, 1994; Das and Das 2000; Ravichandra and

Krishnappa, 2004; Bhosle et al., 2004; Rathour et al., 2006). The distribution and infestation of Meloidogyne spp. in the soils of Pakistan was M. incognita, 52%, M. javanica, 31%, M. arenaria, 8%, M. hapla, 7% and other species about 2%

(Maqbool, 1987) which further proved the present findings. M. arenaria and M. hapla were isolated only from Rawalpindi district. The climate of the district is cool, humid and mild. These results confirmed the findings of Brown (1962) that

M. arenaria and M. hapla are cool, humid and hilly climate species. Gul and Saeed

(1987) also reported M. arenaria and M. hapla from North West Frontier Province

(NWFP) of Pakistan. It is concluded from the present studies that okra is severely attacked by root-knot nematodes and M. incognita is the most predominant species, which warrant that strict control measures should be adopted for its management.

Chapter 5

EVALUATION OF DIFFERENT OKRA CULTIVARS FOR THEIR RESISTANCE AND SUSCEPTIBILITY AGAINST ROOT-KNOT NEMATODE, MELOIDOGYNE INCOGNITA

5.1 INTRODUCTION

Okra (Abelmoschus esculentus L.) is one of the important vegetable crops of the world and popular in many tropical and subtropical countries. Due to present food problems of the underdeveloped countries, vegetables have gained great importance in Pakistan. Several cultivars of okra are cultivated throughout the year. Root-knot nematodes (Meloidogyne spp.) attack different crop plants including vegetables causing severe growth retardation due to formation of typical galls. Sikora and Fernandez (2005) reported severe attack of root-knot disease caused by Meloidogyne spp. on okra. Root knot nematodes are responsible to cause yield losses up to 27 % in okra. The annual yield losses caused by Meloidogyne spp. are estimated to be 16.9 % (Bhatti and Jain, 1977; Sasser, 1989). The yield losses caused by root-knot nematodes are due to build up of inoculum of this pathogen and continuous growing of similar okra varieties in the same field year after year.

Chemicals are being used to control nematodes successfully but due to their high cost and hazardous effects, nematicides are not attractive to farmers. Use of cultivars resistant to nematodes is one of the alternatives which are environmentally benign, secure and economically feasible means of controlling root-knot nematodes. The cultivars resistant to root-knot nematodes have

comparatively better crop yield as compared to susceptible varieties. These can

also be employed as a component of integrated nematode management along with

other control strategies like organic soil amendments, soil solarization, heat

treatment, and crop rotation with non hosts for controlling root knot nematodes. As

the information regarding the availability of resistant cultivars is lacking, the

objective of present studies was to find the resistance source against M. incognita

among commercially available okra cultivars which are widely cultivated in

majority of the okra cultivated areas of the country.

5.2 MATERIAL AND METHODS

Twelve okra cultivars (Section 3.6, Chapter, 3) were screened for resistance

against M. incognita in plastic pots (20-cm-dia.) containing 5 kg formalin sterilized

soil (sand 70%, silt 22%, clay 8% and pH 7.5). Three seeds of each cultivar were

sown per pot. Ten days after germination, one healthy seedling of each test cultivar

was maintained in each pot. The plants of each cultivar were then inoculated with

freshly hatched J2s of M. incognita (Section 3.3, Chapter, 3) by making holes around the plants. The plants of each cultivar which were not inoculated with J2s served as control of that cultivar. Each cultivar was replicated five times. The pots were maintained in a green house in a Randomised Complete Block Design

(RCBD) at a temperature of 25 ± 2 oC for seven weeks. The pots were watered

when required. After six weeks plants were carefully removed from the inoculated

and control pots of each cultivar and their roots were excised from the shoot. The

roots were gently washed and blotted dry. Data were recorded on the following

parameters.

Root and shoot lengths

Fresh and dry shoot and fresh root weight

Number of galls and egg masses

Total nematodes population for determination of reproduction factor

The percent reduction or increase in growth parameters of each cultivar was

calculated by the following formula.

A - B Percent (%) reduction or increase = ------x 100 A Where A = Value of the control plant

B = Value of the inoculated plant

The cultivars were categorized for their relative resistance / susceptibility on the basis of galling index developed by Hartman and Sasser 1985 as below

When 0 = No. of gall Immune or highly resistant

1 = 1-2 Resistant

2 = 3-10 Moderately resistant

3 = 11-30 Moderately susceptible

4 = 31-100 Susceptible

5 = > 100 galls per root system Highly susceptible

All the data were subjected to ANOVA using GENSTAT and means were compared by DMRT.

5.3 RESULTS

M. incognita caused significant reductions in fresh and dry shoot weights and increase in fresh root weight over their respective controls in all the cultivars.

Maximum reduction in fresh shoot weight was observed in Punjab Selection

(38.96%) followed by Selection-31 and Okra Sindha causing 24.37 and 20.35% respectively. The minimum reduction in fresh shoot weight was recorded in case of

Sanam (2.88%). The reductions in fresh shoot weight of the other cultivars ranged from 4.48 to 7.4% as shown in Figure 5.1. Similar trends were observed in dry shoot weight. The individual reductions in this parameter are given in Figure 5.2.

The fresh root weight of all the cultivars was found to be more as compared to their respective control due to formation of galls and egg masses. Maximum increase in fresh root weight was found in Punjab Selection followed by selection-

31 and Okra Sindha resulting into 33.43, 20.16 and 16.42% increases over controls which were statistically different from each others. A minimum increase of 2.61% was observed in case of Sanam. The increase in fresh weight in other cultivars varied from 3.52 to 6.1% as shown in Figure 5.3.

The cultivars also varied significantly in causing decreases in shoot and root lengths over control. Maximum decrease of 21.71% was found in Punjab

Selection; the reductions in Selection-31 and Okra Sindha were not statistically similar. The minimum reduction (2.11%) was recorded in Sanam. The individual reductions in shoot lengths are given in Figure 5.4.

Similar tendency was seen among okra cultivars regarding root length as shown in Figure 5.5.

Likewise, all the cultivars behaved differently regarding formation of galls and egg masses. Maximum galls were observed on Punjab Selection (167) followed by Selection-31 (96) and Okra Sindha (79). Minimum galls were recorded in case of Sanam (12). However galls varied in other cultivars ranging from 18-96 as shown in Figure 5.6.

The formation of egg masses followed the same trends as shown in Figure

5.7. As regards reproduction factor, it is again highest (8.2) in Punjab selection, followed by Selection-31 (5.51) and Okra Sindha (4.88) which differ significantly from each other. The reproduction factor in the remaining cultivars was found below 2 as shown in Figure 5.8. It is clear from Table 5.1 that none of the cultivar was found tolerant or highly resistant, resistant and moderately resistant.

Maximum galls were found on Punjab Selection which proved to be highly susceptible. Also maximum reductions in growth parameters were observed in case of Punjab Selection. The cultivars Selection 31 and Okra Sindha were found to be susceptible. The reductions in growth parameters of these cultivars were less severe as compared to Punjab Selection. The remaining cultivars viz. Sabz Pari,

Sanam, Dikshah, Arka Anomika, Ikra-1, Ikra-2, Super Star, PMS-55 and PMS

Beauty appeared as moderately Susceptible. This is also evident by the reduction in growth parameters of these cultivars.

45 40

g 35 i e w

t 30 o o

h 25 s n i

e 20 s a e

r 15 c

de 10 % 5

i h r a 1 2 1 r n 5 a y m - - a 5 t a a a k 3 t o - h i a a - i d u n h p r r s t S a s n c . n a z m k k o r i e k I I e S i b o i e l M s b a t p e . D n c a S S A e u S P r . l e S b k a .M S a O k j P r n A u P Cultivars Figure 5.1: Effect of M. incognita on shoot weight of different okra cultivars

h 60 g i e

w 50

oot h 40 y s

r 30 d n

e 20

0 % decreas i 1 2 h r a - - 1 r 5 a m a n 5 y a a a ik a a 3 t o - h t h p r r - s i u n t S d a s m k k r . n a k o I I on c i e S i bz i e e s b a n t p l M D c u e a S A e S P. r .S l S k a e b k O M r S a j P. A n Pu Cultivars

Figure 5.2: Effect of M. incognita on dry shoot weight of different okra cultivars

40 t h g i

e 30 w

ot o r

n 20 i e eas 10 cr n i

% 0

i y a 1 2 1 r n 5 a m h r - - a t a a ik 3 t o 5 h u a a a - i - d n h P r r S t a s m n c S n e a k z o k k o r e i S I I i e l M s b b n t e Di a c p P a S S A e u S r . l M e S b k A S P O P Cultivars

Figure 5.3: Effect of M. incognita on root weight of different okra cultivars

h 20

ngt e l t 15 hoo s

n i e

s 10 a e r c

de 5 % 0

h i 1 2 1 r m r ka - - a 5 ty a a i 3 t ion 5 ha h p a a s t - u na s m n- c nd a a k kr kr o r .S i i I I i le s S bz no t be D a c pe M a A u Se . r S S le P . e S b k ka M r S ja O . n P A u P Cultivars

Figure 5.4: Effect of M. incognita on shoot length of different okra cultivars

h 35 t

ng e 30 l

o 25 r

i 20 e s a

e 15

r 10 dec % 5

i r a 1 2 1 r 5 a m h - - n 5 y a a ik a a 3 ta o - h t a p r r - i u n h s t S d s m k k c . n a a k z I I on r i e S i b o i e e s n t p l M b a c e a D S A e P. r S l Su S . a e k k b M r S a O . j P A n u P Cultivars

Figure 5.5: Effect of M. incognita on root length of different okra cultivars

200 180 160 s l 140

gal 120 of

r 100 e b 80

m u 60 N 40 20 0 i a n h r 1 2 1 r a y m a k - - 3 a o 5 h t a a i - t i 5 h p a a s t - d u n s m r r n c n a a k z k k o r e S i e i o e l . S b n I I ti s b a p e D c u .M a S S A e S r . l S P k a e b k a M r S j O . n P A u P Cultivars

Figure 5.6: Effect of M. incognita on Number of galls on different okra cultivars

180 160 s e

s 140 s a 120

gg m 100 e 80 of r

e b 60 m

u 40 N 20 0

h i a 1 r n a y m a r k 1 2 a o 5 h t a a i - - 3 t i 5 h p a a - s t - d u n s m r r n c n a a k z r e S i e i o k k o e l . S b n I I ti s b a p e D c u .M a S A e S r .S l S P k a e b k a M r S j O . n P A u P Cultivars Figure 5.7: Effect of M. incognita on Number of egg masses on different okra cultivars

r 7 o t

c a 6

on f 5 i t 4

oduc 3

pr e R 2 1

i a r n a h r 1 2 1 y m a k 3 a o 5 h t a a i - - t i 5 h p a a - s t - d u n s m r r n c n a a k z k k o r e S i e i o e l . s S b n I I ti b a p e D c u .M a S A e S r .S l S P k a e b k a M r S j O . n P A u P Cultivars

Figure 5.8: Effect of M. incognita on Reproduction factor (Rf) on different okra cultivars

Table. 5.1: Response of different okra cultivars against M. incognita

No. of galls Level of resistant Cultivars

0 Immune None

1-2 Resistant None

3-10 Moderately Resistant None

11-30 Moderately Susceptible Sanam, Dikshah, Sabz Pari, Arka Anomika, Ikra-1, Ikra-2, PMS-55, PMS Beauty and Super Star

31-100 Susceptible Selection-31, Okra Sindha

> 100 Highly Susceptible Punjab Selection

5.6 DISCUSSION

The evaluation of 12 okra cultivars against M. incognita revealed that none

of the cultivar was immune, resistant or moderately resistant. The cultivar Punjab

selection was the highly susceptible as maximum galls were produced on this

cultivar. The galls on Selection-31 and Okra Sindha ranged between 31-100 and

rated as susceptible. On the other hand, nine cultivars, produced galls between 11-

30, hence categorized as moderately susceptible.

Different researchers have investigated the reaction of different okra

cultivars to root-knot nematodes (Sharma and Trivedi, 1990; Singh et al., 1993;

Jain and Gupta, 1996; Rekha and Gowda, 2000; Sheela et al., 2006) and reported varying levels of resistance among various cultivars against root knot nematodes.

The root knot nematodes, M. incognita, a destructive pest of many crops in

tropical and sub tropical regions has a very wide host range including crops and

weeds (Siddique et al., 1973), but not all are equally good at supporting nematode

reproduction. Differences in multiplication rates may be in part, due to genetic

factor in the host which confers susceptibility or resistance as well as genetic

differences between nematode populations (Bingefors, 1982; Griffin, 1982;

Jacquet et al., 2005; Castagnone-Sereno 2006). Various stages in the life cycle of the nematode could be affected by host differences. The juveniles in a resistant plant are either incapable of penetrating the roots or their death may result ensuing penetration, or they fail to develop or females cannot reproduce. The differences in the susceptibility to M. incognita in okra cultivars might be due to differences in their genetic make up which can be explained in terms of number of galls.

‘Punjab Selection’ was found highly susceptible as maximum galls and egg masses were observed on the roots which showed that maximum juveniles penetrated the roots and completed their cycles successfully. On the other hand

Super Star, Anomika and Sanam were the least susceptible which allowed only a limited number of juveniles of M. incognita to penetrate the roots, leading to maturity.

There are contradictory reports regarding differences between resistant and susceptible cultivars in rates of invasion by J2s of root knot nematode. A number of scientists (Fassuliotis et al., 1970; Reynold et al., 1970; Hung and Rhode, 1973;

Griffin and Eligin, 1977) reported that host status made no difference to rate of invasion whereas Sasser (1954) found that the roots of resistant plants were not invaded as rapidly as that of susceptible ones. Dropkin and Nelson (1960) reported

that resistant cultivars contained fewer developed nematodes than susceptible plants. Resistance to invasion of J2s has been attributed to hypersensitive reaction as well as development of less numbers of J2s in the infected roots (Dropkin,

1969). In addition to morphological modifications, molecular and biochemical changes also occur in resistant plants following infection. Increased activity of phenylalanine ammonia-lyase and anionic peroxidase enzymes have been noticed in resistant plants after nematode inoculation (Brueske, 1980; Zacheo et al., 1993).

The development of J2s is also influenced by the type of host (Davide,

1980) with female size positively correlated with host susceptibility (Veena et al.,

1985). Juveniles can express their full developmental potential on susceptible host whereas development can be delayed or curtailed in resistant hosts (Nelson et al.,

1990). Comparative studies on invasion and development of root-knot nematodes are generally limited to cultivars of the same crop (Huang, 1986; Niblack et al.,

1986; Schneider, 1991). For nematode with a wide host range it is also desirable to understand the effect of different host species on invasion and further development of J2s. Such information may be useful in devising nematode management schemes (Johnson, 1985).

Chapter 6

ASSESSMENT OF THE DAMAGE CAUSED BY M. INCOGNITA ON OKRA AT DIFFERENT INOCULUM LEVELS.

6.1 INTRODUCTION

Root-knot nematodes are serious and economically most important pests of all cultivated crops around the world. According to Sikora and Fernandez (2005), root-knot nematodes are particularly damaging vegetables in tropical and subtropical countries of the world and cause losses up to 80% in heavily infested fields.

Okra (Abelmoschus esculentus) ranked high amongst the economically important vegetables of the world. The immature fruits of okra are good sources of vitamin C, and are used for the preparation of certain soups and sauces (Diouf,

1997). In the Tropics, M. incognita very frequently attack okra (Seck, 1991; Singh et al., 1993; Khan and Khan, 1994; Khan et al., 1998). Khan and Saxena (1992) reported that M. incognita causes leaf browning, suppression in plant growth, fruit yield and photosynthetic pigments of okra crop.

Short life cycle of five to six weeks enables root-knot nematodes to survive well in the presence of a suitable host. In susceptible plants, the nematode population build up to a maximum usually as crop reaches maturity (Shurtleff and

Averre, 2000) and in some cases the plants die even before reaching maturity

(Singh and Khurma, 2007).

Various methods are being employed for managing plant parasitic

nematodes which cause reduction of nematode population densities to levels below

damage thresholds (McSorley, 1994; McSorley and Duncan, 1995).

Damage caused by the nematode is determined by relating pre plant

nematode densities (initial population) to growth and yield of annual crops. The

minimal density that causes a measurable reduction in plant growth or yield varies

with nematode species, host plants, cultivar and environment (Barker and Olthof,

1976).

Infections of non efficient or efficient hosts by low densities of

Meloidogyne spp. may enhance growth and yield of host (Madamba et al., 1965

and Olthof and Potter, 1972) or have no effect (Madama et al., 1965), or cause severe damage to the crop (Barker and Olthof, 1976). In the present studies the effect of different inoculum densities of M. incognita has been investigated on the growth and root galling of okra cv. Punjab Selection.

6.2 MATERIALS AND METHODS

The root-knot nematodes, M. incognita used in the experiments was already maintained on tomato cv. Money maker and mass produced from a single egg mass in the greenhouse of Regional Agricultural Research Institute, Bahawalpur. The highly susceptible cultivar of okra (Punjab Selection) was used in to see the effect of different inoculum levels of M. incognita.

Three seeds of okra cv. ‘Punjab Selection’ were sown in plastic pots containing 5 kg of formalin sterilized soil. After germination one healthy seedling was maintained. Ten days after germination the pots were inoculated with 500,

1000, 2000, 4000 and 8000 freshly hatched J2s of M. incognita by making four

holes around the stem. The un-inoculated plants served as control. Each treatment

was replicated five times.

The pots were arranged in a RCBD on the bench of the green house at 25

±2 oC. The pots were watered when required. Six weeks after inoculation the plants were removed from the pots and data were recorded regarding growth parameters, number of galls, egg masses and reproduction factor (Rf). Percent reductions or increase in growth parameters was calculated over control by the formula given in section 3.15, Chapter, 3. All the data were analyzed statistically (Section 3.16,

Chapter, 3).

6.3 RESULTS

All the inoculum levels of M. incognita caused significant reductions over control. The ANOVA regarding root length, shoot length, shoot weight and root weight showed highly significant effects of inoculum levels (Appendices-6.1 to

6.4). A maximum reduction in root and shoot lengths and shoot weight were recorded at an inoculum level of 8000 J2s. The reductions in these parameters caused at a level of 4000 J2s were quite similar to those caused at 8000 J2s, showing that there is no statistical significant difference in these two levels. The reductions in these parameters were recorded 38.21, 36.95 and 43.78 % at 4000, and 42.1, 44.65 and 43.38% at 8000 J2s respectively. The minimum reductions were recorded at a level 500 J2s. The reduction in these parameters increased with an increase in inoculum level. These relationships are shown by trend lines and equations given in Figures 6.1, 6.2 and 6.4. Similar trend was found in dry shoot

weight as shown in Figure 6.5. On the other hand, the inoculum levels resulted in the increase in root weight.

A maximum increase of 33.54% was observed in fresh root weight at an inoculum level of 8000 J2s followed by 4000. While minimum increase was noticed in pots where 500 J2s were applied. The increase in root weight was found to be directly proportional to inoculum levels. The relationship has been shown by trend line and equation in Figure 6.3.

Significant increases in number of galls (204) and egg masses (195.4) were observed at all inoculum levels. Maximum galls and egg masses were produced at a level of 8000 J2s followed by 4000, while the galls and egg masses were the minimum in plants inoculated with 500 J2s. Direct relationships were observed between inoculum densities and number of galls and egg masses and are represented by regression equations in Figures 6.6 and 6.7.

All the inoculum levels varied significantly regarding reproduction factor.

Maximum Rf (12.2) was found at the lowest inoculum level and minimum (4.94) at the highest level. An inverse relationship was found between levels and Rf as shown in Figure 6.8.

45 y = 8.194x - 0.21 40 R2 = 0.94 h t

g 35 n e l

t 30 o

ro 25 n

se i 20

15 ecrea

d 10 % 5

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.1: Effect of inoculum levels on the reduction (%) of root length of okra.

50 y = 11.431x - 9.731 45 R2 = 0.94

h 40 t g n

e 35 l t

o 30 o sh

n 25

se i 20

ecrea 15 d

% 10

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.2: Effect of inoculum levels on the reduction (%) of shoot length of okra

40 y = 8.019x - 4.827 35 R2 = 0.96 t

h 30 ig e w

t 25 o o r 20 in se a

e 15 r c

in 10 %

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.3: Effect of inoculum levels on the (%) increase in root weight of okra

60 y = 11.31x - 7.65 R2 = 0.94 50 t h ig e 40 t w o o

sh 30 in se a e

cr 20 e d % 10

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.4: Effect of inoculum levels on the (%) decrease in shoot weight of okra

45 y = 10.066x - 7.798 t 2 h R = 0.94

g 40 i e

w 35 t o

o 30 sh

y 25 r

d 20 n

se i 15 10 ecrea

d 5 % 0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.5: Effect of inoculum levels on the (%) decrease in dry shoot weight of okra

250 y = 46x - 22.64 R2 = 0.97 200 s l l 150 ga of r e b

m 100 u N

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.6: Effect of inoculum levels on number of galls caused by M. incognita

250 y = 44.6x - 25.6 R2 = 0.96 200 es s s a

m 150 g eg f

100 er o b m

Nu 50

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.7: Effect of inoculum levels on the egg masses of M. incognita

14 y = -1.647x + 14.059 12 R2 = 0.92 f) (R

10 r o t c 8 fa n o ti

c 6 u d o r

p 4 e R 2

0 500 1000 2000 4000 8000 Inoculum levels

Figure.6.8: Effect of inoculum levels on the Reproduction factor (Rf) of M. incognita

6.4 DISCUSSION

The progressive impairment in growth confirms the great damage potential

of M. incognita on okra. The effects of different inoculum densities of different

Meloidogyne species have also been studied by different workers on different hosts

(Siddiqui and Mahmood, 1992; Park et al., 1999; Krzyzanowski and Ferraz, 2000;

Haider et al., 2003; Akhtar et al., 2005; Guo et al., 2005; Sasanelli et al., 2006; El-

Sherif et al., 2007 and 2009; Neog and Bora, 2007 and Jiskani et al., 2008). The

findings of these workers confirmed that the increase in nematode population and

subsequent reduction in yield of crops or other manifestations of pathogenic

effects, physiological responses (total leaf chlorophyll content, CO2 exchange rate) and concentration of sodium, potassium, iron, manganese, copper and zinc are directly influenced by initial density of nematodes in soil (Wallance, 1973; Haseeb et al., 1990).There are reports that the damaging effects of M. incognita population levels were higher on younger plants as compared to older ones. This was due to the tenderness and succulence of tissues of younger plants being more attractive and susceptible for large number of nematodes. The older plants being harder and stronger, suffered less. Choudhury (1985) reported that one week old seedlings of tomato cv. Money maker did not tolerate the attack of M. incognita larvae, while 3 and 5 week old seedlings did. Salares and Capasin (1988) found that percent yield reduction of amplaya (Momordica charania) was lower on 8-week old plants compared with 2-, 4-and 6-week old plants when inoculated with different inoculum densities of M. incognita. Initial densities of M. incognita affected the rate of nematode multiplication; higher rates were observed where initial densities were lower. This might be due to destruction of root system and also due to the

failure the larvae of the subsequent generations to locate the new infection sites of subsequent generations (Ogunfowora, 1977). Similar observations have also been made by Khan (2003); Khan et al. (2004) and Pathak et al, (2000). It is therefore

concluded that M. incognita is pathogenic to A. esculentus at all population levels

and more damaging at higher densities.

Chapter 7

EFFECT OF BIOCONTROL AGENTS ON M. INCOGNITA INFECTING OKRA

7.1 INTRODUCTION

The root-knot nematode, M. incognita, is a sedentary endoparasitic plant

pathogen with a broad range of host-plant species. Damage caused by this

organism often goes unreported or is attributed to other causes with loss of crop

revenue annually amounting to millions of dollars (Choi, 1999). Use of chemicals

for nematode control, though very effective, cannot be adopted by the farmers of

underdeveloped countries due to being expensive and costly. In the developed

countries, nematicides like other chemicals have been under pressure, time and

again due to associated problems of residual toxicity, environmental pollution and

public health hazards (Thomason, 1987) and provide an impetus for the discovery

of innocuous yet efficacious methods of nematode control (Tzortzakakis and

Petsas, 2003). One of the viable alternatives to chemical nematicides is the use of

biological control agents, either alone or integrated with other pest management

strategies (Davies et al., 1991).

In biological control, myriads of antagonists have shown efficacy against

root knot nematodes. (Stirling, 1991). Among these Pochonia chlamydosporia, an egg parasite of root-knot nematodes, ubiquitous in distribution, has controlled root knot nematodes (Kerry, 2000). Another fungus Paecilomyces lilacinus infects eggs and females or Meloidogyne spp. and causes death of embryo in 5 to 7 days. The fungus has given excellent results under varying conditions. Similarly,

Trichoderma harzianum has been found effective biocontrol agent for the

management of root knot and other nematodes (Reddy et al., 1996; Rao et al.,

1998; Saifullah and Thomas, 1996; Sharon et al., 2001; Windham et al., 1986).

Furthermore, Pasteuria penetrans, an obligate parasite has been widely investigated for their efficacy against root-knot nematodes throughout the world

(Stirling, 1991; Oostendorp et al., 1991; Chen et al., 1996a; Duponnois et al.,

1999). It has the potentiality to be used as a bio-control agent both by interfering with nematode migration towards the roots and by limiting their reproduction

(Davies et al., 1991). The objective of the present studies was to evaluate these antagonists with different concentrations for the management of M. incognita in

pots.

7.2 MATERIALS AND METHODS

The bio-control potential of P. penetrans, P. chlamydosporia, P. lilacinus and T. harzianum was tested against M. incognita in pot experiment. The bio-

control agents were mass produced as described in Sections 3.10, 3.11 and 3.12

(Chapter, 3) and were mixed with the formalin sterilized soil at the rate of 2 x 103,

4 x 103, 6 x 103, 8 x 103, and 1 x 104 endospores / chlamydospores / cfus per g of soil. The treated soil was put in plastic pots (2 kg / pot). The okra seeds were sown in these pots and after germination one healthy seedling was maintained per pot.

Ten days after germination, the plants were inoculated with 2000 J2s of M.

incognita. The inoculated pots without antagonists served as control. The pots were

arranged in RCBD in a green house at 25 ± 2 oC for 6 weeks. The plants were watered as needed. After specified period, the plants were carefully removed, shoots were excised from the roots and data were recorded on shoot and root

lengths, root and shoot weights, number of galls, egg masses and reproduction

factor (Rf).

All the data were subjected to ANOVA after calculating percent increase /

reduction over control as described previously (Section 3.15 and 3.16).

7.3 RESULTS

7.3.1 SHOOT AND ROOT WEIGHT

The analysis of variance showed significant variations among different

antagonists, concentrations and their interaction regarding fresh shoot and root

weight and dry shoot weight as shown in appendices 7.1, 7.2 and 7.3.

Maximum increase in shoot weight was recorded where P. penetrans was applied followed by P. chlamydosporia. The increase in shoot weight as a result of application of P. lilacinus and T. harzianum was statistically similar from each other. Similarly, maximum average increase was recorded at a concentration of 1 x

104 which was statistically not different from the concentration of 8 x 103. The concentration of 2 x 103 resulted into minimum increase in shoot weight.

Maximum individual increase of 19.44% was recorded at a concentration of 1 x

104 spores of P. penetrans. The individual increases at each concentration of the antagonists are given in Table 7.1. Almost similar trends were observed in case of dry shoot weight which is given in Table 7.2.

Reduction in root weight was considered as an improvement of root health because root weight is affected by gall formation which results into increase in root weight. P. penetrans proved to be the most effective as it resulted into maximum

reduction in root weight. All the antagonists differed significantly in reducing root

weight. Similarly, concentrations of the antagonists also had significant effects on

root weight reduction. Maximum reduction was noticed at a concentration of 8 x

103 which did not differ significantly from 1 x 104 concentration. The individual

maximum increase was seen in case of P. penetrans at 1 x 104 concentration. The individual and average reductions are given in Table 7.1.3.

7.3.2 SHOOT AND ROOT LENGTH

The antagonists varied significantly in enhancing shoot and root lengths as indicated by their analyses of variance given in appendices 7.4 and 7.5. Maximum increase in shoot length was observed in case of P. chlamydosporia. P. lilacinus and P. penetrans proved equally effective in increasing shoot lengths. Minimum average increase was recorded in case of T. harzianum. Concentrations also affected increase in shoot length significantly. Maximum increase was observed at a concentration of 8 x 103. The average shoot length reduced significantly at 1 x

104 concentration. Minimum increase was recorded at the lowest concentration of antagonists. The average and individual increases are given in table 7.4.

Similarly, root length also increased significantly by the application of antagonists. Maximum increase was noticed in case of P. chlamydosporia and P. penetrans which showed no statistical difference from each other. T. harzianum caused minimum increase. The concentrations also showed variations in improving root lengths. Concentrations of 8 ¯ 103 and 1 ¯ 104 showed no significant differences in increasing root lengths. Similarly, concentration of 4 ¯ 103 and 6 ¯

103 were also statistically similar in their efficacy. The average and individual

increases in root lengths caused by antagonists at different concentrations are given

in Table. 7.5.

7.3.3 NEMATODE INFESTATION

The analysis of variance regarding number of galls, egg masses and

reproduction factor (Rf) showed significant variances amongst biocontrol agents

and their concentrations as shown in appendices 7.6, 7.7 and 7.8.

All the antagonists caused significant reductions in these parameters. P.

penetrans and P. lilacinus caused maximum reduction in number of galls and

proved equally effective. Similarly, T. harzianum and P. chlamydosporia also

showed similar reductions in root galls. Significant variations were also found

among concentrations of the antagonists. Maximum reduction was noticed at

concentrations of 8 ¯ 103 and 1 ¯ 104. The reduction observed at 1 ¯ 104

concentration was not statistically different from 8 ¯ 103. The average and individual reductions are given in Table. 7.6. A similar pattern was seen in case of egg masses. The reductions in egg masses caused by antagonists at different concentration are given in Table. 7.7.

The antagonists and their concentration caused significant decline in reproduction factor. Maximum average diminution was made by P. penetrans, followed by P. lilacinus. Similarly, the concentration of 1 ¯ 104 caused maximum decline in reproduction factor. The average and individual decreases are given in

Table 7.8.

Table.7.1: Effect of biocontrol agents on increase in fresh shoot weight at various concentrations.

Bio Control Agents % increase in fresh shoot weight at concentrations Means 2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 9.14 ± 0.53 c 11.12 ± 0.48 d 11.69 ± 0.97 de 16.12 ± 0.40 hi 15.91 ± 0.70 hi 12.80 ± 0.44 B

P. lilacinus 7.36 ± 0.48 ab 8.27 ± 0.36 bc 8.75 ± 0.63 bc 13.82 ± 0.33 f 14.63 ± 0.33 fgh 10.57 ±0.37 A

P .penetrans 9.44 ± 0.46 c 13 ± 0.58 ef 15.7 ± 0.58 gh 17.34 ± 0.62 i 19.44 ± 0.37 j 14.98 ± 0.74 C

T. harzianum 6.35 ± 0.28 a 8.97 ± 0.32 bc 9.33 ± 0.37 c 14.23 ± 0.40 fg 14.15 ± 0.69 fg 10.61 ±0.33 A

Mean 8.07 A 10.34 B 11.37 C 15.38 D 16.03 D

Data are means of five replicates. Means sharing common letters do not differ significantly from each other (P<0.05) according to Duncan Multiple Range Test Values followed by ± are standard errors of means

Table.7.2: Effect of biocontrol agents on increase in dry shoot weight at various concentrations.

Bio Control Agents % increase in dry shoot weight at concentrations Mean 2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 5.25 ± 0.47 a 7.93 ± 0.61 bcd 8.44 ± 0.57 cde 10.72 ± 0.43 f 10.28 ± 0.43 f 8.524 ± 2.24 B

P. lilacinus 4.17 ± 0.38 a 7.38 ± 0.39 bc 7.55 ± 0.61 bcd 7.93 ± 0.35 bcd 8.17 ± 0.44 bcd 7.040 ± 1.74 A

P. penetrans 6.92 ± 0.28 b 9.53 ± 0.40 ef 9.66 ± 0.43 ef 13.17 ± 0.43 g 14.76 ± 0.32 h 10.808 ± 2.96 C

T. harzianum 4.71 ± 0.25 a 6.82 ± 0.40 b 7.76 ± 0.50 bcd 8.85 ± 0.42 de 8.76± 0.22 cde 7.380 ± 1.73 A

Mean 5.262 A 7.915 B 8.352 B 10.167 C 10.492 C

Table.7.3: Effect of biocontrol agents on decrease in root weight at various concentrations.

Bio Control % decrease in root weight at concentrations Means 3 3 3 3 4 Agents 2 ¯ 10 4 ¯ 10 6 ¯ 10 8 ¯ 10 1 ¯ 10

P. chlamydosporia 10.42±0.28 bcd 10.72± 0.49 cde 12.75 ± 0.74 fg 13.44 ± 0.21 gh 13.62± 0.56 ghi 12.190 ± 0.34 C

P.lilacinus 9 ± 0.31 a 9.24 ± 0.47 ab 10.45± 0.35 bcd 12.75 ± 0.75 fg 12.54 ± 0.25 fg 10.796 ± 0.38 A

P. penetrans 10.92± 0.79 cde 11.9 ± 0.60 ef 13.89 ± 0.33 ghi 14.28 ± 0.33 hi 14.94 ± 0.61 i 13.186 ± 0.39 D

T. harzianum 8.6 ± 0.15 a 9.84 ± 0.60 abc 11.62 ± 0.58 def 13.82± 0.19 ghi 12.9 ± 0.40 fg 11.356 ± 0.43 B

Mean 9.735 A 10.425 B 12.178 C 13.573 D 13.500 D

Table.7.4: Effect of biocontrol agents on increase in shoot length at various concentrations.

Bio Control Agents % increase in shoot length at concentrations Mean 2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 5.13 ± 0.53 a 10.76 ± 0.36 cd 13.27 ± 0.39 f 17.73 ± 0.46 gh 16.82 ± 0.39 g 12.74 ± 0.95 C

P. lilacinus 4.83 ± 0.66 a 7.91 ± 0.40 b 11.58 ± 0.21 de 16.94 ± 0.48 gh 16.8 ± 0.76 g 11.61 ± 1.00 B

P. penetrans 4.35 ± 0.63 a 8 ± 0.44 b 10.73 ± 0.49 cd 18.38 ± 0.67 h 16.41 ± 0.48 g 11.57 ± 1.09 B

T. harzianum 5.21 ± 0.28 a 9.43 ± 0.31 c 9.75 ± 0.43 c 12.65 ± 0.37 ef 12.72 ± 0.74 ef 9.95 ± 0.59 A

Mean 4.88 A 9.03 B 11.33 C 16.42 E 15.69 D

Table.7.5: Effect of biocontrol agents on increase in root length at various concentrations.

Bio Control Agents % increase in root length at concentrations Mean 2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 5.13 ± 0.31 de 6.72 ± 0.26 fghi 6.73 ± 0.38 fghi 7.61 ± 0.50 i 7.44 ± 0.34 ghi 6.726 ± 0.23 C

P. lilacinus 3.83 ± 0.28 bc 4.16 ± 0.50 bcd 4.37 ± 0.50 bcd 5.75 ± 0.26 ef 5.82 ± 0.36 ef 4.786 ± 0.23 B

P. penetrans 5 ± 0.27 de 6.32 ± 0.31 fg 6.44 ± 0.29 fgh 7.31 ± 0.29 ghi 7.53 ± 0.56 hi 6.520 ± 0.23 C

T. harzianum 2.51 ± 0.26 a 3.28 ± 0.21 ab 3.47 ± 0.35 ab 4.81 ± 0.33 cde 4.93 ± 0.26 cde 3.800 ± 0.22 A

Mean 4.117 A 5.120 B 5.252 B 6.370 C 6.430 C

100

Table.7.6: Effect of biocontrol agents on decrease in root galls at various concentrations.

% decrease in number of galls at concentrations Bio Control Agents Mean

2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 12.28 ± 0.84 a 21 ± 0.52 c 22.96 ± 1.08 cd 31.68 ± 0.52 h 30.73 ± 1.28 gh 23.73 ± 1.49 A

P. lilacinus 13.75 ± 0.46 a 23.44 ± 1.01 d 26.82 ± 0.70 e 35.55 ± 0.61 i 36.6 ± 0.70 i 27.23 ± 1.74 B

P. penetrans 16.5 ± 0.29 b 27.33 ± 0.69 ef 27.75 ± 0.53 ef 32.7 ± 0.96 h 34.8 ± 0.83 i 27.82 ± 1.33 B

T. harzianum 14.45 ± 0.24 ab 22.93 ± 0.59 cd 23.65 ± 0.73 d 28.88 ± 0.82 efg 29.2 ± 0.41 fg 23.82 ± 1.12 A

Mean 14.24 A 23.68 B 25.30 C 32.20 D 32.83 D

101

Table.7.7: Effect of biocontrol agents on decrease in egg masses at various concentrations.

% decrease in number of egg masses at concentrations Bio Control Agents Mean

2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 10.62± 0.37 a 22.44± 0.66 c 23.18± 1.42 cd 29.65± 0.74 ef 30.46± 0.58 f 23.27± 1.49 A

P. lilacinus 14.91± 0.41 b 28.93± 0.93 ef 30.94± 0.70 f 37.71± 1.10 hi 36.82± 0.72 ghi 29.86± 1.70 C

P. penetrans 14.94± 0.66 b 22.78± 0.91 cd 35± 0.91 g 38.71± 1.08 ij 40.5± 0.45 j 30.39± 2.05 C

T. harzianum 15.62± 0.34 b 24.85± 0.35 d 27.8± 0.71 e 35.95± 0.57 gh 36.18± 0.57 gh 28.08± 1.58 B

Mean 14.02 A 24.75 B 29.23 C 35.51 D 35.99 D

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Table.7.8: Effect of biocontrol agents on decrease in Rf at various concentrations.

% decrease in reproduction factor at concentrations Bio Control Agents Mean

2 ¯ 103 4 ¯ 103 6 ¯ 103 8 ¯ 103 1 ¯ 104

P. chlamydosporia 20.18± 0.95 a 30.93± 0.80 b 35.47± 0.43 c 42.73± 1.07 ef 43.18± 0.92 ef 34.504 ± 1.77 A

P. lilacinus 19.75± 0.58 a 41.38± 0.69 e 43.91± 0.39 f 52.22± 0.95 h 52.37± 0.78 h 41.93± 2.45 C

P. penetrans 38.41± 0.68 d 43.63± 1.01 ef 47.53± 0.87 g 54.83± 0.74 i 56.73± 0.79 i 48.23± 1.44 C

T. harzianum 20.54± 0.61 a 36.78± 0.91 cd 38.85± 0.52 d 44.64± 0.94 f 50.48± 0.67 h 38.26± 2.08 B

Mean 24.72 A 38.18 B 41.44 C 48.61 D 50.69 E

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7.4 DISCUSSION

All the test antagonists significantly meliorated growth parameters and

caused decline in number of galls, egg masses and reproduction factors. The

antagonistic effects of these biocontrol agents have been reviewed in Section

2.13.2 of chapter 2. The improvement in various growth variables and nematode

infections can be ascribed to a number of mechanisms. The ability of P. penetrans

in the suppression of root-knot nematodes is attributable to (i) reduced root penetration by the spore-encumbered juveniles and/or (ii) failure to form egg masses by the infected females. Many researchers have reported that movement and mobility of juveniles were reduced and their capability to locate host roots was affected when juveniles were encumbered with endospores. Reduced motility probably leads to a high mortality of J2s in the soil (Chen et al., 1996a). Since the reproductive system fails to develop in the infected females of root-knot nematodes, such nematodes do not lay eggs resulting into reduced number of egg masses and reproduction factors. This leads to marked declines in the secondary infection by the second or subsequent generation juveniles.

P. lilacinus has been reported to be a potential biological control agent against root-knot nematodes and other plant parasitic nematodes (Franco et al.,

1981; Jatala, 1982; Adiko, 1984). It parasitizes eggs of Meloidogyne spp. and

Globodera pallida (Dunn et al., 1982; Jatala, 1986). This fungus also invades the females or cysts of a number of nematodes apecies (Franco et al., 1981; Gintis et

al., 1983; Jatala, 1982; Jatala, 1986). It exhibits chitinase activity when grown on

chitin agar medium (Gintis et al., 1983) and produces a peptidal antibiotic which

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has wide antimicrobial activity against fungi, yeast and gram positive bacteria

(Isogai et al., 1980; Isogai et al., 1981). P. lilacinus colonizes M. incognita eggs, preventing them from hatching and leaving fewer juveniles to penetrate root tissues

(Dunn et al., 1982; Jatala, 1986). The present studies indicate that antagonistic organisms certainly exert sufficient action in the soil to suppress the activity of nematodes.

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Chapter 8

EFFICACY OF ORGANIC AMENDMENTS IN CONTROLLING M. INCOGNITA ON OKRA

8.1 INTRODUCTION

The detrimental root-knot nematodes are cosmopolitan in distribution and occur in soil and are rarely exposed. Ways to reduce their damage are not much developed. Many noxious chemicals have been tried over the last few decades but only a few have stood the test of time. Most of these compounds are over expensive and out of the reach of the farmers holding limited means. Of various control strategies, organic amendments have gained much attention of the scientists, as it is safe to use as far as environmental hazards are concerned.

The incorporation of organic material into the soil reduces root-knot nematodes densities, resulting into an increase in yield (Muller and Gooch, 1982).

Oilcakes, sawdust, urea and biogases have been used with some success (Singh and Sitaramaiah, 1966, 1967). Chitin in combination with waste products from the paper industry has been used to reduce root-knot nematodes (Culbreath et al.,

1985). Although the use of organic amendments for effective nematode control is often limited by the large quantities needed, they can reduce nematode population densities to different degrees. In addition to their suppressive effects on nematode density, organic amendments provide better media to grow, result in better soil texture, increase water holding capacity, supply the nutrients to deficient soil and stimulate microbial population of actinomycetes, bacteria, fungi and other elements which might be antagonistic to nematodes (Badra et al.,1979; Godoy et al., 1983;

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Rodriguez-Kabana, 1986). Pallai and Desai (1976) reported that undecomposed

cake of Callophyllum inophyllam gave best control of M. javanica on tobacco but cake was allowed to be decomposed for 15 days before application gave better plant growth and nematode control was directly proportional to the quantity of

cake added. Haseeb et al. (1977) observed highest reduction in nematode

population when soil was amended with chopped leaves of Calotropis procera

while Iserin herbstii was highly effective in promoting the growth of egg plant.

Keeping in view the beneficial effects of organic amendments, the present study

was carried out to evaluate the efficacy of leaves of Azadirachta indica, Calotropis

procera, Datura stramonium and Tagetes erecta as soil amendments at various dosages.

8.2 MATERIALS AND METHODS

The leaves of A. indica, C. procera, D. stramonium and T. erecta were washed and dried under shade. The dried leaves were then mixed thoroughly with formalin sterilized soil @ 25, 50 and 75 g / Kg of soil. The amended soils, 5 Kg, were transferred to 20 cm diameter pots along with 1 g of nitrogen. The pots without organic matter served as control. The pots were watered daily to facilitate decomposition of organic matter. Two weeks after amendment, 3 seeds of okra cv.

‘Punjab selection’ were sown and after germination one healthy seedling was maintained. The plants were inoculated with 5000 J2s of M. incognita (Section 3.3,

Chapter, 3) 10 days after germination. Each treatment was replicated five times.

The pots were arranged in a completely randomized block design in the green house at 25 oC ± 2 for six weeks. The pots were watered when needed. After

stipulated period, the plants were carefully removed, the roots were excised from

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the shoot, carefully washed under water and blotted dry. The data were recorded on

root and shoot lengths, shoot and root weights, number of galls, egg masses and

reproduction factor (Rf = Pf/Pi). The percent increases and reductions in these

parameters were calculated over control as described in section 3.15. All the data

were analyzed statistically as described in Section 3.16, Chapter, 3.

8.3 RESULTS

The analysis of variance regarding effect of organic amendments on growth

parameters of okra and number of galls, egg masses and reproduction factor

showed significant variations.

8.3.1 ROOT AND SHOOT LENGTH

The analysis of variance regarding root and shoot lengths showed

significant effects of organic amendments, their concentrations and the interaction

between amendments and their concentrations as shown in Appendices 8.1 and 8.2.

Root and shoot lengths were found maximum in case of A. indica followed by C. procera. Similarly maximum increase in these parameters was recorded at the concentration of 75 g / kg of soil. The amendment @ 25 g / kg of soil was the least effective. It was observed that with the increase in concentration, there was a corresponding increase in these parameters over control. The individual root and shoot lengths and relationships between these parameters and concentrations of individual amendments are given in Figures 8.1 and 8.2.

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8.3.2 SHOOT AND ROOT WEIGHT

The analysis of variance regarding fresh root and shoot weights and dry shoot weight showed significant effects of the amendments, their doses and the interaction between them (Appendices 8.3, 8.4 and 8.5) All the amendments significantly increased percent fresh and dry weights over their controls being maximum in case of A. indica followed by C. procera. Similarly increases in these parameters were higher at higher concentrations of the amendments. A direct relationship was observed between the concentrations of the amendments and these parameters which are shown with trend lines and equations in Figures 8.3 and 8.4.

On the other hand, a decrease in root weight was observed by the applications of amendments. The reductions in root weights were maximum in case of C. procera and D. stromonium. Similarly, the magnitude of reduction increased with an increase in the concentration of amendments and this increase in reduction of root weight was found directly proportional to the dosage of the amendments. The relationships between doses of individual amendments and reduction in root weight are shown with trend lines and regression equation in

Figure 8.5.

8.3.3 NUMBER OF GALLS, EGG MASSES AND

REPRODUCTION FACTOR

The analysis of variance regarding number of galls, egg masses and reproduction factor showed significant effects of amendments, their concentrations and the interaction between amendments ant their concentration as shown in

Appendices 8.6, 8.7 and 8.8. All the amendments caused significant reductions in

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these parameters. Maximum reductions were observed with A. indica and D. stromonium. Similarly, higher concentrations of amendments caused maximum reductions as compared to others. The reductions in these parameters were found directly proportional to the concentrations. These relationships are shown by trend lines and regression equation in Figures 8.6, 8.7 and 8.8 respectively.

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A. indica C. procera D. stramonium T. erecta

10 y A.I = 2.915x + 0.2033 R2 = 0.9787 9

y C.P = 2.9x - 0.6033 8 R2 = 0.989 e s

a 7

e y D.S = 1.825x + 1.35 r

c 2

n 6 R = 0.995 i

h t

g 5 y T.E = 1.8x + 1.25

n 2 e R = 0.75 l 4 oot

r 3 % 2

0 25 50 75 Concentrations

Figure 8.1: Effect of organic amendments at various concentrations on % increase in root length. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E ( Tagetes erecta)

A indica C procera D stramonium T erecta

30 y A.I = 8.2x + 0.4 R2 = 0.9982

25 y C.P = 4.175x + 0.95 h

t R2 = 0.9999 g n

le 20 y D.S = 2.755x + 3.3733 t R2 = 0.9696 oo h s 15 y T.E = 5.745x + 0.37 in

e R2 = 0.9417 s a e r

c 10 in % 5

0 25 50 75 Concentrations

Figure 8.2: Effect of organic amendments at various concentrations on % increase in shoot length. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E ( Tagetes erecta)

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A.indica C.procera D.stramonium T.erecta

50 yA.I = 14.775x - 2.0833 2 45 R = 0.9697

40 y T.E = 0.5076x - 1.3933 t 2 h R = 0.9646 g 35

wei y = 9.625x + 0.6967

t C.P

o 30 2 o R = 0.9654

sh 25 n y D.S = 0.343x + 3.0867 i e

s 2 20 R = 0.9168 crea

n 15 i % 10

0 25 50 75 Cocentrations

Figure 8.3: Effect of organic amendments at various concentrations on % increase in shoot weight. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta) A indica C procera D stramonium T erecta

14 y A.I = 4.09x - 0.2733 R2 = 1 12

ht y C.P = 4.465x - 1.3567 g i 2 e R = 0.9971

w 10 t o

o y D.S = 2.84x + 1.26 h

s 8 R2 = 0.9999 y r d

n y T.E = 3.035x - 0.2933

i 6

e 2

s R = 0.9988

crea 4 n i % 2

0 25 50 75 Concentrations

Figure 8.4: Effect of organic amendments at various concentrations on % increase in dry shoot weight. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E ( Tagetes erecta)

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A indica C procera D stramonium T erecta

18 y A.I = 5.51x - 2.4133 2 16 R = 0.966

y = 5.39x - 1.82 14 C.P R2 = 0.9977 t h

ig 12 e y D.S= 3.6x + 1.7133 w

t 2

o R = 0.9995

o 10 r

e y = 4.025x - 0.2767 s 8 T.E a 2 e

r R = 0.9989 c

in 6 %

0 25 50 75 Concentrations

Figure 8.5: Effect of organic amendments at various concentrations on % reduction in root weight. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E ( Tagetes erecta)

A indica C procera D stramonium T erecta

70 yA.I = 18.45x + 3.7667 R2 = 1

60 y C.P = 14.95x + 3.5667 R2 = 0.8868

y D.S = 18.45x + 3.2

lls 50 a R2 = 0.9885 g

in 40

s y T.E = 12.65x + 7 n R2 = 0.9996 io t

c 30 u d e

r 20 %

0 25 50 75 Concentrations

Figure 8.6: Effect of organic amendments at various concentrations on % reductions in galls. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)

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A indica C procera D stramonium T erecta

80 y A.I = 22.95x - 9.4 R2 = 0.9912 70 y C.P = 19.9x - 2.2333

s 2 e 60 R = 0.992 ss a y D.S = 20.1x + 8.6 m

g 50 2

g R = 0.9739 e n 40 y D.S = 12.95x + 12.567 s i 2 n R = 0.9862 o ti

c 30 u d e

r 20 %

0 25 50 75 Concentrations

Figure 8.7: Effect of organic amendments at various concentrations on % reductions in egg masses. A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)

A indica C procera D stramonium T erecta

80 y A.I = 21.5x - 3.0333 R2 = 0.9995

70 y C.P = 21.5x - 1.2 R2 = 0.9936 60 y D.S = 19.3x + 15.467

f 2

R R = 0.9568 50 i n

o y T.E = 14.4x + 14.7 i t 40 R2 = 0.9857 duc e

r 30 %

0 25 50 75 Concentrations

Figure 8.8: Effect of organic amendments at various concentrations on % reductions in reproduction factor (Rf). A.I (Azadirachta indica), C.P (Calotropis procera), D.S (Datura stromonium) and T.E (Tagetes erecta)

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8.4 DISCUSSION

All the amendments caused significant reductions in M. incognita infections resulting into increase in growth parameters. Higher doses were more effective as compared to lower ones. The results are in conformity with those of

Muller and Gooch (1982), Ali (1990) and Akhtar and Alam (1990). The use of organic soil amendments is the cheapest and effective way for the control of plant diseases caused by the nematodes. It not only changes the physical properties and other chemical reactions of of soil but also develops a wide variety of antagoniststic microorganism like fungi, bacteria etc., which later on by the phenomenon of competion, antibiosis or parasitism retard the population of the plant disease inciting agents like fungi, bacteria and nematodes etc. The addition of soil ammendments results into considerable increase in the liberation of CO2 by the saprophtic activities of soil saprophytes and thus in return suppress the activities of disease causing agents (Papavizas and Davey, 1992). Due to rapid multiplication of micro-organisms within the soil, the siol nitrogen, which is often scanty in soil, is utilized by the soil saprophytes rapidly and a huge scarcity of nitrogen is created by the soil saprophytes. The nitrogen deficiency reduces the growth of pathogens greatly. It has also been reported that the nematodes population may be reduced due to the accumulation of toxic substances, which are produced by the decomposition of organic amenments in soil (Akhtar et al., 1982 and Khan et al.,

1966). Sayre (1980) postulated two hypothesis, which explain the effectiveness of soil amenments in two ways. Firstly the decomposition products from amendments into soil are directly toxic to plant nematodes and secondly mainipulation of soil microbial population by addition of amendments initiates a succession of events

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favoring the build up of bacteria, microbivorers, nematode trapping fungi and other soil antagonists that destroy plant parasitic nematodes. Dropkin (1980) repoted the action of soil amendments in a different way. According to him organic matter is incorporated into soils by many organisms (from bacteria to earthworms) and ultimately forms humus. The break down of organic matter releases compounds into soil that may be toxic to nematodes. The microorganisms release simple organic acids such as acetic, propionic and butyric acids, which remains for several weeks in concentrations sufficient to kill some phytonematodes, but are not toxic to free living species.

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Chapter 9

GENERAL DISCUSSION

The survey of root-knot nematodes in 17 districts of the Punjab province of the country divulged that these nematodes are widespread on okra in these districts.

Among the four most common root-knot species, M. incognita and M. javanica were found associated with okra in all the districts. M. arenaria and M. hapla were only found in Rawalpindi district. M. incognita was the most predominant species in these areas. The above mentioned four species of root-knot nematodes are considered to be the most common and M. incognita being ranked first with respect to geographical distribution and host range (Taylor et al., 1982). In the present survey too, M. incognita was found to be more widely spread than the other species. On global basis M. incognita has been reported to constitute about

47 per cent of the total root-knot nematode population (Sasser and Carter, 1985) and in the present study the frequency of occurrence of this species was found to be 74.74 per cent. This variance may be attributed to soil characteristics and climate of this particular study area. In other parts of the country such as Karachi and Sindh too, M. incognita has been found to dominate over the other species

(Ahmad and Saeed, 1981). M. hapla was isolated from Rawalpindi district only

which has cool climate. The present finding is in conformity with those of Sasser

and Carter (1985), Taylor et al. (1982) who regarded M. hapla as a cool climate

species. In the present survey, 100 percent prevalence of root-knot nematodes has

been recorded in Multan, Okara, Dera Ghazi Khan, Bahawalnagar, Vehari, Rahim

Yar Khan and Rawalpindi districts and a minimum prevalence of 22.4% was found

in Lodhran district. The incidence was above 60% in Bahawalnagar, Rahim Yar

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Khan, Dera Ghazi Khan and Vehari and was only 4.44% in Lodhran. The severity

of infection of the nematodes was highest in Bahawalnagar and Vehari, while it

was lowest in Lodhran. Earlier survey conducted by Khan et al. (2005) showed 78

% incidence of root knot nematodes in Pakistan. The high incidence and severity of root-knot nematodes in these districts was due to the cultivation of susceptible varieties, high temperature and comparatively less annual rainfall. These conditions favored the multiplication, development and infection of root-knot nematodes. There are reports which showed that nematode populations are influenced by soil type (Kincaid, 1946; Wallace, 1969; Prot and van Gundy, 1981;

Jain, 1992). In the present studies M. incognita and M. javanica were found in all the districts in varying proportions and M. incognita being the most predominant.

Trudgill et al. (2000) reported that M. incognita and M. javanica were the most

widespread root-knot nematode species in all the countries. Similar results have

also been reported by many workers (Khan, et al., 1993; Campos, 1994; Das and

Das, 2000; Ravichandra and Krishnappa, 2004; Bhosle et al., 2004; Rathour et al.,

2006). The distribution and infestation of Meloidogyne spp. in the soils of Pakistan

was M. incognita, 52%, M. javanica, 31%, M. arenaria, 8%, M. hapla, 7% and

other species about 2% (Maqbool, 1987) which further proved the present findings.

M. arenaria and M. hapla were isolated only from Rawalpindi district. The climate

of the district is cool, humid and mild. These results confirmed the finding of

Brown (1962) that M. arenaria and M. hapla are cool, humid and hilly climate

species. Gul and Saeed (1987) also reported M. arenaria and M. hapla from North

West Frontier Province (NWFP) of Pakistan. It is concluded from the present

studies that okra is severely attacked by root-knot nematodes and M. incognita is

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the most predominant, which warrants that strict control measures should be

adopted for its management.

A total of twelve cultivars of okra were screened for their resistance against

M. incognita, and none was found immune or resistant against the pathogen. The cultivar, Punjab Selection was found to be highly susceptible while 9 cultivars viz.

Sanam, Dikshah, Sabz Pari, Arka Anomika, Iqra-1, Iqra-2, PMS-55, PMS Beauty and Super Star were rated as moderately susceptible.

The variations in resistance to M. incognita among various okra cultivars

are due to many mechanisms. Resistant plants prevent the nematodes from

completing their life cycles in their roots. Immune plants prevent all nematode

development in their tissues. The roots of plants resistant to a species or race of M. incognita are invaded by second stage juveniles but the multinucleate syncytia (the giant cells) that form around the heads of sedentary juveniles in the roots of susceptible plants, which are essential to the development of egg-laying females, are absent, fewer or smaller. As a result, only a few females develop to maturity, though males may develop from small syncytia. Second stage juveniles may even vacate resistant roots, soon after entering them. In roots of the resistant peach rootstock ‘Nemaguard’, the syncytia that form in response to feeding of M. javanica are surrounded by a dense layer of cells, followed by degeneration of syncytia, resulting into failure of the females to develop (Meyer, 1978). In resistant cowpea roots invaded by M. incognita, sclereids formed in the root cortex and there were fewer synctia, which were small and had few nuclei. Cells died around the juveniles in a hypersensitive reaction to invasion of the roots of one of the cowpea lines studied (Singh et al., 1984).

119

There have been several comparative biochemical studies of roots of

resistant and susceptible cultivars. Resistance to M. graminicola in three resistant

rice cultivars was associated with an increase in the silica content of the roots as

the plants aged, which did not occur in two susceptible cultivars (Swain and

Prasad, 1988). Most other studies have been on tomatoes. In resistant, but not in

susceptible cultivars of tomato, invasion of the roots by M. incognita stimulated

the synthesis of ascorbic acid. Reduced ascorbic acid in the roots increases their

susceptibility to the nematode (Arrigoni et al., 1979a). Conversely, increasing the

amount of ascorbic acid in the roots of susceptible tomatoes induced resistance in them to the nematode (Arrigoni et al., 1979b). Spraying tomato foliage with

ascorbic acid (4000 ppm), before and after planting, significantly decreased the

number of eggs of M. incognita produced by the roots and increased tomato fruit

yield (Al-Sayed, 1990). When tomato roots were inoculated with M. incognita, the

concentrations of phenol and orthodihydroxyphenol increased more in the roots of

the resistant cv. Anahu than in the roots of susceptible cv. Marglobe.

Polyphenoloxidase increased more in the former than in the later (Hassan and

Sexena, 1979). Accelerated senescence of Polyphenoloxidase isozymes was seen

in susceptible cv. Pusa Ruby but not in resistant SL-120 tomato roots (Ganguly and

Dasgupta, 1984). In resistant cotton roots, there is increased polyphenoloxidase

and peroxidase activity involved in the necrotic reaction to invasion by M.

incognita. Resistance also appears to be linked to the presence of gossypol, a

terpenoid aldehyde, at more than 0.52% of dry root matter (Okopnyi et al., 1983).

In sweet potato, root extracts of resistant plants following invasion by M. incognita

and M. javanica, contained larger amounts of phenolics (principally chlorogenic

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acid, scopoletin and esculin) than did root extracts of susceptible plants (Gapasin et

al., 1988).

Zacheo and Bleve-Zacho (1988) found that infestation of tomato roots by

M. incognita led to the production of superoxide anion (O2). These were scavenged by increased superoxide dismutase (SOD) activity in susceptible cultivars, whereas in resistant cultivars there was less SOD activity, so there was an increase in O2

anions. They suggested this might cause cell necrosis and the hypersensitive

reaction common in resistant plants. Similarly, SOD activity increased in

susceptible cowpea roots inoculated with M. incognita but decreased in resistant

cowpea roots (Ganguly and Dasgupta, 1988). In cultivars of excised tomato roots,

hydroxyurea at 10 or 20 ppm in the culture medium induced resistance to M.

javanica by suppressing the synctyia (Stender et al., 1986). Glyceollin

accumulated (mainly in the stele) in roots of resistant Centennial soybean two to

three days after they had been inoculated with M. incognita. This did not occur in

the roots of the related but susceptible cv. Pickett, nor in either cultivar exposed to

M. javanica, to which both are susceptible. As glyceollin inhibited movement of

M. incognita but not M. javanica, it might be a factor for resistance in Centennial

(Kaplan et al., 1980). Finally, terpenoid aldehydes (polyphenols), which are toxic

to M. incognita, were produced more rapidly in the endodermis and stele of the

roots of cotton cultivars resistant to the nematode (Veech, 1977 and 1979).

However there was no significant negative relationship between the terpenoid

aldehyde content of healthy roots and the number of egg masses of M. incognita in

ten strains of cotton, whereas there was a significant negative correlation between

the terpenoid aldehyde content of 17 other strains of cotton and the incidence of

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Fussarium oxysporum f.sp. vasinfectum (Hedin et al., 1984). More recent work has shown that some resistant lines of cotton produce less terpenoid aldehydes when inoculated with M. incognita than susceptible lines, so terpenoid aldehydes cannot, in general, be the factor governing infestation in resistant cotton (Khoshkoo et al.,

1994).

Cultivar resistance to Meloidogyne spp. may break down when soil

temperatures are high, when the roots are also attacked by certain fungi and, more

commonly, when a cultivar is exposed to a species or race to which it is

susceptible.

At soil temperatures in excess of 30 oC, cultivars may lose their resistance

to root-knot nematodes, so the species or races to which they are resistant at lower

temperatures are able to multiply on the roots. Four tobacco lines highly resistant

to M. incognita at 27 oC became moderately susceptible at 35 oC (Ohashi, 1977).

Similarly, two tobacco cultivars resistant to M. javanica at 27 oC lost their

resistance at 35 oC (Fukudome and Kamigama, 1982). In tomato cv. VFN 8,

resistance to M. incognita decreased progressively as temperature rose from 28 to

37 oC. At 34 oC, 50% of invading juveniles completed their life cycles in the roots, which showed maximum galling. Peroxidase activity ceased after the nematodes had become established in the roots (Zacheo and Bleve-Zacheo, 1984). In this same cultivar exposed to M. incognita, polyphenoloxidase and peroxidase activity

o o increased in infested roots at 27 C not at 34 C. An O2-generating system and lipid peroxidation increased at 27 oC but decreased at 34 oC. In inoculated roots, SOD and catalase activity, which were unchanged at 27 oC, were increased at 34 oC. All this suggests that the higher temperature induced susceptibility to the nematode as

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a result of decline in superoxides and hydrogen peroxide, due to the activity of

SOD and catalase (Zacheo et al., 1993). In nine other cultivars of tomato, however,

little loss of resistance to M. incognita was observed above 30 oC (Roberts and

May, 1986). Although high temperatures have been reported not to affect the resistance of cotton to M. incognita (Jorgenson, 1979), in a later study (Carter,

1982) M. incognita completed its life cycle on Auburn 623 cotton at 35 oC, at which temperature there were as many or more nematodes in the resistant roots as in the roots of susceptible Deltapine 16 cotton. Resistance to M. incognita, which is heat-stable at 25 and 30 oC, has been transferred from Lycopersicon peruvianum

to tomato (L. esculentum) hybrids, from which cultivars with heat-stable resistance

may be produced (Cap et al., 1991). When either Rhizoctonia solani or Pythium

aphenidermatum was inoculated with or after M. incognita in capsicum annum cvs

Jowala or Longthin Faizabadi, resistance to the nematode broke down (Hasan,

1985).

Studies on the influence of inoculum levels of M. incognita on okra

disclosed that significant reductions in various growth parameters have been

caused by the nematode densities. The numbers of galls and egg masses have been

found directly proportional to the inoculum levels. Several studies conducted by

others also confirmed the present findings (Wonang and Akueshi, 1990; Devi and

Das, 1994; Aparajita et al., 1998; Khan et al., 2004a; Yousuf and El-Nagdi, 2004;

Bora and Neog, 2006; Mishra and Usha, 2007; Senthamarai et al., 2006).

All the tested antagonists proved effective in controlling M. incognita. All

the antagonists significantly increased the root and shoot lengths and weights and

caused reductions in number of galls and egg masses. Pochonia chlamydosporia

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and Pasteuria penetrans were equally effective at a concentration of 8 ¯ 103

chlamydospores / endospores per gram of soil.

Paecilomyces lilacinus and Pochonia chlamydosporia (Verticillium

chlamydosporium), can parasitize both the egg and female stages of the nematode

(Morgan-Jones et al., 1982, 1983; Rodriguez-Kabana et al., 1984; Freire and

Bridge, 1985; de Leij and Kerry, 1991; Siddiqui and Mahmood, 1996). P.

chlamydosporia is primarily regarded as an egg parasite; observations have shown

that during the initial stages of infection, it produces a branched mycelial network

that is in close contact with the eggshell (Morgan-Jones et al., 1983; Lopez-Llorca

and Duncan, 1988; Lopez-Llorca and Claugher, 1990). Penetration of the eggshell

occurs both from a specialized penetration peg, an appressorium, and also from

lateral branches of mycelium, and leads to the disintegration of the vitelline layer

and dissolution of the chitin and lipid layers (Segers et al., 1996; Morton et al.,

2004). Enzymes are thought to be important in the infection process, and

experiments indicate that a cocktail of proteases and chitinases are necessary to

initiate infection. Proteases of nematode parasites have been characterized,

including one from Verticillium suchlasporium (Lopez-Llorca and Robertson,

1992) and another from P. chlamydosporia (Segers et al., 1996). Studies of

different isolates of P. chlamydosporia have shown that they produce a range of

different subtilisins (Segers et al., 1998).

P. lilacinus has been tested widely for its potential as a biological control

agent and shown to suppress nematode population densities and increase plant

yields. The fungus appears to be a good root colonizer (Cabanillas, et al., 1988)

and rhizosphere competitor. The fungus first colonizes the gelatinous matrix of

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Meloidogyne and eventually a mycelial network develops and engulfs the

nematode eggs. Penetration of nematode eggs is completed with an appressorium

or simple hyphae (Holland et al., 1999; Jatala, 1986). Both mechanical and enzymatic activities may be involved in the penetration. Morgan-Jones et al.

(1984) reported that the fungal hyphae penetrated the eggshell of M. arenaria through small pores dissolved in the vitelline layer. Following penetration, the fungus grows and proliferates in the eggs in early embryonic development. After depleting all nutrients in the egg, the mycelium may penetrate and rupture the cuticle of the infected egg from within and then emerge to infect other eggs in the vicinity. The fungus may also colonize the juveniles within the eggshell, and the

3rd and 4th stages of juveniles on water agar (Holland et al., 1999). Culture filtrates of P. lilacinus were toxic to nematodes (Cayrol, et al., 1989; Chen, et al.,

2000a; Khan and Goswarni, 2000). Cuticles of nematodes were ruptured, and the nematodes were killed within a few hours after exposure to the culture filtrates. A peptidal antibiotic P-168 has been isolated from culture of P. lilacinus and

characterized (Isogai et al., 1980). This substance has anti microbial activity

against fungi, yeast, and gram-positive bacteria, and therefore may enable the

fungus to compete with soil microorganisms.

Trichoderma is a ubiquitous soil fungus and colonizes the root surface and

root cortex. Several species of Trichoderma viz. T. harzianum, T. viride, T.

atroviride, and T. asperellum provide excellent control of root-knot nematodes

(Sharon et al., 2001 Sharon et al., 2007). Application of Trichoderma results in

reduced nematode galling and improved plant growth and tolerance (Spiegel and

Chet, 1998). The highly branched conidiophores of Trichoderma bear conidia that

125

can attach to different nematode stages. Conidial attachment and parasitism varies between fungal species and strains (Sharon et al., 2007). This process was often associated with the formation of fungal coiling and appressorium-like structures. T. harzianum colonizes isolated eggs and J2 of M. javanica (Sharon et al., 2001).

Successful parasitism of the nematode by Trichoderma requires mechanisms to facilitate penetration of the nematode cuticle or eggshell. The involvement of lytic enzymes has long been suggested in Meloidogyne parasitism and demonstrated

(Spiegel et al., 2005). Besides direct antagonism, other mechanisms involved in

Meloidogyne control by Trichoderma spp. include fungal metabolites and induced resistance (Freitas et al., 1995, Goswami et al., 2008, Rodriguez-Kabana et al.,

1984; Umamaheswari et al., 2004). In general, Trichoderma should be applied before planting to achieve maximum nematode control (Dababat et al., 2006). In all cases, good establishment of the fungus in the rhizosphere seems to be important for nematode control.

Incorporation of leaves of Azadirachta indica, Calotropis procera, Tagets erecta and Datura stromonium in the soil @ 25, 50 and 75 g / kg of soil controlled

M. incognita to varying degree. A. indica and C. procera caused maximum reductions in number of galls, egg masses and reproduction factor (Rf) of the root- knot nematodes resulting into increases growth parameters.

The reduction in galls, egg masses and reproduction factor and increase in growth parameters following the application of organic amendments might be (i) due to production of large number of predators and parasites during the decomposition of soil amendments which may attack the nematodes causing reduction in their population (Linford et al., 1938; Duddington, 1962; Sing and

126

Sitaramaiah, 1969). (ii) The decomposed products of the soil amendments may be directly toxic to nematodes (Patrick et al., 1965; Mankau and Das, 1969; Khan et al., 1974; Sitaramaiah and Singh, 1978). (iii) The microbial metabolites produced during decomposition of amendments may be toxic to nematodes (Tomerlin and

Smart, 1969; Mankau and Das, 1969). (iv) The materials used as soil amendments may be directly toxic to nematodes (Ellenby, 1945). (v) The amendment may change soil pH which may be unfavorable for nematode multiplication (Singh and

Sitaramaiah, 1969). (vi) The soil temperature also changed during decomposition of amendments in soil which interferes in the normal activity of nematodes.

Johnson (1963) observed temperature as a major factor in the control of tomato root-knot with oat straw. Sitaramaiah (1977) observed significant reduction in

Tylenchorhynchus elegans population with the addition of margosa cake in soil at

20 oC and above, indicating the role of temperature on the efficacy of oil cakes as a nematicide. Similarly, production of volatile fatty acids, phenols, amino acids etc. during decomposition of organic soil amendments may cause inhibitory effect to the nematodes. Sayre et al. (1965) and Toussoun et al. (1969) reported the presence of propionic, n-butyric, iso-butyric and phenyl propionic acids during decomposition of crop residues. Sayre (1965) reported 50% killing of nematode larvae in soil by 1350 ppm butyric acid. Sitaramaiah and Singh (1969) observed weak molar solutions of butyric and propionic acids increasing egg release and hatching of M. javanica. (Alam et al., 1979) studied the mechanism involved in the control of plant parasitic nematodes by applying oil cakes of mahua, castor, mustard, neem and groundnut having formaldehyde concentrations of 0.966, 0.088,

0.316, 0.258 and 0.475 and acetone concentration of 0.900, 0.110, 0.220, 0.280 and

127

0,180 mg per 100 mg sample, respectively. They observed 100% mortality of five nematode species in 1000 ppm formaldehyde after 12 hrs but in acetone the mortality varied from 2-4% even at 10,000 ppm. Larval hatch of M. incognita decreased with increasing formaldehyde concentration but acetone did not affect hatch. Considerable amount of phenols was detected in the oil cakes of mahua, castor, mustard, neem and groundnut with highest concentrations in mustard cake

(Alam et al., 1979). In a study conducted to see the effect of ten phenolic and related compounds on the mortality and population of nematodes, they found that all the compounds tested were highly deleterious to Hoplolaimus indicus,

Helicotylenchus indicus, R. reniformis, T. brassicae and Tylenchus filiformis both in vitro and in vivo. However, their effect was selective. Hydroquinone, p-cresol, catechol, pyrogllol and gallic acid were found most toxic. Alam et al. (1977) found suppression in the population of T. brassicae attacking knol-khol by applying oil cakes, bone meal, and horn meal. They observed increase in the level of phenols in the host roots which might have induced certain degree of resistance in host plant.

Nematicidal properties of certain organic chemicals were tested by Hussain and

Masood (1973) against Helicotylenchus species in vitro. Formaldehyde, ammonia, diastase, salicylic acid, propionic acid, acetic acid, isopropyl alcohol and methyl alcohol were found possessing marked nematicidal properties. Khan et al. (1974) reported that ammonia liberated as a result of the decomposition of oil cakes

(mahua, castor, mustard, neem and groundnut) was found to be toxic to varying degrees to several species of plant parasitic nematodes. Krishnan and Valthee

(1984) reported that uninoculated plants subjected to varying doses of groundnut oil cakes showed a gradual increase in free amino acid contents as against control

128

plants. The increase in amino acid content might be due to host response in synthesizing new amino acids through metabolic pathways, and degradation of old protein. In case of M. incognita plants, subjected to varying dosages of oil cakes, the impact of infection was found to be reduced by the nutritional stress as seen through the decreased amount of free amino acid as against the infected plant not subjected to nutritional stress.

CONCLUSIONS

From the present studies following conclusion have been made

• Root-knot nematodes (Meloidogyne species) are commonly prevalent in all

the okra growing areas of the Punjab province of the country.

• M. incognita is the most predominant specie found to be associated with

okra in all the surveyed districts followed by M. javanica.

• M. arenaria and M. hapla was found only in Rawalpindi district.

• A minimum level of 1000 J2s caused significant reductions in growth

parameters of okra. The damaging effects increased with an increase in the

inoculum level.

• No resistant source was identified from the tested germplasm, however

some moderately susceptible cultivars were identified.

• All the antagonists significantly controlled M. incognita to varying degrees.

• All the organic amendments reduced nematodes infestations. A. indica and

T. erecta at a concentration of 75 g/kg of soil gave better control as

compared to others.

129

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APPENDIX

Appendix-5.1: Analysis of variance for % decrease in root length

S.O.V D.F S.S M.S V.R F pr. Blocks 4 1.7827 0.4457 1.22 Variety 11 5194.9755 472.2705 1287.52 <.001* Residual 44 16.1395 0.3668

Total 59 5212.8977 *HS Highly significant

Appendix-5.2: Analysis of variance for % decrease in shoot length

S.O.V D.F S.S M.S V.R F pr. Blocks 4 0.5834 0.1458 0.35 Variety 11 1890.3055 171.8460 415.02 <.001* Residual 44 18.2188 0.4141

Total 59 1909.1077 *HS Highly significant

Appendix-5.3: Analysis of variance for % decrease in dry shoot length

S.O.V D.F S.S M.S V.R F pr. Blocks 4 14.9548 3.7387 5.71 Variety 11 10787.6699 980.6973 1498.24 <.001* Residual 44 28.8009 0.6546

Total 59 10831.42 *HS Highly significant 191

Appendix-5.4: Analysis of variance for % increase in root weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 9.3650 2.3412 6.46 Variety 11 7909.4161 719.0378 1983.28 <.001* Residual 44 15.9522 0.3626

Total 59 7934.7333 *HS Highly significant

Appendix-5.5: Analysis of variance for % decrease in shoot weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 14.9548 3.7387 5.71 Variety 11 10787.6699 980.6973 1498.24 <.001* Residual 44 28.8009 0.6546

Total 59 10831.4256 *HS Highly significant

Appendix-5.6: Analysis of variance for no. of galls

S.O.V D.F S.S M.S V.R F pr. Blocks 4 53.83 13.46 0.76 Variety 11 114124.58 10374.96 582.14 <.001* Residual 44 784.17 17.82

Total 59 114962.58 *HS Highly significant

192

Appendix-5.7: Analysis of variance for no. of egg masses.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 81.167 20.292 2.23 Variety 11 103303.333 9391.212 1030.89 <.001* Residual 44 400.833 9.110

Total 59 103785.333 *HS Highly significant

Appendix-5.8: Analysis of variance for rf.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 0.41068 0.10267 1.76 Variety 11 273.53833 24.86712 425.13 <.001* Residual 44 2.57372 0.05849

Total 59 276.52273 *HS Highly significant

Appendix-6.1: Analysis of variance for % decrease in root length.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 11.350 2.837 1.00 Inoculum 4 3545.406 886.352 313.47 <.001* Residual 16 45.241 2.828

Total 24 3601.997 *HS Highly significant

193

Appendix-6.2: Analysis of variance for % decrease in shoot length.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 2.746 0.686 0.37 Inoculum 4 5348.164 1337.041 728.52 <.001* Residual 16 29.365 1.835

Total 24 5380.274

*HS Highly significant

Appendix-6.3: Analysis of variance for % decrease in root weight.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 9.631 2.408 1.68 Inoculum 4 3339.279 834.820 583.17 <.001* Residual 16 22.904 1.432

Total 24 3371.814 *HS Highly significant

Appendix-6.4: Analysis of variance for % decrease in shoot weight.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 4.521 1.130 0.56 Inoculum 4 6753.865 1688.466 836.24 <.001* Residual 16 32.306 2.019

Total 24 6790.692 *HS Highly significant 194

Appendix-6.5: Analysis of variance for % decrease in shoot weight dry.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 2.746 0.686 0.37 Inoculum 4 5348.164 1337.041 728.52 <.001* Residual 16 29.365 1.835

Total 24 5380.274 *HS Highly significant

Appendix-6.6: Analysis of variance for number of galls.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 875.4 218.8 0.89 Inoculum 4 108776.6 27194.1 110.77 <.001* Residual 16 3927.8 245.5

Total 24 113579.8 *HS Highly significant

Appendix-6.7: Analysis of variance for number of egg masses.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 946.8 236.7 1.72 Inoculum 4 102949.2 25737.3 186.84 <.001* Residual 16 2204.0 137.8

Total 24 106100.0 *HS Highly significant

195

Appendix-6.8: Analysis of variance for Rf.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 4.6692 1.1673 2.15 Inoculum 4 146.3824 36.5956 67.40 <.001* Residual 16 8.6868 0.5429

Total 24 159.7384 *HS Highly significant

Appendix-7.1: Analysis of variance for % increase in root length

S.O.V D.F S.S M.S V.R F pr. Blocks 4 1.8242 0.4561 0.71 Bio-agents 3 148.4054 49.4685 77.30 <.001* Concentrations. 4 74.5988 18.6497 29.14 <.001* Bio-agents ¯ Conc. 12 3.6873 0.3073 0.48 0.920** Residual 76 48.6378 0.6400

Total 99 277.1536

*HS Highly significant **NS Non Significant

Appendix-7.2: Analysis of variance for % increase in shoot length

S.O.V D.F S.S M.S V.R F pr. Blocks 4 3.394 0.849 0.68 Bio-agents 3 98.828 32.943 26.44 <.001* Concentrations. 4 1835.119 458.780 368.24 <.001* Bio-agents ¯ Conc. 12 123.616 10.301 8.27 <.001* Residual 76 94.687 1.246

Total 99 2155.64 *HS Highly significant 196

Appendix-7.3: Analysis of variance for % decrease in root weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 27.1767 6.7942 7.53 Bio-agents 3 81.2838 27.0946 30.04 <.001* Concentrations. 4 245.9099 61.4775 68.16 <.001* Bio-agents ¯ Conc. 12 13.1760 1.0980 1.22 0.287** Residual 76 68.5449 0.9019

Total 99 436.0912 *HS Highly significant **NS Non Significant

Appendix-7.4: Analysis of variance for % increase in shoot weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 2.597 0.649 0.46 Bio-agents 3 332.882 110.961 79.26 <.001* Concentrations. 4 919.451 229.863 164.19 <.001* Bio-agents¯ Conc. 12 44.644 3.720 2.66 0.005 ** Residual 76 106.401 1.400

Total 99 1405.974 *HS Highly Significant **S Significant

Appendix-7.5: Analysis of variance for % increase in dry shoot weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 5.3652 1.3413 1.50 Bio-agents 3 217.4516 72.4839 80.96 <.001* Concentrations. 4 351.5356 87.8839 98.16 <.001* Bio-agents ¯ Conc. 12 51.3834 4.2820 4.78 <.001* Residual 76 68.0426 0.8953

Total 99 693.7784

*HS Highly Significant 197

Appendix-7.6: Analysis of variance for % reduction in number of galls

S.O.V D.F S.S M.S V.R F pr. Blocks 4 10.810 2.703 1.00 Bio-agents 3 355.593 118.531 43.68 <.001* Concentrations. 4 4572.713 1143.178 421.26 <.001* Bio-agents ¯ Conc. 12 170.978 14.248 5.25 <.001* Residual 76 206.241 2.714

Total 99 5316.335 *HS Highly Significant

Appendix-7.7: Analysis of variance for % reduction in number of egg masses

S.O.V D.F S.S M.S V.R F pr. Blocks 4 6.494 1.623 0.55 Bio-agents 3 787.473 262.491 88.63 <.001* Concentrations. 4 6551.211 1637.803 553.00 <.001* Bio-agents ¯ Conc. 12 305.433 25.453 8.59 <.001* Residual 76 225.088 2.962

Total 99 7875.700 *HS Highly Significant

Appendix-7.8: Analysis of variance for % reduction in Rf

S.O.V D.F S.S M.S V.R F pr. Blocks 4 24.753 6.188 2.11 Bio-agents 3 2564.225 854.742 291.04 <.001* Concentrations. 4 8490.878 2122.719 722.78 <.001* Bio-agents ¯ Conc. 12 575.814 47.985 16.34 <.001* Residual 76 223.202 2.937

Total 99 11878.871 *S Significant 198

Appendix-8.1: Analysis of variance for % increase in root length

S.O.V D.F S.S M.S V.R F pr. Blocks 4 1.5571 0.3893 0.53 Concentrations. 2 223.1559 111.5779 151.95 <.001* Amendments. 3 12.5633 4.1878 5.70 0.002** Conc. ¯ Amend. 6 25.2689 4.2115 5.74 <.001* Residual 44 32.3086 0.7343

Total 59 294.8538 *HS Highly significant ** S Significant

Appendix-8.2: Analysis of variance for % increase in shoot length.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 1.9407 0.4852 0.74 Concentrations. 2 1101.7341 550.8670 837.25 <.001* Amendments. 3 595.9311 198.6437 301.91 <.001* Conc. ¯ Amend. 6 174.9402 29.1567 44.31 <.001* Residual 44 28.9497 0.6579

Total 59 1903.4959 *HS Highly significant

Appendix-8.3: Analysis of variance for % decrease in root weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 7.017 1.754 0.77 Concentrations. 2 859.655 429.828 189.71 <.001* Amendments. 3 13.587 4.529 2.00 0.128** Conc. ¯ Amend. 6 37.678 6.280 2.77 0.023** Residual 44 99.692 2.266

Total 59 1017.630 *HS Highly significant ** NS Non-significant 199

Appendix-8.4: Analysis of variance for % increase in shoot weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 4.594 1.148 0.49 Concentrations. 2 5214.248 2607.124 1106.76 <.001* Amendments. 3 567.757 189.252 80.34 <.001* Conc. ¯ Amend. 6 468.096 78.016 33.12 <.001* Residual 44 103.648 2.356

Total 59 6358.343 *HS Highly significant

Appendix-8.5: Analysis of variance for % increase in dry shoot weight

S.O.V D.F S.S M.S V.R F pr. Blocks 4 8.9756 2.2439 4.20 Concentrations. 2 520.7963 260.3982 487.03 <.001* Amendments. 3 39.6185 13.2062 24.70 <.001* Conc. ¯ Amend. 6 19.3157 3.2193 6.02 <.001* Residual 44 23.5252 0.5347

Total 59 612.2313 *HS Highly significant

Appendix-8.6: Analysis of variance for number of galls.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 1726.29 431.57 4.39 Concentrations. 2 10522.69 5261.34 53.53 <.001* Amendments. 3 857.79 285.93 2.91 0.045* Conc. ¯ Amend. 6 423.99 70.66 0.72 0.636** Residual 44 4324.93 98.29

Total 59 17855.69

*HS Highly significant * S Significant ** NS Non- significant

200

Appendix-8.7: Analysis of variance for number of egg masses.

S.O.V D.F S.S M.S V.R F pr. Blocks 4 314.85 78.71 1.02 Concentrations. 2 14412.42 7206.21 93.09 <.001* Amendments. 3 1462.95 487.65 6.30 <.001* Conc. ¯ Amend. 6 741.28 123.55 1.60 0.171** Residual 44 3405.92 77.41

Total 59 20337.42 *HS Highly significant ** NS Non-significant

Appendix-8.8: Analysis of variance Rf

S.O.V D.F S.S M.S V.R F pr. Blocks 4 695.37 173.84 2.40 Concentrations. 2 14717.39 7358.70 101.56 <.001* Amendments. 3 1801.60 600.53 8.29 <.001* Conc. ¯ Amend. 6 561.23 93.54 1.29 0.281** Residual 44 3188.07 72.46

Total 59 20963.66 *HS Highly significant ** NS Non-significant