BIOLOGICAL CONTROL OF NEMATODES : Use of Biopesticides in Management of Root Knot Nematodes

•?* - ? I

ABSTRACT g ; 4 THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF Boctor of pi)tIoSopl)p t m AGRICULTURE / (NEMATOLOGY)

1 -» , .f«'

FAUZIA

INSTITUTE OF AGRICULTURE AUGARH MUSLIM UNIVERSITY AUGARH (INDIA) 1999 ABSTRACT

Root-knot nematodes (Meloidogyne spp.) are polyphagous and worldwide in distribution they infect almost every crop grown for food and fibre. They cause substantial yield losses to various groups of agricultural crops. Four species viz. Meloidogyne incognita, M. javanica, M. arenaria and M. hapla are called major species because of their extensive hosh range and prevalence in agricultural soils all over the world. M incognita is commonest (Sasser, 1980; Khan, 1997).

There has been consistent effort to develop measures for management of this group of nematodes in order to minimize the losses caused to agricultural crops. Use of nematicides is an effective measure but it causes health hazards and environmental risks. They persist in plant parts and contaminate ground water. To overcome these problems, for the last several years efforts are being made to develop biopesticides by using natural bioresources which can replace nematicides. The present study is an effort in this direction where potentials of some indigenous bioresources - some soil fungi and a predatory nematode have been examined which can be used as biopesticides as field dispension.

To achieve this objective root samples of vegetable crops infected with root-knot nematodes were collected and soil fungi infecting egg masses of root- knot nematodes were isolated and their pathogenecity on egg masses was established by artificial inoculations under laboratory conditions. Three fiingi, namely Verticillium chlamydosporium, Acremonium strictum, and Fusarium oxysporum were found to be egg parasites of root-knot nematode, M. incognita and were subsequently used in the experiments.

Culture filtrates of V. chlamydosporium, A. strictum and F. oxysporum inhibited juvenile hatching of M incognita and caused mortality of the second stage juveniles. Higher concentrations of the culture filtrates and greater exposure time were more effective. It was surmised that these fungi produce certain metabolites in the culture medium which prevents the biological activity of the nematode. Seed-soaking of tomato in the culture filtrates of the fungi was also found to be effective in suppressing the disease. All the three soil fungi, when applied to the soil in order to determine their effect on root-knot nematode on tomato, suppressed root-galling and egg mass production and soil and root populations of the root-knot nematode. Plant growth, consequently, showed improvement as all considered parameters of plant growth showed enhanced measurements as compared to control (nematode inoculated) plants.

V. chlamydosporium was apparently more effective than A. strictum and F. oxysporum. Nevertheless, all the three fungi demonstrated their potential as biocontrol agents of the root-knot nematode. To select suitable organic substrate for their growth and sporulation for dispensing of these in field, fungi various organic substrates (agricultural wastes of plant origin) were evaluated. Gram husk, rice husk, mungbean husk, wood charcoal and saw dust were included for the screening, and all the three fungi were inoculated seperately. Though all the materials used supported their growth and sporulation, rice husk was found to be best organic substrate for growth and sporulation of V. chlamydosporium, and gram husk for A. strictum and F. oxysporum. Spore load of V. chlamydosporium was also highest on rice husk and that of ^. strictum and F. oxysporum on gram husk.

Soil application of these organic materials colonized with either of the fungi effectively suppressed root-knot nematodes and disease development on tomato. Plant showed enhanced growth in all considered parameters. Rice husk colonized with V. chlamydosporium and gram husk colonized with A. strictum and F. oxysporum proved to be best for controlling the disease. Performance of V. chlamydosporium grown on rice was better than other two fungi. In sequential inoculation plant growth was much higher in the treatments were either of the fungi colonized with gram husk/rice husk was inoculated 15 days prior to nematode inoculation as compared to the treatment where nematode was inoculated 15 days before the fungus. In sequential inoculation, the percentage of egg infection was higher in the treatments where fungus colonized substrate was applied before nematode inoculation.

Viability of spores of all the three fungi colonizing the various organic substrates were checked at different duration of time. Spores of all the three fungi were viable at the time of observations i.e. 2, 4, 6, and 8 weeks. Aeration of the packets storing the rice husk with V. chlamydosporium and gram husk with A. strictum or F. oxysporum did not affect the viability of the spores for eight weeks. This demonstrated that these fungi can be cultured on their respective suitable materials of plant origin for field application and can be maintained in storage even upto 8 weeks for distribution to farmers or can be transported to other places during this period of time. In the second part of the study, predatory nematode, Mononchoides longicaudatus was tested for its biocontrol potential against root-knot nematode. Several culture media were screened in different combinations (agar, soil and farmyard manure) for culturing the nematode were also carried to determine its predatory behaviour of Mononchoides longicaudatus on Meloidogyne incognita juveniles. Prey catching and feeding mechanism was studied in detail. Effect of prey density, temperature, age of the predator, feeding pattern were also studied.

M longicaudatus was found to predate upon the second stage juveniles of M incognita. M. longicaudatus probed the prey and punctured the cuticle by movable dorsal tooth, feeding by oesophageal suction. There was a positive correlation between the numbers of prey killed and prey density. Predation was greatly influenced by change in temperature.

Adult predator killed a maximum number of the prey. Feeding pattern remained unaltered even upto 10 days. Application of M longicaudatus in soil showed that it had the potential to reduce root-knot disease. It was found that higher number of M longicaudatus can significantly reduce the disease and root- knot nematode population. BIOLOGICAL CONTROL OF NEMATODES : Use of Biopesticides in Management of Root Knot Nematodes

#^ •• • \ r V

THESIS , \ SUBMITTED FOR THE AWARD OF THE DEGREE OF Boctor of ^IfiloiopJjp i m "^ AGRICULTURE . . ' • (NEMATOLOGY) v

FAUZIA

INSTITUTE OF AGRICULTURE AUQARH MUSUM UNIVERSITY AUGARH (INDIA) 1999 .<^ ^

Ct i^w4_ JUij

( ' CC. 1 '

T5575 ^ cAimt 4* January 1999

CERTIFICATE

This is to certify that the entire work contained in the thesis entitled "Biological Control of Nematodes- Use of Biopesticides in the Management of Root-Knot Nematodes" was carried out by Ms. Fauzia as a research scholar in the Institute of Agriculture under our supervision. The work is original and up- to-date. We have allowed her to submit this thesis to the Aligarh Muslim University for the award of the degree of Doctor of Philosophy in Agriculture {Nematology)'.

(ci^xs-*^ (PROF. M. WAJID KHAN) (PROF. M. SHAMIM JAIRAJPURI) Co-supervisor Supervisor Q/ uti&A ta ea^yfieiS iUvcetcecu MI a/}''e

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ao/yt^ia. tne ituau. QT^'^O^H t/ie aeAt/i ai my, neaitt Q/ a-fn t/tatvKlal to mu nuio^atw,

^^a-uea, lo-f nii oa-'fctna ana enco-u^aatng, attitude t?i oanv/tietto-n o-i nvy- tAescd.

QT'Mia/nciat iuAAo^t t/tv t/ie too^m^ o^iu/ni

o-u tne zDeAavtme^ o£ iyOtatecA'naco-aUj ^ov^eicnment ac Q^ttata, Q/Veta ^ec/Uj

tA'ti&aa/i a •yeieaitcA /ifoiect entttlea c/oto^eiticlaei 09t matiaaentent o£ fco-at-Ktiat

'ne/nvatoa&i od i^eaetalUe o^oAi is a^ateMttu aoKtio-io/eaaea.

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du/ym^a tAe cou^e oJf co^mAuatto^ and Avi/ntlna 9nt(Jtt o-i tAii tAeded. LIST OF CONTENTS

Page no.

INTRODUCTION 1

LITERATURE REVIEW 5 PREDACIOUS FUNGI 10 ENDOZOIC FUNGI 13 OPPORTUNISTIC FUNGI 17 Paecilomyces lilacinus 20 Verticillium chlamydosporium 24 AcremonJum strictum and Fusarium oxysporum 28 PREDATORY NEMATODES 30

SECTION-I (OPPORTUNISTIC SOIL FUNGI) MATERIALS AND METHODS 3 4 RESULTS 53 DISCUSSION 106

SECTION-II (PREDATORY NEMATODES) MATERIALS AND METHODS 116 RESULTS 122 DISCUSSION 135

SUMMARY 138

LITERATURE CITED 142

ANOVATABLE LIST OF TABLES

SECTION-l Page No.

Table 1. Infection of eggs in the egg masses of Meloidogyne 54 incognita by some soil fungi.

Table!. Juveniles mortality oi Meloidogyne incognita in 59 different concentrations of culture filtrates of some soil fungi.

Table 3. Juvenile hatching oi Meloidogyne incognita in different 61 concentrations of culture filtrates in some soil fungi.

Table 4. Effects of soil application of Verticillium 66 chlamydosporium on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato.

Table 5. Effects of soil application of Acremonium strictum on 69 plant growth and development of root-knot nematode, Meloidogyne incognita on tomato.

Table 6. Effects of soil application of Fusarium oxysporum on 72 plant growth and development of root-knot nematode, Meloidogyne incognita on tomato.

Table 7. Spore load of some opportunistic soil fungi on various 77 organic materials of agricultural origin after ten days of growth.

Table 8. Viability of spores (spore load/g) of some opportunistic 78 soil fungi on various organic materials of agricultural origin over a period of eight weeks.

Table 9. Effect of aeration on the viability of spores of some 79 opportunistic soil fungi in waste substrate after eight weeks.

Table 10. Efficacy of some organic materials of agricultural origin 83 for soil application of Verticillium chlamydosporium (VC) for the management of Meloidogyne incognita (MI) on tomato.

Table 11. Efficacy of some organic materials of agricultural origin 8 5 for soil application of Acremonium strictum (AS) for the management of Meloidogyne incognita (MI) on tomato.

Table 12. Efficacy of some organic materials of agricultural origin 87 for soil application of Fusarium oxysporum (FO) for the management of Meloidogyne incognita (MI) on tomato.

Table 13. Effect of soil application of rice husk colonized with 92 Verticillium chlamydospornim (VC) on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato.

Table 14. Effect of soil application of gram husk colonized with 94 Acremonium strictum (AS) on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato.

Table 15. Effect of soil application of gram husk colonized with 96 Fusarium oxysporum (FO) on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato.

Table 16. Effect of seed soaking in different concentrations of 100 culture filtrate of Verticillium chlamydosporium on plant growth and root-knot nematode development on tomato.

Table 17. Effect of seed soaking in different concentrations of 102 Acremonium strictum on plant growth and root-knot nematode development on tomato.

Table 18. Effect of seed soaking in different concentrations of 104 Fusarium oxysporum on plant growth and root-knot nematode development on tomato. SECTION-2 Page No.

Table 1. Comparative suitability of some culture media for mass 123 culturing of a predatory nematode Mononchoides longicaudatus.

Table 2. Effect of predatory nematode Mononchoides 132 longicaudatus on plant growth and development of root- knot nematode, Meloidogyne incognita on tomato. LIST OF FIGURES

SECTlON-l Page No.

Figure 1. Juvenile hatching of Meloidogyne incognita in different 60

concentrations of culture filtrates in some soil fungi.

Figure 2. Juveniles mortality of Meloidogyne incognita in 62

different concentrations of culture filtrates of some soil

fungi.

Figures. Effects of soil application of Verticillium 67

chlamydosporium (VC) on plant growth and development

of root-knot nematode, Meloidogyne incognita (MI) on

tomato.

Figure 4. Effects of soil application of A cremonium strictum (AS) 70 on plant growth and development of root-knot nematode, Meloidogyne incognita (MI) on tomato.

Figures. Effects of soil application of Fw^an'Mw oa:75/?orww (FO) 73

on plant growth and development of root-knot nematode, Meloidogyne incognita (MI) on tomato.

Figure 6. Efficacy ofsome organic materials ofagricultiiral origin for soil 84 application of Verticillium chlamydosporium (VC) for the

management of Meloidogyne incognita (MI) on tomato. Figure 7. Efficacy of some organic materials of agricultural origin 86 for soil application of Acremonium sirictum (AS) for the management of Meloidogyne incognita (MI) on tomato.

Figure 8. Efficacy of some organic materials of agricultural origin 88 for soil application of Fusarium oxysporum (FO) for the management of Meloidogyne incognita (MI) on tomato.

Figure 9. Efficacy of soil application of rice husk colonized with 93 Verticillium chlamydosporium (VC) on plant growth and development of root-knot nematode, Meloidogyne incognita

Figure 10. Effect of soil application of gram husk colonized with 95 Acremonium strictum (AS) on plant growth and development of root-knot nematode, Meloidogyne incognita (MI) on tomato.

Figure 11. Table 9. Effect of soil application of gram husk 97 colonized with Fusarium oxysporum (FO) on plant growth and development of root-knot nematode, Meloidogyne incognita (MI) on tomato.

Figure 12. Effect of seed soaking in different concentrations of 101 culture filtrate of Verticillium chlamydosporium (VC) on plant growth and root-knot nematode Meloidogyne incognita (MI) development on tomato.

Figure 13. Effect of seed soaking in different concentrations of 103 culture filtrate oi Acremonium strictum (AS) on plant growth and root-knot nematode Meloidogyne incognita (MI) development on tomato.

Figure 14. Effect of seed soaking in different concentrations of 105 culture filtrate of Fusarium oxysporum (FO) on plant growth and root-knot nematode Meloidogyne incognita (MI) development on tomato. SECTION-2 Page No.

Figure 1. Comparative suitability of some culture media for mass 124 culturing of a predatory nematode Mononchoides longicaudatus.

Figure 2. Effect of predation by Mononchoides longicaudatus on 127 the prey density of Meloidogyne incognita..

Figure 3. Effect of temperature on the rate of predation by M. 128 longicaudatus.

Figure 4. Effect of different stages of M longicaudatus on M. 129

incognita..

Figures. FeedingpattemofM. longicaudatus. 130

Figure 6. The biocontrol potential of predatory nematode, 133

Mononchoides longicaudatus for Meloidogyne incognita on tomato. PLATES

Page No.

1. Penetration and infection of egg masses/eggs of Meloidogyne 55 incognita by Verticillium chlamydosporium.

2. Penetration and infection of egg masses/eggs of A/e/o/c/ogy«e 56 incognita by Acremonium strictum.

3. Penetration and infection of egg masses/eggs oiMeloidogyne 5 7 incognita by Fusarium oxysporum.

4. Effect of Verticillium chlamydosporium on plant growth. 68

5. Effect oi Acremonium strictum on plant growth. 71

6. Effect oi Fusarium oxysporum on plant growth. 74

7. Eiitctoi^T^dziXory ntmoXoAt, Mononchoides longicaudatus 134 on pljint growth. INTRODUCTION

Root-knot nematodes (Meloidogy?7e spp.) are world wide in distribution.

They show an extensive hostrange and interact with other plants pathogens, resulting in an increase in the disease severity. They are considered as one of the major plant pathogens affecting the world's food production. They infect an array of economically important crops affecting them both quantitatively and qualitatively. Vegetables, cereals, pulses, oilseed crops fibre-yielding crops, fruit trees, plantation crops, ornamentals etc. are affected by root-knot nematodes in different countries across the world. Estimated crop losses due to root-knot nematodes in major geographical regions of the tropic range from 5 to 43%

(Sasser, 1979). This, however, varies depending upon the crop, species, and the geographical region and climatic conditions. In areas where management practices of root-knot nematodes are not practicized, average crop yield losses are estimated to be about 25% with damage in individual fields, ranging as high as 60% (Sasser,

1980; Sasser and Carter, 1982).

In India, occurrence of root-knot nematodes on a variety of cultivated and wild plants has been recorded in different states of the country. Existence of the four major species of root-knot nematodes viz., Meloidogyne incognita, M. javanica, M. arenaria and M. hapla has been also recognized to occur in the country. Populations of the species have been characterized for the host races. Four races (races 1-4) in M incognita and race 2 of M arenaria have been differentiated in their populations (Khan, 1997). Attempts have been made for successful management of root-knot nematodes using different management measures from time to time. Several nematicides (chemicals that kill the nematodes) were found to be most effective against major plant parasitic nematodes including root-knot nematodes and were successfully used in the different parts of the world. Use of nematicides has been found to cause health problems in human beings and environmental risks are involved in their manufacturing and application. Several nematicides have been shown to persist in soil or leave their residue in edible plant parts and contaminate groundwater. For these reasons attention has been diverted to look for measures that are free from health hazards and environmental concerns and can be used effectively for the management of plant-parasitic nematodes including root-knot nematodes. Global efforts are now being made to analyse various natural bioresources for their nematode biocontrol potential, so that they can be artificially exploited and can be developed as biopesticides. Biocontrol of plant parasitic nematodes and development of biopesticides is now a thrust research area in Nematology. Biopesticides a newly coined word is used in different context, different from biocontrol. In biocontrol, biocontrol agent is released in pest population to establish itself and control the target pest but biopesticides refer to microbial pathogens that uniformly kill or suppress the target pest in a manner similar to chemical pesticides. They generally show a high degree of specificity on target pest. Many natural enemies that attack the plant parasitic nematodes in agricultural soils are bioresources and can be developed to be used as biopesticides. The nature and extent of activity in relation to nematode multiplication in order to establish biopesticidial potential of the indigenous bioresources need to be investigated.

Several micro-organism are reported to be predators of plant parasitic nematodes. Nematode trapping fungi, nematodes, turbellarians, enchytraeides, mites, oligochaetes, tardigrades, collembolans are predators while endozoic and opportunistic fungi, bacteria, protozoans, viruses and ricketssae are parasites (Sayre, 1971). Regulation of nematode populations under natural conditions by these parasites and predators and research on these aspect have been reviewed (Mankau, 1980;Jatala, 1986; Stirling, 1991).

Research at Aligarh has shown that root-knot nematodes particularly, Meloidogyne incognita, M. Javanica and M. arenaria infest vegetable fields in the state of Uttar Pradesh of India. M hapla is also present in the hilly districts of the state (Khan, 1988; Khan, 1990 and Khan 1991). Occurence of all the four races inM />2Co^«/7flf populations of the state has also been recognized. The level of infestation of these root-knot nematodes species in vegetables fields is exceedingly high and crops suffer substantial yield losses. Therefore, management of root-knot nematodes through biocontrol agents and development of pesticide is an essential component of agricultural development in this parts of the country as in other parts of the country or world.

Keeping this aim in view, the present study was planned to explore the possibilities of developing biopesticides using indigenous bioresources, particularly soil fiingi (opportunistic soil hyphomycetes) and predatory nematodes. The study presented in the thesis is divided into two sections — Section-I related to opportunistic soil fungi and Section-Il related to predatory nematodes. Introduction, Literature Review, Summary and Literature Cited for the two sections are given jointly. Materials and Methods, Results and Discussion are seperate for both the sections. LITERATURE REVIEW

The nematodes are highly diversified, perhaps most numerous multicellular animals on earth. They are either free-living in marine or freshwater, in soil or parasitic on different kinds of plants and animals. The species parasitic on plants are of considerable agricultural importance. They show three different kinds of parasitic behaviour-ectoparasitism, semi-endoparasitism and endoparasitism. Endoparasites cause significant crop losses. Among the endoparasites, species oi Meloidogyne, Heterodera and Globodera are major pests of agricultural importance. Four species of Meloidogyne namely M. incognita, M. javanica, M. arenaria and M. hapla described by Chitwood (1949) are considered as most destructive species and in recent years they have been termed as major species of root-knot nematodes because of their wide spread occurrence and extensive host range (Sasser, 1980).

Plant diseases induced by nematodes cause significant economic losses. According to an estimate, yield losses caused by plant parasitic nematodes amounted to 1.6 billion dollars. The estimated crop loss in vegetables such as tomato, potato, egg plant and pepper was 267 million dollars (Sasser and Freckman, 1987). In India, crop losses of Rupees 30 millons in barley and Rupees 40 millions in wheat are caused in the state of Rajasthan alone by cereal cyst nematode, Heterodera avenae. The disease in India is known as "Molya" disease (Van Berkum and Seshadri, 1970). The ear cockle disease of wheat has been estimated to cause an annual loss of over Rupees 75 millions at low infestation level (Seshadri, 1970). The infestation of coffee plantation by the root-lesion nematode Pratylenchus coffee causes an annual loss around Rupees 20 millions (Van Berkum and Sheshadri, 1970). In the Nilgiri hills of Tamil Nadu, about 2500 acres under potato cultivation are badly infested by the cyst nematodes, Globodera rostochiemis and G. pallida (Sheshadri and Sivakumar, 1962; Thangaraju, 1983).

The root-knot nematodes, Meloidogyne spp. are destructive plant parasitic nematodes. They are widely distributed and cause annual loss upto the extent of 29% in tomato, 23% in eggplants, 22% in okra, 28% in beans and it varies from country to country (Sasser, 1979). In vegetables, the crop losses caused by root-knot nematodes may vary from 25 to 90% depending upon the cultivar, season and cropping system followed (Sasser, 1980).

Since plant-parasitic nematodes reduce the growth and yield of plants and cause severe damage to cultivated plants, there has been consistent effort to introduce the nematode management programmes in order to minimize the losses suffered by grower. The management of plant parasitic nematodes started with the introduction of chloropicrin in 1919 (Singh and Sitaramiah, 1994). Since then several kinds of nematicides, both fumigant and non-fumigant were formulated and recommended to reduce the population of plant-parasitic nematodes in agricultural soils. Majority of the nematicides cause adverse impact on environment and these are harmful to man and animals. Nematicides have some disadvantages as well such as residue problem in crops and contamination of ground water (Khan, 1997). To overcome these adverse problems and impacts, scientific interest has been stimulated, in the recent years, in the development of safer and potent non-chemical pesticides for the management of plant parasitic nematodes. In this endeavour, mainly the efforts are concentrating on developing biocontrol measures for their management or integrating biocontrol measures into overall nematode management strategies.

The biological control strategy is based on exploitation of living organisms for reducing the pest population. Over the past several years, the potential of microorganisms for biocontrol of nematodes has attracted attention of researchers. Lohde (1874) suggested for the first time about the possible implication of parasites and predators attacking soil inhabiting nematodes in regulating nematode populations in soil. Since then several observational records of presence of parasites and predators attacking nematodes in soil have been made (Kuhn, 1877; Zopf, 1888; Cobb, 1920; Thorn, 1927;Dolfus, 1946; Lowenberg ef al. 1959). Some pot trials (Linford, 1938) and field trials (Duddington, 1957) were also conducted to determine efficiency of biocontrol agents for management of plant parasitic nematodes.

Biopesticides have been suggested an effective substitute for chemical pesticides. Biopesticides are microbial pathogens that uniformly kill or suppress the growth of target pest in a manner similar to chemical pesticides (Khan, 1997). Bacterial, fungal and viral pathogens that are virulent, host specific and genetically stable but constrained naturally by low inoculum production and poor dissemination were considered as good candidates for development as biopesticide (Templeton et al., 1984). Only those organisms can be regarded as potential biopesticides which can be mass cultured, easily and safely transported and have the ability to adjust the ecological and physiological compatibility at the site of release. The potent biopesticides possess the advantage of high degree of specificity for the target pest, no adverse effect on non-target and beneficial organisms or man and absence of pest resistance problem and residual build-up in the environment.

However, some drawbacks associated with biopesticides include the expensive methods of development and production, their lengthy process of registration, stability under field conditions etc. (Kulshreshtha, 1995).

Several predaceous and parasitic organisms such as fungi, bacteria, predaceous nematodes, viruses, protozoa, tardigrades, turbullarian, collembolans, mites, enchytraeides, oligochaetes etc. are known to reduce the nematode population in natural conditions (Webster, 1972; Sayre, 1971, 1980; Norton, 1978;

Brown, 1978; Kerry, 1980, 1981, 1984, 1986, 1987; Jatala, 1986; Jairajpuri et al. 1990).

In recent years, research has been focussed on the utilization of biocontrol agents such as nematophagous fungi, bacteria and predatory nematodes for the management of plant parasitic nematodes.

Nematophagous fungi are potentially most useful biocontrol agents. These fungi are either obligate endoparasites or predaceious trap-forming species. Some hyphomycetous soil fungi which in recent years have been reported to be parasitic on nematodes, particularly, the reproductive stages of endoparasites are called opportunistic fungi. Activities of these fungi are influenced by various ecological conditions like soil pH, moisture, temperature etc.

Several nematophagous fungi are known to reduce the population of plant parasitic nematodes. Important genera are Paecilomyces, Verlicillium,

Nematophlhora, Hirsutella, Arthrobotrys, Drechmeria, Fusarium, Acremonium and Monacrosporium (Jatala, 1986; Nigh, 1980; Zaki and Maqbool, 1996). The important genera of predatory nematodes like Mononchus, Mylonchulus (Cobb,

1917) several species of Aporcelaimus, Nygolaimus, Labronema and

Actinolaimus {Cobh, 1929; Thorne, 1930 and 1939; Linford and Oliviera, 1937), several species of Seinura, Butlarius and Mononchoides (Linford and Oliviera,

1937; Hechler, 1963; Grootaert et al., 1977; Small and Grootaert, 1983) are reported as enemies of plant parasitic nematodes.

Among bacteria, Pasteuriapenetrans, a prokaryotic obligate parasite of nematodes is known to be potential biocontrol agent. First time it was described by Throne (1940) as a protozoan and named as Doboscqia penetrans. Mankau

(1975 a and b) renamed it as Bacillus penetrans. Starr and Sayre (1985, 1988) made several observations on this bacterium and placed it under the genus

Pasteuria. It ia a specific obligate parasite of root-knot nematodes.

Nematode destroying fungi

Nematode destroying fungi are known to be detrimental to plant parasitic nematodes. Most of these fungi belong to the order Moniliales of Fungi Imperfecti but some are from Zoopagales in Zygomycetes. More than 200 species possess the capacity to destroy or deleteriously affect the population dynamics of plant- parasitic nematode (Barron, 1977, 1981; Mankau, 1980; Morgan-Jones and Rodriguez-Kabana, 1987; Khan, 1987; Kerry, 1990).

Morgan-Jones and Rodriguez-Kabana (1987) have categorized them into three groups : predaceous, endozoic and opportunistic fungi. Jatala(1986) placed the endozoic and opportunistic fungi into four groups : (a) parasites of sedentary females, (b) parasites of eggs, (c) parasites of vermiform nematodes and (d) parasites of eggs, sedentary females and vermiform nematodes.

Predaceous fungi More than 100 species of predaceous fungi are known to be living on free-living nematodes. Nematode-trapping habit is found in several fungi belonging to the family Moniliaceae (Hyphomycetes) in the class Deutromycetes. A few species are also found in the order Zoopagales in class Zygomycetes. The most important genera are Arthrobotrys, Trichothecium, Dactylaria, Dactylella and

Monacrosporium.

These fungi develop special structures from their hyphal branches to trap or catch the nematode. Death of nematodes occur due to mechanical damage caused by the trap and also due to release of certrain nematotoxic secretion (Sayre,

1971; Mankau, 1980; Jatala, 1986).These special structures are either sticky traps such as sticky branches in Dactylella lobata, or sticky network in Arthrobotrys oligospora or mechanical traps such as constricting ring e.g. Dactylella bembicodes (Dreschler, 1937; Duddington, 1960). The fungi that trap and prey on nematodes were first reported by Zopf (1888). Later efforts were made to control plant parasitic nematodes with the use of nematode-trapping fungi (Dreschler,

1937). The first attempt to exploit the fungi for nematode control was made by

Linford and Yapp (1938, 1939) in Hawaii against root-knot nematode on pine­ apple. They used five fungi (Arthrobotrys oligospora - 2 isolates, A. musiformis,

10 Daclylaria rhaumasia, Dactylella ellipsospora), but except for a small measures of control with D. ellisospora, no effect was recorded with the other fungi.

Other studies were also carried out to determine the efficiency of predacious fungi in controlling the root-knot nematode infection. Deschien et al.

(1943) used A. oligospora and D. bembicodes against root-knot nematode of begonia and obtained considerable control. Later, it was found that increase in the dosage of the fungi resulted in greater control of nematodes (Gorlenko, 1956).

Nematode-trapping fungi were found to be controlling the root-knot nematode on tomato and okra (Muller, 1961). Dactylella oviparasitica was found naturally controlling root-knot nematodes on a highly susceptible variety "Lovell" of peach, in California (Stirling and Mankau, 1977). The fungus was able to survive without the nematodes (Stirling et al., 1979). The fungus D. megaspora is reported to trap and assimilate 18 genera of plant parasitic nematodes (Esser et al, 1991). An isolate of

Arthrobotrys robustus named 'Royal 300' for the control of Ditylenchus myceliophagus on mushroom (Cayrol et al., 1978) and another isolate of

Arthrobotrys named 'Royal 350' for the control of Meloidogyne on tomato (Cayrol and Frankwoski, 1979) are commercially available.

Sayre (1971) described the details of various other type of trapping devices formed by these fungi. Sunderland (1990) has briefly reviewed the entrapping and assimilarion of nematodes by fungi like Arthrobotrys, Monacrosporium and Dactylaria spp. by employing constricting rings. The nature of nematode entrapment and their biological control potential were also studied. Nakasono and Gaspard (1991) determined effecriveness of Arthrobotrys

11 ciactyloidcs and Dactylella haploiyk for Meloidogyne incognita and free living nematodes in soil. Boag et al. (1988) found a species of Arthrohotrys, A. dasguptae attacking a range of plant-parasatic nematodes. Another species A. irregularis protected plants against light and moderate infestation by Meloidogyne spp. (Cayrol, 1988).

Arthrohotrys oligospora was found to produce extracellular proteases in liquid culture and involvement of proteases in infection and immobilization of nematodes was investigated by Tunlid and Jansson (1991). According to Tunlid (1992) lectin-carbohydrate interaction leads to a adhesion of nematode to A. oligospora, which is the first step in the infection of nematodes. A. oligospora was found to develop the traps in response to living nematodes (Dackman and Nordbring-Hertz, 1992). Jaffee et al. (1992) investigated trap formation in A. dactyloides, A. oligospora, Monacrosporium ellipsosporum andM cionopagum from parasitized nematodes. Conditions that induce traps may normally prevail when these fungi grow from nematodes in soil. Hirsutella rhossiliemis produce infective structures when submerged and non-infective spores in the atmosphere; therefore allocation of resources to traps or spores may be affected by soil water. When introduced into untreated loamy sand in the form of parasitized nematodes, Hirsutella rhosiliensis, M. ellipsosporum and A. dactyloides parasitized significant proportions of assay nematode Heterodera schachtii. These fungi parasitized fewernematodes if the soil was disturbed before the assay nematodes were added. In contrast, parasitized assay nematodes by ^. obligospora and M cionopagum was not detected in loamy sand.

\2 Six geographic isolates of Monacrosporium ellipsosporum a predatory fungus varied in relative predacity towards Meloidogyne ju\em\&s emerging from egg masses. A most effective (aggressive) isolate was tested for its efficacy to protect tomato against M. incognita on tomato, in vitro, greenhouse and field conditions. An aggressive isolate oiMonacrosporium ellipsosporium chosen for rapidity and abundance of trap formation in vitro, was tested in greenhouse and field tests for protection of tomato from M incognita. A test on tomato in potted field soil inoculated with 1, 5 and lOg fungal inoculum/15 cm pot, 15 days before nematode inoculation, showed significant reduction in plant damage at 5 and lOg levels, but not at Ig levels. Root galling was reduced 42% and 49% respectively, in the 5 and lOg fungal treatments. Larvae in the soil and females in the plants were also reduced. The reduction in plant damage was associated with a reduction in the larvae in the soil and females/plant. In a field trial, the improved plant growth and M. incognita reduction was directly related to the amount of fungus used, but treatment did not differ statistically from controls (Mankau and Xuiying, 1985).

Endozoic Fungi Endozoic fungi are obligate parasites of nematodes and attack plant parasitic nematodes in soil, but their biocontrol potential has not been fully determined because they can not be easily grown in axenic culture.

Lohde (1874) was first to suggest the possibility of using an endozoic fungus, Harposporium anguillulae for regulating nematode population. Some endozioc fungi which are hyphomycetous such as Acrostalagmus, Harposporium and Meria have been cultured artificially while phycomycetous fungi such as

13 Catenaria, Haptoglossa and Nematophthora are zoosporic form which pose great difficulty to grow axenically.

The endozoic fungi {Meria, Hirsutella) produce spores which are generally sticky and adhere to the body of nematodes when come in their contact and penetrate the cuticle to enter the nematode body. Hyphae ramify within the nematodes body and utilize the body contents as their food. Spore bearing hyphae emerge later on from the body to help in dispersal of the fungus.

Barron (1979) reported that Harposporium arthrosporum parasitized the nematode only when its arthro-conidia become lodged in nematode's oesophagus. Nematodes ingest the conidia and conidiophores emerge through their cuticle at the tail end (Barron, 1980). These fungi form mycelium inside nematode body and after its death fungal hyphae comes out and start producing conidia.

Meria coniospora infects nematodes (Jansson and Nordbring-Hertz, 1983).

Jansson et al. (1984) gave an information onM coniospora at different stages of nematode infection using light scanning and transmission electron microscopy. It was shown that conidia of M coniospora infect Panagrellus redivivus mainly in mouth region. Jansson et al. (1985) observed the different patterns of adhesion of conidia to the cuticle of nematode. Hisutella rhossiliensis, an endozoic fungus produces spores that adhere to and penetrate the nematode cuticle and assimilate the body contents prior to its emergence and sporulation (Jaffee and Zehr, 1985).

Drechmeria coniospora also shows similar adhesion of conidia on the cuticle and infection of plant parasitic nematodes.

14 Proteins are found to be involved in the adhesion process (Jansson et al., 1987). Mycelial growth and rate of sporulation of D. coniospora and

Harposporium anguillulae in different liquid cultures were tested by Lohman and Sikora (1989). Conidia produced in liquid cultures retained their parasitic capabilities. Dijksterhuis et al. (1991) studied the colonization and digestion of nematodes by D. coniospora. They reviewed the ultrastructural studies on infection of nematodes by conidia of D. coniospora (Dijksterhuis et al., 1990).

The potential of Meria coniospora for reducing the root-galling caused by root-knot nematodes was evaluated. The endoparasitic nematophagus fungus

Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trails. The fungus introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematode infected with M. coniospora to the soil, both methods reduced root-galling by root-knot nematode (Jansson et

al. 1985). Hirsutella rhossiliensis suppressed the population of Criconemella xenoplax to non-injurious level on peach seedlings (Sayre et al., 1987). Jaffee

and Zehr (1985) concluded that//, rhossiliensis was better parasite than competive

saprophyte. The saprophytic ability ofH. rhossiliensis was greatly influenced by

substrate and environment.

Catenaria auxiliaris and Nematophthora gynophila are two zoosporic fungi that have been found to attack plant parasitic nematodes. C. auxiliaris was first observed infecting females of Heterodera schachtii by Kuhn (1877). It infects the females of H. avenae and Globodera rostochiensis (Kerry, 1975; Kerry et al., 1976). A^. gynophila has been found paratisizing females and eggs of

15 Heiewdera avenae in nature (Kerry, 1974; Kerry and Crump, 1977); Crump el al, 1983). The infected females tend to lose turgor and their bodies become completely filled with resting spores within four days at 13°C (Kerry,

1974). When body of such females are ruptured, the spores are dispersed in the soil. Soil moisture greatly governs the parasitic ability of this fungus

(Kerry et al., 1980). This fungus also infects H. corotae, H. cruciferae, H. gottingiana, H. schachtii and H. trifolii. This is, however, not parasitic on

G. ro5?oc/2/e«>s'/5 (Kerry and Crump, 1977).

Voss and Wyss (1990) determined the potential of Catenaria anguillulae as a control agent against several plant parasitic nematodes. Variation between the strains of the fungus and its attack to Heterodera schachtii was also studied

(Voss et al., 1992). Two of three isolates (C^, C,p) significantly reduced the number of cysts produced while one isolate (Cjj) showed no effect on the H. schachtii population. Infection of Meloidogyne incognita eggs by the zoosporic fungus C. anguillulae was reported by Wyss et al. (1992) in artificial inoculation. The embryo was killed within a few minutes following mass aggregation and encystment of zoospores of C. anguillulae. Jansson and Thiman (1992) studied chemotaxis of zoospore of C anguillulae. Out of 21 L-amino acids tested only one serine, attracted the zoospore. Furthermore, among the serine - containing peptides tested, only the dipeptides, but not obligopeptides with serine inside the molecule gave a chemotactic response in zoospores. Serine was the only amino acid tested which gave a chemotactic response and serine dipeptides attracted the zoospores. Serine is a very strong and specific attractant.

16 Opportunistic fungi

Discovery of opportunistic soil fungi as parasites of females, cysts and eggs of endoparasitic nematodes intensified and increased an enormous interest in using fungi as biocontrol agents. The important genera of opportunistic fungi are Cylindrocarpon, Fusarium, Paecilomyces, Exophiala, Gliocladium, Phoma,

VerticilUum and Acremonium. These fungi colonize the reproductive structures of the nematodes. They are mainly egg parasites. Eggs are more susceptible to infection by these fungi and embryogenesis is inhibited and first stage juvenile fail to develop. Second-stage juveniles are also colonized (Khan and Esfahani,

1996). Infected young females show decreased fecundity. Eggs of cyst and root- knot nematodes are more vulnerable to attack by opportunistic soil fungi than those of migratory endoparasites (Jatala, 1986). Once in contact with cyst or egg- masses, these fungi grow rapidly and eventually parasitize all eggs that are in early embryonic developmental stages. Apparently when juveniles are formed the parasitic activities of these fungi are generally reduced. These fungi possess greater biocontrol efficiency for nematodes than the predatory and endozoic fiingi (Jatala,

1986; Siddiqui and Mahmood, 1996).

The efficiency and adaptability of these fungi vary in soil and in different environmental conditions. Some of these fungi have limited reproductive potential (Mankau, 1981; Tribe, 1977), while others require enriched laboratory media for sporulation or have limited saprophytic growth (Mankau, 1981). There are reports of some studies carried out during 1929-1932 on these fungi. Korab (1929) and Rozypal (1934) reported a fungal disease of eggs and larvae in Hetewdera schachtii caused by Torula hetewdera (probably the fungus Phialophora

17 molarum) while, Rademacher and Schmidt (1933) found Metarrhizium auisopliae highly deleterious to the cyst of H. schachtii. It was found that embroyonic development of Hetewdera avenae was hindered by Cylindrocarpon radicicola

(Goffart, 1932).

Lysek (1963) for the first time observed invasion and destruction of nematode eggs by Fusariiim and Cephalosporium. In a later study, he found that egg shells were perforated by Acremonium bacilosporum, Helicoon farinosum,

Mortierella nana, Paecilomyces lilacinus, VerticilUum chlamydosporium and V. bulbillosum (Lysek, 1966). A large number of fungi are reported to occur in the cyst of Hetewdera avenae and H. schachtii. This confirmed the capability of fungi for invading the cysts and eggs of nematode to induce the disease (Bursnall and Tribe, 1974).

Jatala et al. (1979) reported that eggs of Meloidogyne incognita on potato roots in Peru were found to be infected by P. lilacinus and this fungus was shown to be capable of invading mature females and cyst of Globodera pallida. Nigh (1979) found that eggs of Hetewdera schachtii were attacked by other fungi also such as Acromonium strictum and Fusarium oxysporum in California sugar beet fields. In total 14 fungal species were isolated as biocontrol agents from the Meloidogyne incognita egg masses. Of these fungal species, Paecilomyces lilacinus was found to be most effective (Villunueva and Davide, 1984). Morgan- Jones et al. (1984) observed the development of P lilacinus on Meloidogyne arenaria. This fungus penetrated the eggs through minute pore which are dissolved in vitelline layer. The infection causes the splitting of the vitelline layer into 3

18 distinct layers, where the chitin layer becomes vacuolated with the disappearence of lipid layer.

Several studies have been carried out to determine the potential of opportunistic soil fungi as a biocontrol agents of nematodes. Price ei al. (1980) reported that root-galling by Meloidogyne hapla decreased in the presence of F. oxysporum f.sp lycopersici and Verticillium dahliae. Watanabe (1980) found that Verticillium spaerosporum was detrimental to nematodes such as Aphelenechoides, Cephalorobus and Panagrolaimus. Another species of Verticillium, V. chlamydosporium was found parasitizing the females of Meloidogyne arenaria and cyst of Heterodera glycines (Morgan-Jones et al., 1981). This fungus reduces the egg hatching of M arenaria with the help of metabolites (Morgan-Jones etal., 1983). The percentage of the infected juveniles and eggs of Globodera pallida was increased as the time of exposure to P. lilacinus was increased. The hatching decreased due to the infection of eggs (Franco era/., 1981).

Musa and Haque (1988) found that Fusarium spp. reduced the number of females of Meloidogyne incognita and increased the proportion of males. Soil samples from 31 fields where cereals were grown for four successive years and 26 fields of permanent pastures were sampled for both plant-parasitic nematodes and nematophagous fungi. Four potentially important fungal parasites of Heterodera avenae were identified. These were Verticillium s^., Nematophthora gynophila, Paecilomyces carneas and Cylindrocarpon destructans (Boag and Llorca, 1989).

Qadri and Saleh (1990) studied the impact of several nematode

19 antagonists on Heterodera schachtii and Mehidogyne javanica by inhibiting the fungus in fields soil and by their addition to nematode culture in pots. On agar,

Acremonium sclerotignum a sterile fungus, Preussia sp. Verticillium and

Fusarium solani parasitized 72-82% eggs of//, schachtii, V. chlamydosporium

and F. solani destroyed 72-82% eggs of M javanica.

Paecilomyces lilacinus The genus Paecilomyces was described by Bainier (1907) as a relative

of Penicillium. Paecilomyces lilacinus was found infecting egg masses of

Mehidogyne incognita acrita infecting potato roots (Jatala et al., 1979). Earlier

perforation of egg shell by Paecilomyces lilacinus and other fungus has been

observed by Lysek (1966). P. lilacinus was found to be highly parasitic on eggs of

Mehidogyne incognita and females and cysts of Ghbodera pallida on potato

(Jatala era/., 1979).

Morgan-Jones and Rodriguez-Kabana (1984) found P. lilacinus and P. variotii as parasites of cysts and eggs of Heterodera glycines, Mehidogyne arenaria andM incognita. They also reported control of these nematodes with P. lilacinus. Friere and Bridge (1985) studied parasitism of eggs, females and juveniles of M. incognita by P. lilacinus and found that 50% of the live M. incognita eggs in egg masses became infected. Effectiveness of P. lilacinus against M. javanica on tomato was studied by Croshier et al. (1985). They found that at the highest inoculum level (1.1 g), 76.25% of M javanica eggs were parasitized. Cyst nematodes were also found to be attacked by the fungus. The fungus was also found parasitizing eggs of M arenaria in Alabama

20 soil (Godoy et al, 1983). Gintis et al. (1983) reported association of P. lilacinus with developmental stages oiHeterodera glycines from Alabama soybean field soil, while Morgan-Jones et al (1986) found the fungus colonizing cysts of potato cyst nematodes, G. pallida and G. rostochiensis in Peru.

Biochemical activities of the fungus for nematode parasitism have been investigated. P. lilacinus is a chitin degrader. Chitin constitutes the largest portion of the nematode egg-shell, while the larval cuticle lacks it (Okafor, 1967). This perhaps explains the effectiveness of the fungus as an egg destroyer, which becomes almost ineffective once the nematode larva is formed. Endreeva et al. (1972) have observed proteolytic activity of the fungus. The serine protease produced by P. lilacinus possibly plays a role in the penetration of the fungus through egg shell of the nematode (Bonants et al, 1995).

Paecilomyces lilacinus penetrates the eggs of Meloidogyne at much faster rate than it does in Globodera or Naccobus (Siddiqi and Mahmood, 1996). P. lilacinus also causes egg deformation in M. incognita with the help of diffusable toxic metabolites (Jatala, 1986; Jatala et al., 1985). The fungus causes alteration in egg's cuticular structure by enzymatic activity. This helps hyphal penetration. This either changes egg-shell permeability or causes perforation in the cuticle which allows seepage or free movement of diffusable metabolic compounds (Jatala, 1986). Isogoi et al. (1980) reported that P. //7ac/«M5 produces a peptidal antibiotic P-168 which has a wide antimicrobial effect on fungi, yeast and gram positive bacteria, while Khan et al. (1988) found that P. lilacinus does not produce aflatoxins, ochratoxin, T-2 toxin and zearalenone on test substrates.

21 Roman and Rodriguez Marcano (1985) investigated the effect of P. lilacimis on the larval development and root-knot formation by M incognita on tomato. The fungus controlled the nematodes and reduced root knot formation.

Fewer larvae were found in roots and soil of plants inoculated with the fungus.

Culbreath et al. (1986) found no effect of P. liJacinus on M. arenaria infecting

Cucurbitapepo. However, root galling decreased when chitin was mixed with P.

lilacinus. Hewlett etal. (1988) while assessing the efficacy of P. lilacinus against

M. javanica infecting tobacco found no difference in root-galling in the presence

or absence of fungus.

Jimenez and Gallo (1988) found that P. lilacinus infects the eggs and

sometimes females of M. incognita, M. javanica and M. arenaria under glass

house conditions. Sharma et al. (1989) stated that P. lilacinus as biocontrol agent

of M incognita acts better than any commonly used nematicides under field

infestations. Khan and Esfahani (1990) studied the efficacy of P. lilacinus for

controlling Meloidogyne javanica on tomato in greenhouse conditions. It was

found that root-galling and egg mass production were greatly reduced. The fungus

was more effective when both organisms were inoculated simultaneously or the

fungus preceded the nematode in sequential inoculation. A high percentage of

eggs were found to be infected. Khan and Husain (1990) reported that P. lilacinus

reduced the damage to cowpea caused by M. incognita and Rotylenchulus

reniformis.

Growth behaviour and effect of some growth factors have also been examined. The growth and sporulation in P. lilacinus was well between 10-30°C

22 and optimum at 20-25°C (Stephen and Al-Din, 1987). The fungus also tolerates a wide range of pH from 3.5 to 8.5 (Jatala, 1986).

Stephen and Al-Din (1987) evaluated various agricultural materials for its growth and showed that unpealed rice grains, peeled rice grains, wheat and barley were good growth media but corn was not. Sharma and Trivedi (1987) noted that maximum and rapid growth of P. lilacinus on oilcake of sesamum followed by cotton, linseed, mustard and groundnut oil cakes. While among the agricultural waste materials maximum fungal growth was noted on mungbean husk followed by cotton seed, guar powder, gram powder and rice husks. Bansal et al. (1988) have screened some agro-industrial wastes for mass propagation oi P. lilacinus. Zaki and Bhatti (1991) evaluated different whole grain substrates for the sporulation of P. lilacinus. The fungus sporulated profusely on maize, gram, oat, rice and wheat grains. Maximum number of spores/gram were observed on rice (52.8 X 10*) followed by gram (24.5 x 10'). P. lilacinus cultured on gram seed was most effective in reducing the gall index (78.6%) and second-stage juveniles of Meloidogyne javanica (94.2%) and increasing plant growth. The percentage of eggs destroyed by fungus was maximum with the fungus cultured on gram. The suitability of wood charcoal powder as a carrier of P. lilacinus for field application was studied in vitro by Bansal et al. (1992)^ The storage of wood charcoal packets at constant (28 ± l°C)/ambient (14-39°C) temperature or under aerated or non- aerated conditions did not influence the fungal spore viability. Maximum number of spores/gram of wood charcoal was 1 x 10^

Cameiro et al. (1991) showed that population of M. arenaria was

23 significantly reduced by P. lilacinus when applied in different doses. Zaki (1994) determined optimum and effective dose of P. lilacinus for the control of M. javanica on tomato as 4 g of gram seed infected with the fungus in pots.

Jonathan et al. (1995) studied the biological control of root-knot nematode on betelvine (Piper betel) by P. lilacinus. Rice grains colonized with the fungus were inoculated in different doses per plant and compared with carbofuran as control. Better reduction in nematode population was observed where the rice grains colonized with the fungus were added at 8 g/kg soil. The fungus found to penetrate the egg-masses. Effect of delay in planting after application of chicken manure on M. javanica and P. lilacinus was studied by Odour Owine and Wands (1996). The greatest decrease in nematode population was obtained with six or eight week delay in combination with P. lilacinus. P. lilacinus along with V. chlamydosporium and Trichoderma harzianum has been found to cause greater reduction in nematode multiplication in chickpea (Siddiqui et al, 1996). Dube and Smart (1987) found that nematode control was more effectively obtained when both organism P. lilacinus and Pasteuria penetrans (bacteria) were applied together.

Verticillium chlamydosporium Verticillium chlamydosporium is a facultative parasite of the cysts, females and eggs of Heterodera, Meloidogyne and Globodera (Willcox and Tribe, 1974; Morgan-Jones et al. 1981; Friere and Bridge, 1985). In a study, Bursnall and Tribe (1974) examined presence of fungi in the cysts oiHeterodera schachtii. V. chlamydosporium was found to be present in 43 cysts out of 118. It has also

24 been found frequently as egg-parasite of cereal cyst nematode, Heterodera avenae and other cyst nematodes (Kerry and Crump, 1977). Biocontrol potential of V. chlamydosporium has been reviewed by Carbera ei al. (1987) and Leij and

Kerry (1991).

Lysek (1963) examined ovicidal activity of V. chlamydosporium.

Chalupova etal. (1977) also observed ovicidal activity of a morphological mutant of V. chlamydosporium on nematode. They described 57 mutants of V. chlamydosporium which penetrated and destroyed the nematode eggs. Morgan-

Jones et al. (1983) found that V. chlamydosporium was capable of preventing hatching of M. arenana juveniles and colonizing eggs by hyphal penetration. Both

egg shell and juvenile cuticle were found to be physically disrupted and fungal hyphae readily proliferated within eggs and juveniles. Seagre (1994) reported that

V. chlamydosporium produces a chymoelastase-like protease (VCPI) which

hydrolysed protiens in situ from outer layer of the egg shell of M. incognita and

exposed its chitin layer. VCPI was secreted by several isolates of V.

chlamydosporium and V. lecanii, pathogens of nematode and insects, but not by plant pathogenic species of Verticillium. These observation suggested that VCPI

or similar enzymes may play a role in infection of invertebrates.

Irving and Kerry (1986) studied the infection of Hetrodera avenae and H. schachtii egg by six strains of V. chlamydosporium. Strains differed in their photogenicity but all were parasitic and capable of colonizing the viable eggs including those containing second stage juveniles. Kerry et al. (1986) also found that the strains of V. chlamydosporium differed in their average growth rate.

25 optimum temperature and production of chlamydospores. The variation within the strains indicated that careful selection is essential for the development of this fungus in a biocontrol program. Mayer et al. (1990) assayed 20 fungi in vitro for antagonism to eggs of Heterodera glycines, only Phoma chrysanthemecola, one strain of V. chlamydosporium and one strain of V. lecanii caused a decrease in the number of viable eggs, although no hyphae were observed colonizing the live eggs.

Trichoderma polysporum infected live eggs but en chanced egg survival. Dackman and Nordbring-Hertz (1985) found that out of 15 different fungi isolated from the different stages of Heterodera avenae, V. chlamydosporium was found to be common in young cysts.

Stirling and West (1991) studied the fungal parasites of eggs of root- knot nematodes from the tropical and sub-tropical region of Australia and identified the isolates with the biocontrol potentials. Twenty six isolates of

Paecilomyces lilacinus and 13 isolates of V. chlamydosporium were screened

for activity against Meloidogyne Javanica eggs in 3 different tests. Two isolates

of V. chlamydosporium and one isolate of P. lilacinus were highly parasitic in all

3 tests. Kerry et al. (1994) evaluated grow^th and survival of different isolates of

the V. chlamydosporium in field soil. Some isolates proliferated in soil and

survived in considerable numbers for at least 3 months.

Effect of soil temperature and soil texture on the efficiency of V. chlamydosporium has been examined. According to Leij et al. (1992) optimal growth and sporulation on agar of an isolate of V. chlamydosporium occurred between 22-32''C. Total count of eggs and juvenile showed that greatest control

26 of Mcloichgyne incognita was achieved at 25''C while control at the temperature

20°C and 30°C was less i.e. 60-70%. Leij et al. (1993) showed that V. chlamydosporium multiplied in peaty sand but not in loamy sand. In microplot experiment, the control of root-knot disease caused by M. incognita and M. hapla was greater in peaty sand than in comparison to the other two soil types. In greenhouse experiments in pot, V. chlamydosporium controlled the population of M. hapla on tomato by more than 90% in sandy loam soil. V. chlamydosporium was found to depend on external nutrient in soil for its establishment and colonization of nematode egg masses (Leij et al. 1992).Rodriguez and Kabana

(1984) tested the effectiveness of the species of Paecilomyces and Verticillium for the control of M arenaria in field soil. P. lilacinus was found to be given a good control of M arenaria.

Leij et al. (1992) studied the effectiveness of V. chlamydosporium as a biocontrol agent and its effect on the density of M. incognita. They used V. chlamydosporium and Pasteuriapenetrans alone and in combination, to control

M. incognita on tomato. V. chlamydosporium was found to be most effective.

Majority of the egg masses were colonized by P. penetrans and both resulted in

92% control.

According to Kerry and Bourne (1994), control of root-knot nematode population depends on the density of V chlamydosporium in the rhizosphere, plant species and its cultivar. Kerry and Leij (1994) tested the key factors involved in the development of fungal agents for the control of cyst and root-knot nematodes. They also discussed the use of V. chlamydosporium as a biocontrol agent.

27 Clyde (1992) found that females of//, scbachtii are subjected to attack by V. chlamydosporium. The nematode population was significantly reduced but the suppressive effect of the fungus was host dependent. More juveniles were killed on oilseed rape but a reduction in fecundity attributed to fungus was more apparent in females from beet root.

Fusarium oxysporum and Acremonium strictum Fusarium oxysporum and Acremonium strictum have been found to be parasitic on eggs and cysts of plant-parasitic nematodes (Nigh et al., 1980).

First time the destruction of nematode eggs by Fusarium and

Cephalosporium was noted by Lysek (1963). In a later study, he found egg shell perforation by Acremonium bacillosporum, Helicoon farinosum, Morterella

nana, Paecilomyces lilacinus, V. chlamydosporium and V. bulbillosum (Lysek,

1966).

Verticillium chlamydosporium, V. falcatum, Cylindrocarpon destructans, Humicola grisea, Fusarium oxysporum and F. solani have also been found on cysts of Heterodera schachtii (Vinduska, 1979; Heijbrook, 1983). Nigh et al. (1980) found that eggs ofH. schachtii were attacked by Acremonium strictum and Fusarium oxysporum in sugarbeet fields in California, U.S.A. They also studied the effect of temperature and moisture on parasitism ofH. schachtii eggs by A. strictum and F. oxysporum. They reported that parasitic activity of y^. strictum was greater at 24°C than 28°C, that ofF oxysporum was similar at 24 and 28°C. A. strictum was parasitic when soil moisture was near about the saturation, the reverse was true for F oxysporum. Ownley (1983) isolated F oxysporum and other fungi

28 from young cysts and newly exposed females oiHeleroJcra glycine from soyabean fields in Alabama, U.S.A. Al-Hazmi and Razik (1991) found that out of six fungal species, a species of Acrenwnium, A. persicinum and Aspergillus ochareceous were effective against M. javanica.

Effect of culture filtrates of Fusarium oxysporum and Acremonium strictt^m on the nematodes have also been studied. Nordemeyer and Sikora (1983) determined the effect of culture filtrate of Fusarium avenaceum on the penetration of Heterodera daverti into roots of Triofolium subterraneum. Mani and Sethi

(1984a) found that culture filtrate of Fusarium solani showed toxic effect on M. incognita. Mani and Sethi (1984b) also tested the efficacy of culture filtrates of

Fusarium oxysporum f.sp. ciceri and F. solani at different concentrations and exposure time. They concluded that the culture filtrates of these fungi have a profound effect on hatching and mobility of M incognita. Culture filtrates of

Acremonium striatum, Acrophialophora fusispora, Alternaria alternata,

Aspergillus flavus, A. niger and Pencillium spinulosum were found to be inhibitory against M /«cog«/7(7 (Shabana and Khan, 1992).

Mani et al. (1986) isolated and identified the nematoxins produced by Fusarium solani and found that culture filtrate resulted in colourless, highly non­ volatile oil comprising 5 components, M,-Mj constituting 77, 8, 7, 4, and 3% respectively of the total metabolites. Nematoxicity to Z'^'-stage juveniles of M. incognita was in descending order M -M .

Cayrd (1989) found that toxins produced in culture liquid media or m

29 solid gel media by Fusarium, Trkhoderma and Aspergillus niger were effective against larvae and adults of Meloidugyne.

Mousa and Haque (1988) observed that Fusarium oxysporum reduced the number of females of M. incognita and increased the proportion of males. Giant cells were invaded by the fungus and destroyed. Nematode invasion of the roots, however, was not affected. Qadri and Saleh (1990) tested the impact of several nematode antagonists on H. schachtii and M. javanica in field soil. In vitro on agar, Acremonium sclerotignum a sterile fungus, Preussia spp., Veriicillium and Fusarium solani parasitized 72-82% eggs of H. schachtii. V. chlamydosporium and F solani destroyed 72-82% eggs of M. javanica.

Crump and Flynn (1995) isolated and screened several fungi for the biological control of potato cyst nematode, Globodera pallida. Cylindrocarpon destructans, Fusarium oxysporum and Fusarium sambucinum were particularly effective in decreasing nematode population.

Predatory nematodes The predaceous nematodes are of considerable significance for limiting population levels of nematode pests of agricultural crops, in particular those of the plant parasitic species. Biological control of plant parasitic nematodes is advantage over other methods like physical and chemical controls in being less expensive and ecologically more acceptable. Preliminary studies have been carried out on different aspects of predatory nematodes, viz. their biology, feeding behaviour and ecology and this has attracted considerable attention of

30 nematologist for using them as biocontrol agents.

The predatory nematodes fall under three categories depending upon their mode of feeding and the type of feeding apparatus. The first type is of those which feed by cutting the body of prey and then sucking the body contents as they are unable to engulf intact preys. These predators possess small but well developed buccal cavities and belong mainly to the order Diplogasterida. e.g. Monochoides spp. and Builarius spp. The second type of predators feed by a combined action of cutting and sucking as well as at times engulfing a prey whole. The latter group of nematode predators belong to the order Mononchida and possesses a strong and comparatively larger buccal cavity that is provided with teeth and denticles.

The entire group of mononchs is predaceous. e.g., Mononchus, Mylonchulus, lotonchus, Parahadronchus. The third group is of those that feed only by puncturing the cuticle of prey and then suck their body contents. The feeding apparatus of the nematodes of the latter group, which belong to the suborders

Dorylaimina, Aphelenchina and Nygolaimina is of piercing and sucking type. They may possess a stylet or spear or a mural tooth, e.g. Dorylaimus, Labronema,

Discolaimus, Nygolaimus, Aqualides, Seinura etc.

Cobb (1917) was the first who had discussed the influence of predatory nematodes on plant parasitic nematodes. Cobb (1920) and Steiner and Heinly (1922) suggested the use of Clarkus papillatus in controlling populations of nematodes in sugarbeet fields. Thorne (1927) studied the life history and population dynamics of some mononchs in sugarbeet fields infested with Heterodera schachtii. Cassidy (193 1) found that a large number of Heterodera

31 spp. eggs and juveniles in culture media were devoured by lolonchus hrachylaimus.

Christie (1960) suggested that predatory nematodes should be assessed for determining the practicability of their use in the control of plant parasitic nematodes. Further studies on predatory mononchs were made by Mulvey (1961),

Esser(1963), Esser and Sobers (1964), Nelmes (1974) and Ritter and Laumond

(1975). Mononchs generaly feed on a variety of soil micro-organisms including nematodes (Banage, 1961; Arpin and Kilbertus, 1981). According to Cohn and

Mordechai (1974) Mylonchulus sigmaturus predates upon citrus nematode,

Tylenchulus semipenetrans and whenever the predator's density is high, generally the population of T. semipenetrans is low. Research on predatory mononchs has gained momentum in the last few years.

Maertens(1976),Grootaert and Wyss (1979), Small (1979), Small and

Grootart (1983), Azmi and Jairajpuri (1979), Nelmes (1974) have worked on predatory behaviour of mononchs.

Yeates (1969) observed the predatory behaviour of a diplogasterid nematode, Mononchoides potohikus. Grootaert et al. (1977) made observation on the feeding behaviour of Bullarius degrissei. While observing the predatory abilities of Butlarius degressei. Small and Grootaert (1983) found Panagrellus redivivus and the first and second stages of Rhabditis oxycerca as most susceptible prey species. Osman (1988) also evaluated the efficacy of Diplogaster sp., as predator of plant parasitic nematodes. He conducted experiments in vivo as well in vitro conditions and found that populations of plant parasitic nematodes,

Meloidogynejavanica and Tylenchulus semipenetrans were significantly reduced by the addition of predatory nematodes in pot experiments.

32 Several observation have been made on the different aspects of predatory behaviour of Mononchoides longicaudalus and Mononchoides fortidens, in vitro conditions, by Jairajpuri and his co-workers. Jairajpuri et al. (1990) speculated the possibilities of some dorylaims like Eudorylaimus,

Mesodorylaimus, Aporcelaimns, AporcelaimeJhis, Nygolaimns and Sectonema to be predaceous. Khan et al (1991, 1994 and 1995) have provided a detailed account of predatory behaviour of Aporcelaimellus nivalis, Allodorylaimiis americanus, Discolaimus silvicolus and Neoactinolaimus agilis.

Potential of predatory nematodes in the management of plant parasitic nematodes needs to be fully explored and utilized. These nematodes are natural bioresource and if found effective in the nematode management, may be artificially exploited on large scale. With this aim in view, experiments have been conducted to evaluate efficacy of a predatory nematode, Mononchoides longicaudalus in the management of root-knot nematode M. incognita on tomato in pots under greenhouse conditions by artificial inoculations.

Although several fungi, bacteria and predatory nematodes are known to reduce the nematode populations in vitro and in vivo conditions, their potential is either not fully assessed or utilized and biocontrol of nematodes particularly of endoparasites is still in developing stage.

In this research program for Ph.D., emphasis has been laid on utilization of biocontrol entities for the control of root-knot nematodes principally by nematophagous fungi and predatory nematodes.

33 SECTION-I MATERIALS AND METHODS

Collection of root- knot nematode population Surveys were conducted to collect the root samples of vegetable crops from vegetable fields infested with root-knot nematodes in different parts of the country. Root and soil samples collected in properly labelled polythene bags, were brought to laboratory for exmination. Roots from each sample were washed under tap water and examined for the presence of galls and egg masses.

Identification of nematode species The perineal pattern of mature females (10-20) from each sample was prepared and examined under the microscope for their characterstics and identification of species (Eisenback et al 1981). North Carolina differential host tests (Taylor and Sasser, 1978) were conducted for identification of the species. After species identification, single egg mass culture of Meloidogyne incognita was raised on tomato and maintained for experimental use (Taylor and Sasser, 1978).

Observation of egg masses for fungal infection Egg masses of the root-knot nematodes present in the root samples were examined for their natural infection by fungi. Egg masses randomly collected from the roots of each sample were directly observed under the microscope for the presence of fungi especially soil hyphomycetes.

Eggs from the infected egg masses were spread over a glass slide by pressing the egg mass with a coverslip and stained with 0.1% cotton blue in lactophenol and examined under the microscope for fungal infection.

34 Isolation and identification of fungi associated with egg massess A few egg masses found to be naturally infected with fungi were plated on patato dextrose agar (PDA) for isolation of fungi. These egg masses were first treated with 0.1% HgClj for 1-3 minutes and then washed thoroughly three times with sterilized distilled water. The egg masses were plated on PDA in sterilized petriplates under asceptic conditions on a laminar flow bench.

The composition of PDA are as follows -

Peeled and sliced potatoes 200 g Dextrose 20 g

Agar agar 20 g Distilled water 1000 ml

The medium was autoclaved at 15 p.s.i for 30 min. Small amount of streptomycin was added to PDA to check the bacterial contamination. Twenty ml of the medium was poured in each petriplate. Egg masses with fungal infection were placed on the surface of the medium in petri plates and gently appressed. The petriplates containing egg masses were then incubated at 25±2°C for a week in the incubator and fungal colonies developed on the egg masses were examined. The fungi pennetrating the eggs and egg masses were then isolated, multiplied and maintained on PDA slants.

Effect of some soil fungi by artiflcial inoculation into the egg masses of Meloidogyne incognita in vitro

Efficiency of some soil fungi were determined by artificial inoculation

35 of the egg masses of M. incognita. Egg masses of M incognita v^ert picked from tomato {Lycopersicon escidentum Mill. cv. Pusa Ruby) roots maintaining single egg mass culture of M incognita. These egg masses were dipped in mercuric chloride (0.01%) for 1-3 minutes and washed three times with sterlized distilled water to remove the traces of mercuric chloride. These surface sterlized egg masses were placed on cooled PDA in petri plates. The fungi, VerticiUium chlamydosporium, Acremonium strictum and Fusarium oxysporum were inoculated separately near the egg masses. The whole procedure was carried on laminar flow bench. Petriplates having sterilized egg masses served as control. The petriplates were incubated at 25±2°C temperature. After one week of incubation, the egg masses were examined for the infection of eggs.

For determining the percentage of infected eggs in egg masses, the egg masses were stained with cotton blue in lactophenol. The egg masses were gently pressed over a glass slide to seperate the eggs. The number of infected eggs with test fungus were counted under the microscope and percentage of infected eggs was calculated.

Juvenile hatching of Meloidogyne incognita in culture filtrates of some soil fungi

Effect of culture filtrates of VerticiUium chlamydosporium, Acremonium strictum and Fusarium oxysporum on juveinle hatching of Meloidogyne incognita w^s evaluated. The culture filtrates of these fungi were obtained by growing them separately in sterilized potato dextrose broth m 250 ml

36 Erlenmeyer flasks for seven days in an incubator at 25±2°C. After incubation period the mycelial mats were removed and the media were filtered through

Whatman paper No. 1 and 2. The filtrate obtained was designated as standard solution (S). Further dilutions (S/2, S/10, S/100 and S/1000) were prepared by adding requisite amount of sterlized distilled water.

Five average sized egg masses of the Meloidogyne incognita obtained from the roots maintaining single egg mass culture of the nematode were placed in 5 ml of the different dilutions of culture filtrates of the above mentioned fungi contained in sterlized petri plates (3cm diam.). The plates having only sterlized distilled water served as control. Each treatment was replicated five times. The plates were examined after 24, 48 and 72 h of incubation and the numbers of juveniles hatched in each treatment were counted.

Juvenile mortality of Meloidogyne incognita in culture filtrates of some soil fungi Three soil fungi namely, Verticillim chlamydosporium, Acremonium striclum and Fusarium oxysporiim were used in this experiment. The source of these fungi was same as detailed in the previous experiment. Different concentrations of each culture filtrates were prepared as described in the previous experiment.

The second stage juveniles (J2) of Meloidogyne incognita used for the experiment were obtained from egg masses of the nematode maintained as single- egg mass culture on tomato. The egg masses were kept on double layered facial

37 tissue paper supported by a coarse sieve. The sieve was placed over the petri plates having sufficient sterilized water to touch the bottom of the support. A small amount of the water was poured over egg masses. After 24 h the hatched juveniles were collected from the petri-plates and were used for the experiment.

One hundred second stage juveniles (J^) of M incognita-were placed in sterlized 3cm diam. petriplates containing 5 ml of different concentrations of culture filtrates of each selected soil fungus. For each concentrations, there were five replicate plates. The petriplates were incubated at the temperature 25±2°C. The petriplates were examined after 24, 48 and 72 hours.

The numbers of dead juveniles in each treatment of each time interval were counted under the stereoscopic microscope.

Effect of soil application Verticillium chlamydosporium I Acremonium strictum / Fusarium oxysporum on plant growth and development of root-knot nematode Meloidogyne incognita on tomato

Raising and maintenance of test plants Seeds of tomato {Lycopersicon esculentum Mill. cv. Pusa Ruby), selected as test plant for the experiment, were surface-sterilized with 0.01% HgCl2. Seeds were put in the HgCl2 solution for 1 to 2 min. and washed three times with sterilized distillised water to remove traces of mercuric chloride. Seedlings were raised in autoclaved field soil (66% sand, 24% silt, 8% clay, 2% organic manure, pH 7.7). Three-week-old seedlings were transplanted singly into 9cm pots containing sterilized field soil, sand and compost in the ratio of 7:3:1.

38 The pots were arranged in complete randomized block design in greenhouse. Each treatment was replicated five times and the pots were watered regularly.

Nematode inoeulum and seedling inoculation In experimental pots, the tomato seedlings were inoculated with freshly hatched second stage juveniles (J2) ofMeloidogyne incognita in water suspension. Second stage juveniles were obtained from single egg mass culture of the nematode as described earlier.

For inoculation, small holes were made in the soil around the roots of the seedlings and measured quantity of the water suspension with second stage juveniles of M incognita was pipetted out uniformly on the exposed roots. Each seedling was inoculated with 2000 J^. Then the roots were covered with the same soil and light watering was done.

Fungalinoculation Verticillium chlamydosporium, A. strictum and F. oxysporum isolated from the egg masses and maintained as pure cultures on PDA slants in culture tubes were used as inoculum source. PDA broth was used for culturing these fungi in order to obtain inoculum for inoculation purpose.

After preparation of PDA broth, 20 ml of the medium was transferred into 250 ml Erlemneyer flasks. Flasks after proper plugging with cotton were sterilized in autoclave at 15 p.s.i. for half an hour. Flasks were then aseptically inoculated with pure cultures of the V. chlamydosporium /A. strictum I F. oxysporum.

39 Inoculated flasks were incubated at 25±2"C for a week. At the end of incubation period the mycelial mats were removed from the flasks, washed with sterilized distilled water to remove the traces of the medium. Fungal mat was gently pressed between sterilized blotting papers to remove the excess amount of water. Ten gram of fungal mat was macerated in a waring blender in 100 ml sterilized distilled water. The fungi were added at the rate of 1 g mycelium/pot. Therefore, 10 ml of this suspension containing 1 g of the fungus was poured near the roots, exposed by removing the top layer of the soil. The roots was then covered with the same soil again. The inoculated pots were kept moist for few days for stablization of the fungus.

To determine efficacy of the each fungus {V. chlamydosporium/A. strictum'F. oxysporum) for controllingM. incognita, following treatments were used in the experiment.

Tj = Plant (Uninoculated) - control T^ = Plant + Nematode (M incognita)

T, = Plant + Fungus {V. chlamydosporium/A. strictum/F. oxysporum)

T^ - Plant + Fungus {V. chlamydosporim'A. strictum/F. oxysporum) +

Nematode (M. incognita)

Tj = Plant +Nematode 10 days before fungus {V. chlamydosporium/A.

strictum/F. oxysporum) inoculation

T^ - Plant + Fungus {V. chlamydosporium 'A. strictum/F oxysporum) 10 days

before nematode inoculation

Each treatment was replicated five times and the pots were arranged on

40 glasshouse benches in completely randomized block design.

Recording of data The experiment was terminated after 60 days of inoculation. At termination plants were uprooted and roots were washed gently with tap water. The following parameters were used to assess the efficacy of each fungus (1) Root and shoot length (2) Fresh weight of root and shoot (3) Dry weight of root and shoot (4) Gall index (GI) (5) Egg mass index (EMI) (6) Nematode population (root population - 3rd and 4th stage juveniles and males) + (soil population - 2nd stage juviniles and males) (7) Reproduction factor (Rf) (8) Infection of nematode eggs by the fungus (9) Number of eggs per egg mass (Fecundity)

The parameters of plant growth in all the replicates were determined and mean values were calculated. For dry weight, plants were dried in an oven at the temperature 50°C for 48 h. in paper envelopes. Gall index (GI) and egg mass index (EMI) were rated on 0-5 scale (Taylor and Sasser, 1978).

For determining the number of eggs per egg mass (fecundity), 10 egg masses were placed in a container with 100 ml of 0.5% sodium hypochlorite

41 (NaOCl) solution. The container was tightly capped and shaken vigorously for three minutes. Shaking partially dissolved the gelatinous matrix, freeing eggs from the egg masses. The suspension of eggs was poured through a 200 mesh sieve nested upon a 500 mesh sieve. Then after eggs were washed free of residual NaOCl solution under a slow stream of tap water. The concentration of eggs per ml was standardised by counting the eggs from ten, 1 ml samples and the total number of eggs was estimated and average was determined (Taylor and Sasser, 1978 ).

Infection of eggs in the treatments receiving the fungal inoculum was determined and percentage infection was calculated. Egg masses, randomly selected from the roots of various treatments recieving the nematode and fungus, were stained with cotton blue in lactophenol and pressed on glass slide to separate the eggs. The slides were examined under the microscope and the number of eggs infected with fungus was counted and percentage of infected eggs was calculated. In addition to it, deformity and abnormal development of juveniles and their infection in the eggs was also examined and noted.

For counting the nematode population, roots from each replicate of the treatments were weighed and cut into small pieces (2-3 cm). One gram of the root pieces was stained with acid fuchsin and lactophenol solution (Byrd et al., 1983). Each root piece was gently pressed between two glass slides and examined under a stereoscopic microscope and number of Jj, J3 and J^ were counted, from which the total number for the whole root system was calculated. To count the number of females, 1 g of root pieces was transferred into 5% HN03 and inoculated at 25°C. After 72 h the root pieces were gently teased to release the

42 females. The number of the females thus released were counted and the total for the whole root system calculated (Khan and Haider, 1991). The second stage juveniles and males which represent the migratory stages were isolated by Cobb's sieving and decanting and Baermann's funnel methods and counted in a counting dish. The reproduction factor (Rj.) was calculated as Rj.=Pj/P, where Pj,is the final population and P; the initial population (2000 J^) (Oostenbrink, 1966).

Statistical analysis Experiments were conducted in a complete randomized block design. The data obtained were subjected to analysis of variance (ANOVA) to determine significance and critical difference (CD) at P= 0.01 and P = 0.05 to separate the means of replicate for significance (Pame and Sukhatame, 1954). The ANOVA model adopted for analysis of variance are as follows -

CF = Gr/RXT S.S (Treatment) = TF+r+TS^+TA^TSl../No.of replicate - CF S.S (Replicate) = RF+R22+R32+R42+R52.../No. of treatment-CF S.S. (Total) = Sum of square of every value then add - CF S.S (Error) = S.S (Total) - (ss(treatment) + ss(replicate)) df=(R-l)(T-l) MSE = SS(Error) / d,

CD at 5% or 1% = [MSE x 2/R x T^5, T0 1 ANOVA TABLE SV dy SS MSS = SS/df F = MSS(T)/MSE Replicate Treatment Error Total

43 Abbreviations used

CD = Critical difference

GT = Grand Total

R = Replicate

T = Treatment

CF = Correcton factor

SS = Sum of squares

MSE = Mean standard error dj. = Degree of freedom

SV = Source of variation

Effect of seed soaking in different concentrations of culture filtrate of Verticillium chlamydosporium/Acremonium strictum/ Fusarium oxysporum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Methods of raising seedings, preparation of the nematode inoculum in the form of J^ and culture filtrates of the three fungi were same as in the previous experiment. Three concentrations of the culture filtrate of the each fungus, standard (S), undiluted culture filtrate, S/2 and S/10 were used in the experiments.

S/2 and S/10 concentrations were prepared by adding requisite amount of sterilized distilled water.

The seeds of tomato cv. Pusa Ruby were soaked in the concentrations of culture filtrate of V. chlamydosporium/ A. strictum^ F. oxysporum for an hour.

Untreated seeds (unsoaked) served as control. After soaking the seeds were allowed

44 to dry under shade and 4 soaked seeds were sowed in each pot (9 cm), filled with autoclaved soil, sand and compost in the ratio of 7:3:1.

Two - weeks after germination thinning was done to keep only one seedling per pot. Three- week- old seedlings were inoculated with 2000 J^ / pot.

The recording of data was same as in the previous experiment.

Following treatments were used in this experiment-

Tl = Plant (Unsoaked - uninoculated) control

T2 = Plant (Unsoaked) + Nematode (M. incognita)

T3 = Plant + seeds soaked in standard solution (S) of V. chlamydosporium .

A. strictum /F. oxysporum + Nematode (M incognita)

T4 = Plant + seeds soaked in S/2 concentration of V. chlamydosporium / A.

strictum /E. oxysporum + Nematode (M incognita)

T5 = Plant + seeds soaked in S/10 concentration of V. chlamydosporium / A.

strictum /F. oxypsorum + Nematode (M. incognita)

T6 = Seeds soaked in standard solution (S) of V. chlamydosporium ' A.

strictum / F. oxysporum

Each treatment was replicated five times and the pots were arranged on glasshouse benches in complete randomized block design. The recording of data for all the considered parameters were done in the same way as mentioned earlier.

Screening of various agricultural wastes for the growth of fungus This experiment was carried out to find out a suitable and cheap organic

45 substrate to serve as a good nutrient source for field dispensing and establishment of the fungi in the soil ecosystem.

Five organic substrates of agricultural origin, wood charcoal, sawdust, husks of rice {Oryza sativa L.), mungbean [Vigna radiata (L.) Wilczek] and gram husk {Cicer arietinum L.) were tested. The husk of rice, mungbean and gram were collected from their respective factories. The wood charcoal and sawdust are used as fuel in house holds. These materials were powdered and placed in Erlenmeyer flasks separately. Tap water was added to these substrates to attain 20% w/w moisture and autoclaved for 20 min. at 15 p.s.i.

Ten-day old fungal colonies of all the three fungi V. chlamydosporium, A. strictum and Fusarium oxysporum, were flooded with sterilized distilled water. The agar surface was lightly rubbed with a sterile loop to release the fungal spores. The resulting spore suspension was collected in a sterilized flask and homogenized with 2 drops of Tween-20. The autoclaved substrates in each flasks (15g) were inoculated with 3 ml of spore suspension separately and incubated at room temperature (25±2°C) for ten days. The flasks were shaken for the first two days to promote the uniform growth. The concentration of spores in the suspension were determined by a haemocytometer. Spore load per gram was calculated using the following formula-

SLPG = NxVx 10,000/W where, N = Number of spores in the central square V = Volume of the mounting fluid added to the sediment and

46 W = Weight of the substrate (Mathur and Bennum, 1974)

Viability of spores (spore load/g) of some oppotunistic soil fungi on various organic materials of agricultural origin over a period of 8 weeks.

For the study, Verticillium chlamydosporium was cultured on rice husk and Acremonium strictum and Fusarium oxysporum on gram husk. The type of substrate was selected because of highest spore load.

The preparation of spore suspension was same as described in the previous experiment.

For culturing the soil fungi on the substrates, the required quantity of tap water was added to rice husk and gram husk to achieve 20% w/w moisture level. The moist substrate was autocalved at 15 psi for 30 min. and cooled . Fifty gram of the cooled substrate were aseptically transferred to low density polyethylene pouches (10 x 20cm) and sealed. Five ml of the respective spore suspension were injected into each packet using a sterlized syringe and needle and the holes were blocked with cellophane tape. The contents were mixed throughly and the packets were stored in an incubator at the temperature 25±2°C.

The contents of the packets were assayed for fungal spore viability at start of the experiment and after 2, 4, 6 and 8 week intervals. One gram of sample from packet was used for assay by dilution plate method. Serial dilution were prepared in sterile water until an estimated spore concentration of 1 x 10' was reached and 0.1 ml sample was spread on PDA in each petri plate. The plates were

47 incubated in an incubator at 25±2°C for seven days. Germination of the spores was checked under the microscope.

Viability of spores of some opportunistic soil fungi on some organic materials of agricultural origin

For studying the effect of aeration polyethylene bags were punctured at five places with sterlized needle to facilitate gaseous exchange (aeration) during storage. The packets were stored in an incubator at the temperature 25±2°C. Spore load/g of the puncutured and unpunctured packets were examined after 8 weeks. Each treatment was replicated four times.

Efficacy of some agricultural wastes for soil application of V. chlamydosporium /A. strictum/F. oxysporum in the management of M incognita on tomato

Methods of raising and maintenance of seedlings of tomato, preparation of nematode inoculum in the form of J^ were same as described in previous experiments.

For fungus inoculation, the soil fungi, V. chlamydosporium/A. strictum F. oxysporum were cultured on the waste substrates. The method of culturing these fungi was also same as described in previous experiments. Five grams of the substrate with or without fungus per pot were used as dosage for inoculation.

For studying the efficacy of agricultural wastes for soil application of V. chlamydosporium/A. strictum/F. oxysporum in the management of M incognita

48 on tomato following treatments were employed :

T, = Plant (Uninoculated) - control

T^ = Plant + Nematode (M incognita)

T^ = Plant + Mungbean husk (inoculated with V. chlamydosporium/A.

slrictum^F. oxysporum) + Nematode (M. incognita)

T = Plant + Gram husk (inoculated with V. chlamydosporim/A. strictum F.

oxysporum) + Nematode (M. incognita)

Tj = Plant + Rice husk (inoculated with V. chlamydosporium/A. strictum/F.

oxysporum) + Nematode (M incognita)

Tg = Plant + Saw dust (inoculated with V. chlamydosporium/A. strictum/F

oxysporum) + Nematode (M incognita)

T^ = Plant + Wood charcoal (inoculated with V. chlamydosporium/A.

strictum/F. oxysporum) + Nematode (M incognita)

Each treatment was replicated five times and the pots were arranged on the glasshouse benches in completely randomized block design. The recording of data was done in the same manner as described in the previous experiments.

Effect of soil application of rice husk colonized with V. chlamydosporium on plant growth and root-knot nematode development on tomato

Methods of raising and maintenance of seedlings and preparation of nematode inoculum in the form of J^ were same as described in the previous experiments.

49 V. chlamyciosporhim was cultured on rice husk, i.e. the substrate with highest spore load. The method of culturing of the fungus on rice husk was same as described in the previous experiments.

Five grams of rice husk colonized with or without the fungus per pot was inoculated.

For studying the effect of soil application of rice husk colonized with

V. chlamydosporium on plant growth and development of M incognita on tomato the following treatments were used :

Tj = Plant (Uninoculated) - control

Tj = Plant + Nematode (M. incognita)

T3 = Plant + Rice husk colonized with V. chlamydosporium

T^ = Plant + Rice husk colonized with V. chlamydosporium + Nematode (M

incognita)

Tj = Plant + Uninoculated rice husk

Tg = Plant + Uninoculated rice husk + Nematode (M incognita)

T, = Plant + Fungus {V. chlamydosporium) 15 days prior to Nematode (M

incognita) inoculum

Tg = Plant Nematode (M. incognita) 15 days prior to Fungus {V. chlamydosporium) inoculum

There were five replicate for each treatment the pots were arranged on the glasshouse benches in completely randomized block design. The recording of data at termination of this experiment was done similarly as described in previous experiments.

50 Effect of soil application of gram husk colonized with A. strictum/ F. oxysporum on plant growth and root-knot nematode development on tomato Methods of raising and maintenance of seedlings and preparation of

nematode inoculum in the form of J^ were same as described in previous

experiment.

A. strictum/F. oxysporum were cultured on gram husk. The method of

culturing the fungi on gram husk was same as described in the previous experiment.

Five gram of substrate i.e. gram husk colonized with or without fungi

per pot was inoculated.

For studying the effect of soil application of gram husk colonized with

A. strictum/F. oxysporum on plant growth and development of M incognita on

tomato the following treatments were used :

Tj = Plant (Uninoculated) - control

Tj = Plant + Nematode (M incognita)

T3 = Plant + Gram husk colonized with Fungus {A. strictum/F. oxysporum)

T^ = Plant + Gram husk colonized with Fungus {A. strictum/F. oxysporum) +

Nematode (M. incognita)

Tj = Plant + Uninoculated gram husk

Tg = Plant + Uninoculated gram husk + Nematode (M incognita)

T, = Plant + Fungus (A. strictum/F. oxysporum) 15 days prior to Nematode (M incognita) inoculum

51 T^ = Plant + Nematode (M. incognita) 15 days prior to Fungus (A. strictum F. oxysporum) inoculum

Each treatment was replicated five times and the pots were arranged on the glasshouse benches in completely randomized block design. The recording of data was done in the same way as described for previous experiments.

52 RESULTS

Opportunistic soil fungi Root samples of different vegetable crops were collected and examined under the laboratory for the presence of root-knot nematode. Egg masses of root- knot nematodes present in the root samples were examined for the infection of eggs. The fungi infecting the egg in the egg masses were isolated and their pathogenecity on egg masses was established in artificial inoculation under aseptic conditions. Three soil fungi namely Verticillium chlamydosporium, Acremonium strictum and Fusarium oxysporum were found to be parasitic on root-knot nematode, Meloidogyne incognita. These fungi were maintained on the stock culture and were regularly transfered on new slants. These three fungi were used in the experiment given in the Section-I of the thesis (Table 1; Plate 1, 2, 3).

53 si •*cj &) u C CI. 00 c o o u iJ u

E/3 0) V3 IM rt a b£ ON 01 V C/5 u (4- (^ ^c •J o o • o "a. z (^ >

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at/i> V3 e es •o E V m DXI b> - o o o OX) a

1/1

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s v. o O -s: w S 3 OX) a 5 c o ^ 3

O 'J C/3 :^ PLATE!

A. Some eggs of Meloidogyne incognita infected with Verticillium chlamydosporium.

B. Mycelium of V. chlamydosporium colonizing the interior of an egg of M. incognita.

C. Second stage juveniles of M. incognita infected with V. chlamydosporium.

D-E. Second stage juveniles of M. incognita infected with V. chlamydosporium. PLATK-l PLATE-2

A. An egg of Mel oidogyne incognita infected with Acremonium strictum.

B. Magnified view of A.

C. An egg of M. incognita filled with mycelium and spores of A. strictum.

D. A second stage juvenile (J2) of M. incognita infected withyi. strictum.

E. A J2 of M. incognita infected with A. strictum and hypae of the fungus. PI ATr:-2 PLATE-3

A. Some eggs of Meloidogyne incognita infected with Fusarium oxysporum.

B. A second stage juvenile of M incognita infected with F. oxysporum.

C. An egg oiMeloidogyne incognita infected with F oxysporum.

D. An egg of M. incognita filled with the mycelium oiF. oxysporum.

E. Magnified view of an egg of M. incognita infected with F oxysporum. PI VIF-3 Juvenile hatching and mortality of Meloidogyne incognita in different concentrations of culture filtrates of some soil fungi

The effect of culture filtrates of Verlicillium chlamydosporium, Acremonium strictum and Fusarium oxysporum on juvenile hatching and mortality of M. incognita was examined using graded concentrations for varying time durations. The toxic effect of the culture filtrates varied with the fungus involved and increased with the lapse of time. Higher concentrations and extended time durations were more effective. The culture filtrate of V. chlamydosporium was more effective than A. strictum and F. oxysporum (Table 2, 3, Fig. 1, 2).

Juvenile hatching of M. incognita was adversely affected by the culture filtrates of all the three fungi, and the number of hatched juveniles decreased as concentration of the culture filtrate increased. The increase in exposure duration decreased juvenile hatching in each concentration of the culture filtrates (Table 2, Fig. 1).

Mortality of second stage juveniles (Jj) of M. incognita occurred in the culture filtrates of all the three fungi, and the mortality increased as the concentration of the filtrate increased. Like juvenile hatching, increase in the exposure duration increased the juvenile mortality in each concentration of the culture filtrates (Table 3, Fig. 2).

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<— saijueAnf^o jaquinN Effect of soil application of Verticillium chlamydosporium / Acremonium strictum / Fusarium oxysporum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

The efficacy of soil application of Verticillium chlamy dosporium / Acremonium strictum /Fusarium oxysporum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato was studied in artificial inoculation treatments in greenhouse conditions.

Plant growth Inoculation of tomato seedlings with second stage juveniles (J2) of M incognita suppressed plant growth in relation to length, fresh and dry weights of both roots and shoots. When tomato plants were inoculated with V. chlamydosporium /A. strictum /F. oxysporum alone, plant growth did not differ significantly from control plants. When either of the fungi andM. incognita wfere added simultaneously, plant growth showed improvement and considered parameters were greater than those in the plants inoculated with M. incognita alone. Plant growth also improved when either of the fungus was inoculated first followed byM incognita. This treatment was better than simultaneous inoculation (N+F) or where fungus followed the nematode (N -> F) in sequential inoculation (Table 4, 5, 6, Fig. 3, 4, 5, Plate 4, 5, 6, A-B).

In general, the presence of above mentioned fungi reduced the adverse effects of M incognita on plant grovi^h, irrespective of the mode of inoculation.

63 In this respect, VerliciUium chlamydosporium was more effective than Acremonium siriclum and Fusarium oxysporum.

Root-galling and egg mass production Rating of the root galling and egg mass production in the form of gall- index (Gl) and egg mass index (EMI) showed that all the three fungi suppressed root galling and egg mass development. Supressions were greater in sequential inoculation where nematode followed either fungus. V. chlamydosporium was found to be most effective among the three fungi in reducing the root galling and egg mass production (Table 4, Fig. 3).

Nematode population and reproduction factor Meloidogyne incognita reproduced well on tomato plants inoculated with its juveniles alone. Root and soil populations of the nematode in fungus inoculated plants and reproduction factor of the nematode declined, when either fungus was added either in simultaneous or sequential inoculation. In sequential inoculation, nematode reproduction was greater on plants, where nematode was inoculated 10 days prior to either of the fungus. The nematode reproduction was lowest when V. chlamydosporium /A. strictum /F. oxysporum was inoculated 10 days before M. incognita in sequential inoculation (Table 4, 5, 6, Fig. 3, 4, 5).

Infection of egg Verticillium chlamydosporium /A. strictum /F. oxysporum reduced the fecundity (number of eggs/egg mass) of the nematode. The number of eggs/egg mass was significantly less (P = 0.01) on roots of the plants receiving both M

64 incognita and V. chlamydosporium /'A. siriclum /F. oxysporum compared to the plants inoculated with the nematode alone. A higher percentage of eggs obtained from roots of plants where V. chlamydosporium /A. strictum /F. oxysporum was

inoculated were infected with the fungi. Highest infection was obtained in

sequential inoculation where fungus preceded the nematode. V. chlamydosporium was most effective as greater percentage of eggs were infected compared to other

two fungi F. oxysporum was least infective of the eggs (Table 4).

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u JD o o d d 1 II II o 1 73 o "o i PL. a. O <—' in •4-c* 1C y~~. '»»S• + o ^^^ c 'C H>4 Q II O o > U 1 ^ ^ <— (luo/B) ii)6ua|/mB!3M PLATE-4

A-B. Effect of soil application of Verticillium chlamydosporium on plant growth and development of Meloidogyne incognita on tomato.

C-D. Effect of soil application of rice husk colonized with V. chlamydosporium on plant growth and development of Meloidogyne incognita on tomato. ^ VC 00 o ly^ 1 1 o Z d d d o d oo NO iMM vO ON ON oo oo V C8 f^ 00 rs Tt ' ' fN NO £ e f2 i oo a o Os •aioM _« '/-I c^ V cc n 1 (N 1 f—1 fN _^ o o > "o3. Co S d d ii o o V 00 fN -a a u "cS "* '^ i—< NO 1 o 1 •tr vd r~-" «-) o •c E fN fN "5 '5 01 NO —; B tb, 5"^^ •N«^ R es E •* oo fN rt '^ NO o 1 O rr ON -^tf Z r^ 1 00 ON ;f « i-» VD fN TT r-4 '•" ^~' o O o O O E »S o -* NO (S£ o i-S 1 1 fN fN NO ""; "^ n r^ "^ .^ ^ m c •*-> VO fs fN m o 0© 00 C IZ) 00 vO r- t- •— tN a& ^1 R rn -^ m' fN fN fN d d es 5^ •m o TT iri •* fN uo '4^ fi£ 1/-) fN o 00 Tt od NO CA >ri ^^ d d (^ 00 fS fN o o 'it fN fN W3 ON fN NO V3 ON ON d •M d u 0^ c (^ 5 < o 2

C 0) x> S3 c — to w — w a> 5 < < 5 < E a o a> > "aO> •a c (0 o JZ 4-1 TO 1 F o o O) 4-r> o 10 ^• as c o ••SS.^ r/^)^ i: < o u

a Q> o C C ^ V) o E 5 a o c i> o S; E e •o u <£o5 'I (0 o E c 0) o c <

<— (luo/B) q)6u3|/)q6!aAA PLATE-5

A-B. Effect of soil application of Acremonium strictum on plant growth and development of Meloidogyne incognita on tomato.

C-D. Effect of soil application gram husk colonized with A. strictum on plant growth and development of Meloidogyne incognita on tomato. its i

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,o I •••» t»5 (*: y^ II ^ O o §, C/3 CO d d 5^ 5 MH ^ ^ II II "o a •o "O &-, b •*w S ^1*^ c C ^..^ o o I f2 O .—N O + O d 1 1 o C/3 0) 1 10 v> (0 >. •o 2 S •D o O oc O + — D 0 = O k u. nu. k J. U. S D B D D • El

<— (iuo/6) qjBuaiflqBiBM PLATE-6

A-B. Effect of soil application of Fusarium oxysporum on plant growth and development of Meloidogyne incognita on tomato.

C-D. Effect of soil application gram husk colonized with F. oxysporum on plant growth and development oiMeloidogyne incognita on tomato. &4 Screening of various agricultural wastes for the growth of the biocontrol fungi

Four agricultural wastes and organic materials of plant origin were used to determine their suitability for fungal growth for field dispensing of the biocontrol fungi. All the three fungi showed growth on the materials and colonized them. V. chlamydosporium showed good growth all the materials used. Its growth on the basis of spore load was, however, best on rice husk followed by mungbean husk, gram husk, saw dust and wood charcoal in this order. Growth oiA. strictum on the basis of spore load was highest on gram husk followed by rice husk, mungbean husk, saw dust and wood charcoal. Similar treand in the growth of F. oxysporum was also observed (Table 7).

Viability of spores of some opportunistic soil fungi on some organic materials of agricultural origin

Viability of spores (spore load/g) of three soil fungi V. chlamydosporium /A. strictum / F. oxysporum grown on various organic materials of agricultural origin was determined over a period of eight weeks. The spore load of the fungi increased during the period when determined after 2, 4, 6 and 8 weeks of storage.

In V. chlamydosporium spore load increased gradually and peaked after 6 weeks. The spore load determined after 8 weeks was almost equal to the spore load observed after 6 weeks.

In case of Acremonium strictum, increase in the spore load was observed upto 4 weeks after which it declined gradually being 1.74 x 10^ after 6 weeks and

75 1.5 X 10^ after 8 weeks in comparison to 2.45 x 10'' after 4 weeks which was highest.

Similar trend in increase of the spore load upto 4 weeks and decrease afterwards was also observed in F. oxysporum.

When viability of spores was also checked after 2, 4, 6 and 8 weeks by the method described by Bansal et al., (1992), it was found that spores of all the three fungi were viable at different durations (2, 4, 6, and 8 weeks) for observation (Table 8, 9).

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I u C 01 (J c 3 o 5 O Co 5/3 ± Efficacy of some agricultural wastes for soil application of Verticillium chlamydosporium / Acremonium strictum / Fusarium oxysporum in the management of Meloidogyne incognita on tomato

Evaluation of various organic materials of agricultural origin for supporting growth and sporulation of V. chlamydosporium / A. sirictum / F. oxysporum revealed that these fungi colonized and sporulated on gram husk, rice husk, mungbean husk, saw dust and wood charcoal. Maximum number of spores/g (or SPLG) were observed on rice husk in case of V. chlamydosporium and on gram husk in case oiA. strictum /F. oxysporum. All the three fungi cultured on these organic materials when added to soil improved plant growth, reduced root galling and egg mass production and resulting population of the nematode declined considerably.

Plant growth Tomato plants inoculated with root-knot nematode, M incognita showed better plant growth (length of root and shoot, fresh and dry weight of root and shoot) when treated with A. strictum and F. oxysporum cultured on gram husk. The fungi grown on rice husk, mungbean husk and sawdust were also effective in improving the plant growth. Minimum improvement in plant growth was obtained when wood charcoal acted as carrier of either fungus. For V. chlamydosporium rice husk was most suitable material as its application improved plant growth of the nematode inoculated tomato plants better than gram husk, mungbean husk, sawdust and wood charcoal (Table 10).

80 Root galling and egg mass production Root galling and egg mass production was suppressed by application of all the three fungi cultured on various substrates. When V. chlamydosporium cultured on rice husk and A. slrictum and F. oxysporum cultured on gram husk were applied the gall index was 1.38, 1.52/2.98 respectively as compared to the nematode inoculated plants i.e. GI 4.92. Similarly egg mass index was 1.0, 1.02/ 2.5 respectively as compared to the nematode inoculated plants i.e. EMI 4.48 (Table 10, 11, 12).

Nematode population and reproduction factor Nematode population significantly declined in the treatments where either of the fungus cultured on various organic substrates were added to the soil. The lowest nematode population and reproduction factor were found in the treatment where K chlamydosporium cultured on rice husk, andy4. strictum and F. oxysporum cultured on gram husk was most effective in suppressing the nematode population and reproduction factor. The fungi grown on other materials also effectively suppressed the nematode population and reduced reproduction factor of the nematode. Rice husk for V. chlamydosporium and gram husk for^. strictum and F. oxysporum were better materials for their growth, sporulation and suppression of the nematode subsequently improving the plant growth in comparison to other materials tested (Table 10, 11, 12; Fig. 6, 7, 8). Infection of eggs Fecundity was found to be lowest in the treatments with a V. chlamydosporium cultured on rice husk and^. strictum /F. oxysporum cultured

8} on gram husk. It was followed by other materials used for culturing of the fungi.

The number of eggs infected with the fungi varied in different treatment. The highest percentage of infected eggs (87.2) was found when rice husk colonized by V. chlamydosporium was applied. Percentage of egg infection hy A. strictum /

F. oxysporum cultured on gram husk was 78.8% / 68.64%. It was followed by other substrates (Table 10, 11, 12; Fig. 6, 7, 8).

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to — — to •^ O o 4U O 3 to to ij> 'u> vo •o VO to 00 00 ' tt -s ~J o ON 00 to s as 3 n &9 II ft o o to to .— — to -Pi. M TO QTQ !/) 1 TO en to U) to to '•p>. 50 3 tsJ b .&. rt o VO "^ to •t>. ON 00 00 00 2 o o O 3 o -^ i-t) p 3 n3 f» f6 » Wi M TO a. P U) Ul to to OJ TO P' S (TO '->i to -J to CT\ 00 M o a TO <^ M«•n« N) '-t^ (O to v^O^ -[^ ~J 1 •o1 OO ^ °^ 3 TO E" era 'jj °UI bo -J^ o rs fp b o TO O Wl OL *^ B P s •*- • p 3 _, Q. 3 ^ o (% •1 00 3 CD o p o p to ^ as -J 1 MMM p b\ to • ^. era o to io n ON ON a. Si. O

to -P". U) to ^ CM -a o 00 ON 4i. 1 «-i '•-J p NO a 3" OO to s 'O 00 io bo -c^ Ln to •^ (s> •^ P NJ to . ^^ _ 15^ - o era '-/I o 1>J z 1 o 00 •_^ to 00 to to 1 n •—•• OO to *~^••o ^ -P> -o •4^ 'as ?i (^ c 3 QTQ < p o 3 11 o d »-« to (/) u> to OO OO o a ^• &9 ON 3 n >—* '-/I to •^ o\ ON 1^ t'^o O • CO ;; "S •*. to •*.. + •o '*. b ^—s •a n o1 o o •a o F^ c_ ^ n' — to ^^ O o , . 'vJ 2 1 )-N to to 00 00 to p a -t- -u r^ •a n ^—^ •i 3' o O O 3 Q. 'J1 -J 3 3 C ON ON 1 bo to OO -4 o to OC ON IO O OO to O o ON 3 o p p o O p o JO b b to '-ft be ' o o 00 to -p. O -o Weight/length (g/cm)

(Q (l> m 3o ST fil 3 o •^ »< §• o w o o a. 3 e 3 O ^^ oa < D> o 3 ^^^1 n o 3 "1 fi) 3 17 01 3 -^ fit u (Q 3 (Q r* "^ O o ^» c 1 c 5" 3 a. o o ^ a (Q •3^ 3 A ^1 O 51 T O (0 (oQ o 3 B> sr •o ^^ ^ ^ o ^•^ !-fi)•

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to "» 2. U) to •o CTv L« Ijj ?;• (JQ I 0 Ul to o 4^ 00 C-i to o s bo o 4i. 00 to p to C-I TO VO to o to to o ON , ON to n t—i. UJ oo C o •*>. ON 00 4k 3 » O o /^^ p en 0 /-^ »— 3 -P>- -C>. •n 4^ U) to U) Ui rt 0 a. ~-j '^ to VO Ov \J\ ^* vO 'o\ to 00 Ov vyi to 10 s to to oo 00 00 to ON 00 p •+n 73 rt 0 0 p 0 •a bo to o o o 1 p* ^ as o o o 0 to VO (^ «^ o VO 9 o. -J Ln 4^ (J> to 0 3 s c to to to to 1 o •-/I 00 ON to 4^ •4:* o" o "ON o "^ O O o o o 0 0 1 . 3 » o o -~i o\ 4^ to <-ft ~J 1 o -J OS ON -p>. O u> to •o Weight/length (g/cm) —>

Ik •n (fi o 7«j a c: m 3 o (A 01 r* o J •< o o c -* 3 (A o > 3 CO -* oT (Q •^o 0} r^ 3 3" o () q 3 0) 01 3 01 -» (Q 01 (D ^ w (D 3 o-•» r* 01 o (Q ^1 ~t s n (D r* O Cr *•• T 01 a ^" (oQ To •3s (Q Q. o Sl^ll^ (/) a. cu + a —^ o. • o o o Sg- 3- o 3 o Q) N (D F3' II tD Q. Q. en 0 ^ S n b b 0 o s- s- 3 c Tl 3- O en 0 e I TT o + r/i 70 0 o § 0. I 3 0* 3 N 8 s. a. o P ere VO to to C/3 00 to o p to to to

3 O p ON 4^ VO M o. •p>- o ^-^ n to to to to 'A -o 00 0 tn 0•n 3 O to to to ""—^ rt 2 o o \o to VO (M 0 00 O ? OS to •1 •n (ra (re p p o o ^ <-»• TO -o 00 VO o VO to e3v 1 » 3 4^ o n en to to to 3 en — o to -o VO to 00 (/} 5' o o O '-0 LA M3 o. o 00 Ov to s o to 3 m 21 10 CTv to -J (IQ to bo to bo 00 to to to 00 O o o 3 SL p 4^ to O W tn to VO to OS VO to ^ P 00 00 00 to 00 to re O tt m 11 0 o O to W *^ (JQ P to TO pB en to to -J 4^ -J b 3 to 00 00 0 0 3 % ^ f^ J? 2 03 "-h Q. t/i en TO ^ E P en TO W 1 to to to 00 •O = *-»• n -J VO to 4^ s ore P o to to •^ a* TO bv U en "3- UJl to en '~- o en <->- p —• o 3 2 en •o (Jv Ov D. - o a\ to p 00 to 5* era* P bo bv re TO b 4i. to n o 00 O

VO to c to to o 00 to VO bo 00 o s. -« a. VO o to 3- to bv 4:>. ft O P C-l TOo to 2: to o VO <1 re ON bo c< bo 3 w ^ o 3 O — U) 00 re *^ 00 -o U) to •o 2. o. to •o 4^ VO a n 00 4i. 3 as 00 p "^ 7i CTv p O O o a -*> O T3 II as •= o 00 ON ON 00 Ov io •a o o o ^ 3 o OS 00 VO -p>. Ov 9 c o 10 -o 00 00 o to 4i. o ft 00 VO bo 4^ o' to ft 3 "1 0 O — ST O o o o O o bo Ov -J 00 r+ to VO -J Ov 4i. o Weight/length (g/cm) —>

•n (Q QB m S! 3 o t 01 O o 5 o =i w o •n 3 O (D O -*i T (Q oT 01 r* T 3" 9 O 3 3 ni 01 3 01 1 (Q 0> (D V) 3(D 3 o^1 r* 0) (Q ^o1 1 1 o lic* go sc o 0 •S^ -1 (O a

This experiment was conducted using the most suitable materials (rice husk for V. chlamydosporium and gram husk iox A. striatum /F. oxysporum) for application of the fungi in soil in order to determine their efficacy for control of root-knot nematode, M. incognita. Application of the three fungi grown on their respective materials improved plant growth of tomato and suppressed the root- knot disease.

Plant growth Plant growth of tomato (lengths, fresh and dry weights of roots and shoots) in the three treatments : control, (without nematode, fungus, gram husk/ rice husk). Gram husk, rice husk (without fungus) alone (uniniculated) and rice husk/gram husk inoculated with the fungus alone was more or less equal.

All the plant growth parameters of the nematode inoculated plants treated with rice husk colonized with V. chlamydosporium, showed significant increase in comparison to the plants innoculated with the nematode alone.

Similarly when the nematode infected plants were treated with gram husk colonized with ^. striatum /F. oxysporum, plant growth was found to be improved as compared to the nematode inoculated plants (Plate 4, 5, 6, C-D).

In sequential inoculation plant growth was much higher in the treatments

89 where either of the fungi colonized with gram husk/rice husk was inoculated 15 days prior to the nematode inoculation as compared to the treatment where nematode was inoculated 15 days before the fungus (Table 13, 14, 15).

Root galling and egg mass production

Root galling and egg mass production were suppressed by application of gram husk/rice husk irrespective of inoculation with V. chlamydosporium ; A. strictum F. oxysporum. Gl on plants treated with gram husk colonized with A. strictum F. oxysporum and rice husk colonized with V. chlamydosporium was

2.4,2.54 and 1.5 and EMI 1.96, 2.26 and 1.0 respectively as compared to 4.9 (GI) and 4.8 (EMI) on plants inoculated with the nematode alone (Table 13, 14, 15).

In sequential inoculation where fimgi {V. chlamydosporium /A. strictum

/ F oxysporum) colonized substrates were applied 15 days before nematode in the infected plants the Gl and EMI were significantly low in comparison to the treatment where nematodes were inoculated 15 days prior to fungus (Table 13,

14, 15).

Infection of eggs Application of gram husk colonized by ^4. strictum / F oxysporum or rice husk colonized by V. chlamydosporium reduced fecundity of the nematode significantly. A large number of eggs were found infected with the fungi. Per cent infection of eggs by V. chlamydosporium /A. strictum / F oxysporum was 80.14/78.0/73.6.

In sequential inoculation, percentage of egg infection was higher

90 in the treatments where fungus colonized substrate was applied 15 days before nematode inoculation. The percentage of infection by V. chlamydosponum A slrictum F. oxysporum was 90.44/85/82% (Table 13, 14, 15).

Nematode population and Rf value The nematode population recovered from soil and roots decreased significantly when compared with the treatment where plants were inoculated with M. incognita alone. The nematode population declined and was lowest when the infected plants were treated with the organic substrate colonized with fungus compared to the plants inoculated with nematode alone. Lowest Rf values were observed in the sequential inoculation treatment where fungus was inoculated 15 days before the nematode, the Rf value in case of K chlamydosponum ^ A. strictum ' F. oxysporum v/ere 0.22/0.39/0.42 (Table 13, 14, 15).

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(uio/B) MjBu8|flM6iaM Effect of seed soaking in different concentrations of V. chlamydosporium /A, strictum /F. oxysporum on plant growth and development of root-knot nematode on tomato

The present study indicated that seed soaking in culture filtrates of the

fungi V. chlamydosporium A. strictum F. oxysporum significantly reduced the

root galling caused by M incognita on tomato plants and consequently improved

plant growth. Nematode population also showed a decline. The effects were more

evident and significant when S and S/2 concentrations of the fungi were used for

seed soaking.

Plant growth The plant growth of nematode-inoculated plants receiving higher

concentrations (S and S/2) of the culture filtrates of all the three fungi were used

for seed soaking compared to control (nematode-inoculated plants). This effect

decreased with decrease in the concentration of the culture filtrates. Although

similar trend was observed in all the three fungi, seed soaking in the culture filtrate

of V. chlamydosporium was more effective than A. strictum or F. oxysporum.

Root galling and egg mass production Seed soaking in culture filtrates of all the three fungi also significantly reduced development of root-knot nematode. The root galling and egg mass production decreased with the increase in concentration of culture filtrate of all the three fungi.

Pure concentration (S) was relatively more inhibitory for the root-knot

98 development than other concentrations (i.e. S/2, S/10) of V. chlamvdosporium A. strictum / F. oxysporum. V. chlamydosporium was however, more effective than other two fungi. The fecundity- of the nematode was also reduced by soaking of the seeds (Table 16, 17, 18).

Nematode population and reproductive factor The population density of nematode was lower in plants treated with pure concentration of culture filtrates of all the three fungi separately. Population also declined in S/2 and S/10 as well. The reproduction factor of the nematode in the treatment where seeds soaked were in pure concentration of culture filtrate of V. chlamydosporium was 0.12. The values for A. strictum and F. oxysporum were 0.10 and 0.16 respectively (Table 16, 17, 18).

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<— (UJ3/6) i()eua|/)i46j3M DISCUSSION

Root-knot nematodes have a very extensive host range and are prevalent in diverse kinds of habitats. All the four major species, viz. M incognita, M. javanica, M. arenaria and M. hapla are world wide in distribution and cause significant losses to an array of agricultural crops. Vegetables are their most preferred groups of crops. Therefore, management of this kind of plant parasitic nematodes is essential for successful cultivation of a number of crops (Khan,

1997). In recent years biocontrol of root-knot nematodes has become a thrust area research. Because of prohibitive costs of nematicides and environmental concerns researches are in progress in various laboratories to develop biopesticides which can replace nematicides. Natural bioresource which act antagonistically against plant parasitic nematodes is supposedly rich in soil. Among fungi, saprophytic fungi are natural inhibitants of soil and decompose organic materials of plant or animal origin to derive their nutrition. Sometimes, fungi are nematophagus by nature and are known to have developed specific devices for nematode-trapping (Sayre, 1971). Efforts to exploit this category of nematophagous fungi for nematode control failed because of the demanding nature of their nutrition. Saprophytic soil fungi have not been fully assessed for their efficiency in this context. The present study is an effort in this direction where some indigenous saprophytic fungi have been used to assess their efficiency for biocontrol of root-knot nematodes. Some related aspects of these fungi in order to develop bio pesticides have also been explored. The three fungi Verticillium chlamydosporium, Acremonium strictum and Fusarium oxysporum, used in the

106 study, were isolated from egg masses of root-knot nematodes present in the root samples collected from vegetable fields. Their pathogenicity on egg masses was established on egg masses in artificial inoculations. Various aspects of their biocontrol efficiency have been examined under controlled conditions. Most of the soil fungi which has been christened as opportunistic soil fungi are basically egg parasites. These three fungi demonstrated their parasitism on eggs of M incognita in artificial inoculation under laboratory conditions and apparently belong to this category of soil fungi (opportunistic soil fungi). They invaded egg masses and perforated egg shells and colonized the interiors of the eggs inhibiting development of the juveniles. V. chlamydosporium was most effective compared to other two fungi.

Culture filtrates of all the three fungi demonstrated toxic effect on the eggs on juveniles of Meloidogyne incognita. Hatching of juveniles from the eggs was suppressed when incubated in the culture filtrates of the fungi. Hatched juveniles failed to survive in the culture filtrates as mortality was induced by the culture filtrates. Both the effects were, however, concentration dependent.

Suppression of hatching and killing of juveniles was greater in the higher concentrations of the filtrates. In this respect, culture filtrate obtained from V. chlamydosporium was more effective than other two fungi. In continuation of this aspect when seeds of tomato were soaked in the culture filtrates of V. chlamydosporium 'A. strictum F. oxysporum and planted in pots, the developing seedlings showed resistance against the root-knot nematodes, and as the root galling, egg mass production and population density of the nematode were

107 suppressed. Toxic substances present in the culture filtrates may have been absorbed and were present in the roots which possibly acted as deterrent for root invasion by the juvenile at the seedling stage which was reflected in the form of suppressed root gallmg, and egg mass production and reduced population density of the nematode. Plants in consequence, showed improved growth. Khan (1984) reported that culture filtrate of Aspergillus niger and Rhizoctonia soloni moderately improved plant growth, reduced larval penetration, suppressed reproduction and gall formation on tomato roots.

Fungi are known to produce certain chemical substances as metabolic products in the culture medium which are toxic for nematodes (Sayre, 1971).

Culture filtrates of some of the fungi such as Sclerotium rolfsii (Shukla and

Swaroop, 1971), Helminthasporium nodulosum, Trichoderma lignorum,

Curvularia tuberculata, Penecillium corylophilum and Aspergillus niger (Alam et ai, 1973) were found to be toxic for juveniles of root-knot nematodes and inhibited juveniles hatching of nematodes.

Nematicidal effects are also reported from mycelial extracts of Allernaria spp., Penicillium spp., Aspergillus spp. and other soil fungi (Krizkova et al., 1979; Singh et al., 1983; Mankau, 1967). Ciancio et al. (1988) reported that species of Fusariuni produce some compounds that are able to interfere in the embryonic development of the eggs and reduce the viability of first stage juveniles. V. chlamydosporium is reported to reduce the egg hatching of M. arenaria (Morgan-Jones et ai, 1983). Species of PaecHomyces are also known to produce a peptidal antibiotic (Isogoi et al., 1980). P. lilacinus, a biocontrol

108 fungus of root-knot nematode demonstrates proteolytic and chitinolytic activities by which fungal metabolites seep into the eggs and interfere in the embryogenesis resulting in partial or complete inhibition of juvenile development. P. lilacinus is also reported to produce antibiotic like leucostatin and lilacin (Arai, 1973).

Possibly quality of the substrate influences the quality of filtrates and metabolites.

It is possible that V. chlamydospormm, A. striclum and F. oxysporum may be producing naturally some toxic substances in the presence of suitable substrate in the root zone of the plants which may be suppressing the inoculum potential of root-knot nematodes in the field. The bio control fungus and substrate relationship in relation to production of the metabolites which may or may not be toxic to plant parasitic nematodes is unexplored area. Further information is required to exploit the situation for biocontrol of root-knot nematode by artificial adding of fungus and substrate combination.

Root-knot disease on tomato was suppressed by all the three fungi viz.

V. chlamydospormm A. striclum F. oxysporum. They were effective in reducing root galling, egg mass production and root and soil populations of the root-knot nematode. Consequently, growth of tomato plants improved showing enhanced root and shoot lengths and weights. V. chlamydosporium was comparatively more

effective than^. striclum and F. oxysporum. Sequential inoculations of the fungi and the nematode showed that prior inoculation of the fungus (biocontrol fungus) was more effective in suppressing the disease and improving the plant growth.

Prior inoculation of the nematode was not so effective. Even this mode of treatment prior inoculation of the nematode was less effective than simultaneous

109 inoculation of the either fungus and the nematode. Time lapse between the inoculation of the fungus and nematode inoculation possibly provided adequate time for establishment of the fungus in the soil and rhizosphere zone of the plants.

When the nematode inoculum was added around the root the biocontrol fungus was already in established in the zone of action. A number of reports indicate that

some soil fungi like P. lilacinus (Jatala, 1986; Khan and Esfahani, 1990), V. chlamydosporium (Leij and Kerry, 1991), F. oxysporum (Price et al., 1980), F. solani (Khan and Husain. 1986) remain associated with the eggs or females of root-knot nematodes and suppress root-knot disease. The study demonstrates that

indigenous isolates of these fungi obtained from natural habitats are also effective biocontrol agents They can be exploited to develop biopesticides for effective management of root-knot nematodes.

As discussed earlier in the introduction, the potential biopesticides need

to be mass cultured and should adjust the ecological and physiological

compatabilit>' at the site of release, to achieve the target some agricultural waste

materials of organic origin were evaluated for mass culturing of the fungi {V.

chlamydosporium A. stnclum F. oxysporum). These fungi profusely grew well

and colonized the materials used but the spore load varied. Spore of V.

chlamydosporium was maximum on rice husk (8.2 x 10''/g), and those oi A.

strictum that of (1.74 x lOVg) and F. oxysporum (1.14 x lOVg) on gram husk.

Rice husk, therefore, emerged as most suitable agricultural waste

material on which V. chlamydosporium produce maximum spore load. Rice husk

is easily available material as rice is widely grown in India and several other parts

10 of the world. Rice husk, therefore, can be used for mass culturing of V. chlamydosparium for field dispension. For the other two fungi, which are however, less effective biocontrol agents than V. chlamydosporium gram husk supported better spore load. Rice husk too was a good substrate for them.

Soil application of V. chlamydosporium, A. strictum, F. oxysporum on various agricultural waste of origin was found to be effective in reducing root- knot disease. Since these fungi are saprophytic they may require ensured supply of substrate base for growth sporulation and establishment in soil, therefore, for their dispensing in the fields suitable organic materials on which they can grow efficiently would be required. V. chlamydosporium grown on rice husk showed better performance in controlling root-knot disease in comparison to other organic materials of agricultural or plant origin. Similarly A. strictum and F. oxysporum performed better in soil application when grown on gram husk. This confirms their growth performance and spore production as observed under laboratory conditions These materials as suggested earlier can be used for field dispensing of the biocontrol of fungi for management of root-knot nematodes. There are reports of use of various materials for growing P. lilacinus. Sharma and Trivedi (1987) used sesamum oil cake for P. lilacmus and found most efficient substrate base for growth and sporulation of the fungus. Bansal et. al. (1988) evaluated some agro-industrial products like gram bran, rice bran, wheat bran with molasses for mass production of P. lilacmus. Sporulation off. lilacinus was maximum on wheat bran + molasses followed by rice bran + molasses and gram bran + molasses. Zaki and Batti (1991) found whole rice grain most efficient grain where the

111 maximum number of spores/g was observed followed by gram seed. Bansal ei al (1991) found wood charcoal powder as efficient base for growth and sporulation of P. lilacinus.

The experiment in which soil application of rice husk colonized with V. chlamydosporium and rice husk alone were compared, the rice husk colonized with V. chlamydosporium was more effective in suppressing the root-knot disease

and improving the plant growth. Similar results were obtained when gram husk colonized with A. strictum ' F. oxysporum was compared with gram husk

uncolonized withv4. strictum 'F. oxysporum.

Application of these fungi grown on their respective most efficient

organic material was, therefore, a better alternative as compared to application of

these biocontrol agent grown on culture media. These biocontrol agents apparently

work well when applied in this manner because the rice husk in the case of V.

chlamydosporium and gram husk in case of other two fungi apparently serve as

organic base for the establishment of the fungi in the soil and from the

strengthened base which supports them with nutrient supply, these fungi they may

have achieved better growth and colonization of rhizosphere zone and there by

protected the roots from the nematode. The organic materials may have also

channelized some nutrition to the plants as well and this may have been a

contributing factor in enhancement of the shoot and root growth and suppression

of root galling and eggmass production.

A number of organic matters have been found to be effective in

112 suppressing nematode population and improving plant growth (Singh and

Sitaramiah, 1973; Sitaramiah, 1990). When the rice or gram husk added to the soil there was a improvement of plant growth and suppression of population of nematodes. Such reduction in nematode population suggest that organic amendments of these organic material acted independently for improved plant growth and nematode control. It can be surmized therefore that application of these fungi grown on suitable organic material would be better for field dispensing as these materials would support the fungus as well as the plants, resulting in suppressed root-knot disease. These local isolates of the fungi can be, therefore, used as biopesticides after mass culturing on their respective most suitable organic material of agricultural origin for field application.

Storage and transport is one problem that is considered most important in case of biopesticides. Efficient biopesticide would be one which can be stored for extended period and maintained its potentiality of biocontrol when applied. In case of the present biopesticidal fungi, effect of storage on the viability of fungi was determined. Viability of spores of the fungi grown on various organic materials and effect of aeration when tested, it was found that that they can be stored for a extended period of time i.e. two months or possibly more (not determined in the experiments) as they maintained adequate spore load, the spores remained viable during the period When the fungi stored in punctured packets and unpunctured packets were compared, the difference in their spore load and viability was marginal. Bansal (1992) while studying the suitability of wood charcoal as a carrier of P. lilacinus determined the effect of storage of wood charcoal with the fungus

113 at constant (28±rC)/ambient (14-39"C) temperature or under aerated and non- aerated conditions. He found that these factors did not influence the viability of fungus.

This study demonstrated that these indigenous isolates of these three fungi are efficient biocontrol agents with varying ability and efficiency and may be involved in natural biocontrol process of root-knot disease in vegetable fields. These fungi are saprophytic deriving their nutrition by decomposition of organic materials. In addition, these opportunistic biocontrol agents of root-knot nematodes are primarily egg parasites. These two attributes can be exploited jointly for developing them as biopesticides and they can be mass cultured on suitable organic materials of agricultural origin as observed in the experiments and can be stored for an extended period of time which may be required for their transport from place of their production to the area of their distribution or sale for their field application.

114 SECTION-II PREDATORY NEMATODES

Predatory behaviour and potential of Monochoides longicaudatus as biocontrol agent on Meloldogyne incognita The diplogasterid predators are important and may prove to be promismg biological control agents of plant parasitic nematodes Yeates (1969) observed predatory abilities of Monanchoides potohikus with a bacterium The rate of predation was dependent on prey density and affected by presence of water on agar plates Grootaert et al (1977) studied prey selection by Butlarius sp , and elucidated some aspects of its feeding habit and possible application of the same in the biological control of plant parasitic nematodes While observing the predation abilities of Butlarius degnssei, Small and Grootaert (1983) found

Panagrellus redivivus and the first and second stages of Rhabditis oxycerca as most susceptable prey species Several in vitro observations on the various aspects of Mononchoides sp have been made by Jairajpun et al (1988, 1989) Bilgramil et al (1989 a & b) studied the predatory abilities of Mononchoides longicaudatus and Mononchoides fortidens on several nematodes such as second stage juveniles of Meloldogyne incognita and Anguma tritici, adults of Acrobeloides sp ,

Cephalobus sps and Hirchmanniella oryzae and Helicotylenchus indicus in vitro conditions These studies included pre-, and post-feeding attraction and aggregation around the prey at feeding sites, resistance and susceptibility of prey to predators

Osman (1988) studied potential of Diplogaster, as a predator of some plant parasitic nematodes In vitro studies were carried out to assess the efficacy

115 of Diplogaster sp.as a predator of second stage juveniles of Meloidogyne javanica and Tylenchulus semipentram at 25°C and 25% relative humidity in an incubator. In addition to this experiment, a pot experiment was also carried out to explore the role of the predatory nematode in suppression of the population of the nematodes. It was found that the population of M javanica and T. semipenetram were significantly reduced.

In the following experiments, potential of Mononchoides longicaudatus as a predator of Meloidgyne incognita is assessed and its efficacy as an agent for biological control of the root-knot nematode is evaluated.

MATERIALS AND METHODS

In vitro experiments

Culturing nematodes The predatory nematode, M longicaudatus was isolated from sewage sludge by Cobb's sieving and decanting and Baermann's funnel methods. The predatory nematode and prey Rhabditis spp. (saprophagous) were cultured in 5.5 cm. diameter petri-dishes containing 1% water agar. To prepare the culture medium, 1 g of agar was boiled in a beaker containing 100 ml of water for a few minutes over an electric heater. The medium was then poured in petriplates and was left undisturbed for about an hour to allow it to cool down and solidify. Predatory nematodes were then inoculated along with the prey nematodes (saprophagous) with the help of a picking needle or bamboo splinter. Avery small

116 amount of milk powder was spread over the surface of the agar to encourage bacteria to grow which served as food for prey (saprophagous) nematodes. When culture plates became old and dry, the nematodes were transferred to fresh petriplates containing agar of the same concentration. Rhabditis spp. which were used as prey were reared in separate petriplates on 1% water agar.

Five individuals of the predator, M. longicaudatus and twenty five of the second stage juveniles of Meloidogyne incognita were used as prey separately. All experiments were carried out in small cavity blocks containing 1% wateragar at 25±2°C and replicated five times. Observations were made every 24h after inoculation of the predators. All conditions remained same for each experiment unless mentioned otherwise.

Screening of culture media for the mass production of a predatory nematode, Mononchoides longicaudatus Three substrates in three different combinations were screened for mass production of the predatory nematode, M. longicaudatus The population of predatory nematode was determined first after 15 days and then after 30 days.

(1) Soil agar medium In this type of culture medium 1% water (1 g of agar dissolved in 100 ml of distilled water) and soil (10 g) were autoclaved for 20 min. at 15 psi.

(2) Farm-yard manure agar medium In this type of culture medium 1% wateragar and farm-yard manure (lOg) were autoclaved for 20 min. at 15 psi.

17 (3) Farm-yard manure + soil agar medium In this culture medium 1% water agar, 10 g of farm-yard manure and 10 g of soil were mixed together and autoclaved for 20 min. at 15 psi.

Rhahditis sp. were used as prey in all petriplates. A small amount of infant milk powder was spread on the surface of all culture media to encourage bacterial growth. Bacteria served as food for prey nematodes. The constituents of soil were 66% sand, 24% silt, 8% clay. All experiments were replicated 5 times.

The culturing was done at 25±2°C.

Handling of nematodes The nematodes when required in small numbers, were hand-picked either from the culture dishes or from a few drops of nematode suspension in water.

When required in large numbers, the nematodes were obtained from the culture plates with the help of modified Baermann's funnel method. The agar was cut into

pieces and was placed on small sieves lined with moist tissue paper. Most of the

active nematodes migrated from the agar into the clean water of the funnel within

24 h.

Observation chamber To study in detail the prey catching and feeding mechanisms of predatory nematodes at higher magnification, observation chambers as described by Bilgrami el al. (1985) were designed. A plastic ring of 1 cm diameter and 2-3 m thickness was fixed to a metallic slide. A cover slip was fixed on one side of the ring. Hot agar suspension was poured into the chamber and was allowed to cool. The predator and prey nematodes were placed together in chambers containing agar and another

118 cover slip was placed on the top and edges of the cover slip were sealed with vaseline to avoid drying of the agar.

Prey catching and Feeding mechanisms The prey catching and feeding mechanisms of predatory nematodes were studied at 60x magnification by inverting the petriplates containing predator and prey nematodes on the stage of the stereoscopic microscope. To study feeding mechanism in detail, observations were made at 450x or even higher magnification in the obsevation chambers Ten adult predators were placed separately in observation chambers containing water agar with 100 individuals of M incognita. Nematodes were allowed to get acclimatized for 30 min. before observations were started.

Effect of prey density The effect of prey numbers on the rate of predation was detemined by releasing five individuals of predators in cavity blocks containing 25, 50, 75, 100, 125, 175 and 200 individuals of prey nematodes.

Effect of temperature To determine the influence of temperature on predation, the predators were placed in cavit>' blocks containing prey nematodes at different temperatures ranging from 5 to 40°C (with gaps of 5°C).

Effect of age of predator To find out difference in the rate of feeding by adults and juveniles stages of predators, each stage was placed in separate cavity blocks containing water

119 agar along with prey. Predation by each stage was tested separately.

Feeding pattern

The feeding pattern of predators was tested over a period often days.

The predators and prey nematodes were placed together in cavity blocks containing water agar and the observations were made after every 24 h. Each day after observation, the predators were transferred to another cavity block containing fresh agar and prey nematodes.

In vivo experiment Biocontrol potential of Mononchoides longicaudatus for Meloidogyne incognita on tomato plant was studied. The predatory nematodes, M. longicaudatus were isolated from sewage sludge by Cobb's sieving and decanting and Baermann's funnel methods. These predators were cultured in petri-plates containing 1% water agar using Rhahditis sp. as prey. Rhahditis sp. were maintained in the laboratory in the same way as described earlier. The second-stage juveniles of Meloidogyne incognita were obtained from single egg mass cultures maintained on tomato plant {Lycopersicon esculentum Mill.).

Three-week-old seedlings of tomato var. Pusa Ruby were transplanted singly into 9 cm pots containing sterlized field soil, sand, compost (7:3:1). After five days of transplantation, predatory nematodes were added in each pot by making small holes in the soil around roots of the plants. Three inoculum levels of predatory nematodes, viz. 100, 200 and 400 individuals of M longicaudatus were used separately. The control pots remained uninoculated. Two days later each pot was inoculated with 1000 freshly hatched second stage juveniles (J^) of M incognita. These pots were kept in

120 greenhouse in randomized block design Each treatment was replicated five times. Sixty days after the inoculation of M. incognita the plants were carefully uprooted from the pots and roots were washed gently with tapwater. Gall index and egg mass index were rates on 0-5 scale (Taylor and Sasser, 1978). The method of counting of developing stages (3rd, 4th stage juveniles) were same as given in the first section of the thesis. The second stage juveniles and males which represent the migratory stages were isolated by Cobb's sieving and decanting and Baermann's funnel methods and counted in a counting dish. Fresh and dry weight of plants were recorded. For dry weight, plants were dried at 50°C after recording the fresh weights of roots and haulms.

12: RESULTS

In vitro experiments

Screening of culture media for the mass production of a predatory nematode, Mononchoides longicaudatus In soil agar medium, all individuals oiMononchoides longicaudatus were found to be dead. In these media, nematodes got aggregated and died.

In the farmyard manure agar medium, population of the prey (saprophagous) nematode, Rhahditis spp., increased as compared to population of the predatory nematode (M. longicaudatus).

In farmyard manure + soil agar medium, the population of the predatory nematode, M. longicaudatus, significantly increased as compared to the other culture media (Table l,Fig. 1).

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apoieuiau Ajojepajd jo ON Prey catching and feeding mechanisms

Predator and prey nematodes moved randomly in agar. During the random movement M. longicaudatus made contact and attacked their prey. Predators attacked prey only when a lip contact was established with any part of prey body. No preference was shown in attacking any particular region of prey body. Probing was done by moving the head of predatory nematodes after establishing contact.The duration of probing in

M. longicaudatus was 1 -2 minutes. During probing a few oesophageal pulsations were

seen in the predators. If the type of prey and texture of prey cuticle suited to the predator then the prey was attacked, otherwise predators probed other regions

immediately after finding that the first contact was unsuitable. After selecting a suitable

spot, the predator attacked the prey with the movable dorsal tooth. During attack

predators made thrust, with the help of their movable dorsal tooth and oesophageal

suction repeatedly against the cuticle of the prey. M longicaudatus required one or

two attack puncturing the prey cuticle. Predators made their prey inactive by high suction

force created by the oesophagus and rendered the prey immobile.

During ingestion oesophageal secretions were released by predators for extra-corporeal digestion. The predators sucked the body contents of prey and these were seen passing through the lumen of oesophagus. Predators punctured the cuticle and sucked the body contents of prey. Only body cuticle of prey was left unconsumed when the feeding was completed.

Sometimes the prey escaped out of the grip of the predator which led the predator to move again in search of another prey. However, an injured prey became susceptible to attack of another predator.

125 Effect of prey density There was a positive correlation between the number of prey killed and prey density. M. longicaudatus killed maximum number of prey when tested with 200 individuals of the prey that were subjected to predation. Minimum predation was reached when 25 individual of the prey were placed for predation (Fig. 2).

Effect of temperature Predation was greatly influenced by change in temperature. There was a significant increase in the number of prey killed by M longicaudatus as the temperature increased. Maximum numbers of the prey were killed at 40°C (Fig. 3).

Effect of age of predator All stages except the first stage juveniles of the predators fed on prey nematodes. The adult predators killed a maximum number of prey whereas the second stage juveniles the least (P<0.05) (Fig. 4).

Feeding pattern The number of M. incognita juveniles killed by M. longicaudatus remained unaltered (P>0.05) when tested for over a period often days (Fig. 5).

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<~ P^im Aajd|o 'OH In vivo experiment

The biocontrol potential of predatory nematode, Mononchoides longicaudatus for Meloidogyne incognita on tomato plant The population of A/ incognita significantly decreased at all inoculum levels of predatory nematodes. Significant difference in the mean weight of shoot and root and the total weight between treated and control plants were recorded (P>0 05) Similarly, fewer root galls were found on roots of treated plants than those inoculated with M incognita alone (P>0.05) (Table 2).

The number of developing stages of M incognita in tomato roots was significantly low in pots that recieved different inoculum levels of M longicaudatus The decrease was related to the inoculum level of predatory nematodes With the increase in inoculum levels of M. longicaudatus, there was corresponding decrease in the multiplication of M incognita and number of root galls (Table 2, Fig 6, Plate 7)

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The present observation on the predatory behaviour of A/, longicaudatus revealed that this nematode is an efficient predator of M. incognita In diplogastend predators chemoattraction as well as chance encounters bring about predator-prey contacts The ability of these predatory nematodes to perceive prey attractants could lead predators to move directly towards prey and enhance chances for more frequent predator-prey contact resulting in higher rate of killing This could be one of the reasons for these predators possessing a high rate of predation

(Jairajpun et ai, 1990). The prey catching and feeding mechanisms of M laungicaudatus is similar to the other diplogasterid nematodes, e.g., Butlarius degressei and Mononchoides potohikus {Groot&ert, 1977, Yeates, 1969). These predators get hold of their prey with the help of their high suction force created by their oesophagus These predators attack and puncture the cuticle of their prey with their movable dorsal tooth (Yeates, 1969, Grootaert et al 1977; Small and

Grootaert, 1983) The oesophageal suction enables predators to take a firm grip on their prey and prevent its escape (Bilgrami, 1989). The rate of predation by predator increased with increasing number of prey, a phenomenon which has been observed in Mononchoides potohikus and A. nivalis (Yeates, 1969; Khan et al ,

1991) Temperature also influenced the rate of predation of M longicaudatus as the number of prey killed at different temperatures was different. The difference in the rate of predation at different temperature may be attributed to the predators which IS affected by variuos temperatures (Grootaert, 1976)

135 The differences in number of prey killed by adults and juvenile stages

(second, third and fourth stage juvenile) reflects their predatory potential. The adults were more efficient predator than their juvenile stage (Bilgrami and

Jairajpuri, 1988; Khan e/a/., 1991).

The rate of feeding of predator on their prey remained more or less constant over a period often days. Khan et al, (1991), while working with A. nivalis also found consistent predatory pattern. Some factors affected the growth of diplogasterid predators like temperature (Grootaert, 1976; Yeates, 1970) moisture and organic amendments which support the bacterial growth (Jairajpuri et al., 1990). Mononchoides potohikus fed on the bacteria as the source of food

(Yeates, 1969). The population of diplogasterid predators can be elevated to desired levels by supplementing with organic materials which support bacterial growth

(Jairajpuri et al., 1990). In the present observations, population of M longicaudatus increased in the culture media having farmyard manure and soil. It may be that the farmyard manure and soil proved to be a good source of organic material. In pot experiments lowest number of galls were recorded in those pots which had received higher inoculum levels of M. longicaudatus (400 individuals).

Large number of predators perhaps increased in the probability of encounters between predators and plant parasitic nematodes (prey). Pot trials conducted by

Cohn and Mordechai (1974) found that small population of Tylenchulus semipenetrans were associated with large number of Mylonchulus sigmaturus.

Similarly, in pot experiment, population of Globodera rostochiensis (prey) was

significantly reduced in soil in the presence of Prionchulus punctatus (Small,

1977).

136 The population densities of many effective biocontrol agents are linked to those of the pests (Doutt and De Bach, 1964). This appear unlikely to occur with predatory nematodes although evidence is accumulating which indicates that the generation line and fecundity of predatory nematodes compares favourably with that of plant parasitic nematodes (Maertens, 1975; Grootaert, 1976; Small,

1977) Mononchoides sp has a generation time 4-7 days at 30°C (Tahseen et al,

1990). Jairajpuri et al. (1990) have reported that M. longicaudatus and M. foriidens are quite capable of perceiving their prey (nematodes) in agar plates

137 SUMMARY

Root-knot nematodes (Meloidogyne spp.) causes substantial losses to a variety of crops. Therefore, management of this group of plant-parasitic nematodes is an essential component of over all strategy for management of various pests and parasites. In the present context of environmental concerns and health risks involved with the use of nematicides, focus has shifted to look for effective biocontrol agents and to develop biopesticides for the management of plant-parasitic nematodes including root-knot nematodes. Researches are in progress and a number of promising biocontrol agents have been categorized as biopesticides candidates. In the present work, emphasis has been to assess biocontrol potential of some fungi a and predatory nematode in order to develop them as biopesticides for management of root-knot nematodes. To achieve this aim root samples of vegetables crops infected with root-knot nematodes were collected from various vegetable fields. Fungi associated with egg masses of the root-knot nematodes were isolated. Three fungi, Verticilhum chlamydospormn,

Acremomum strictum and Fusarium oxysporum found infecting eggs in the egg masses were selected for further studies Their parasitism on the eggs of root- knot nematode, Meloidogyne incognita were confirmed by artificial inoculation on the healthy surface sterilized egg masses.

Experiments were conducted to determine the efficacy of the culture filtrates of the three fungi on juvenile hatching and mortality of M incognita. All the three fungi, Verticilhum chlamydosporium, Acremonium strictum and

Fusarium oxysporum proved to be toxic as the hatching of juveniles was inhibited

138 and mortality of the juveniles(J2) occurred. Concentration of the culture filtrates and time duration were determinant of these effect. Higher concentration and extended time duration were more effective Among the three fungi, culture filtrate of V. chlamydosporium was more toxic than other two fungi. Efficacy of culture filtrate on plant growth and development of root-knot nematode, M. incognita on tomato was determined by soaking the seeds of tomato in different concentrations of the culture filtrates. Tomato plants developed from such seeds in general showed improved growth and reduced root-knot disease. There effects were more obvious and prominent when 'S' concentration of the culture filtrates were used for seed soaking.

All the three fungi (V. chlamydosporium ' A. strictum /F. oxysporum), when applied in soil in water suspension improved plant growth and reduced the severity of the root-knot disease. Though all the three fungi significantly reduced the disease, V. chlamydosporium was apparently more effective. The plant growth

(lengths and fresh and dry weights of roots and shoots of the plants) showed an increase when either fungus and nematode (M. incognita) were applied. The adverse effect of the nematode on plant growth was reduced. Root galling and egg mass production were low. Root and soil population of the nematode declined.

For mass culturing of the three fungiagricultural waste materials of plant origin which can serve as a better substrate for their growth and which can be easily available for field dispensing, were screened by artificial inoculation. Gram husk, rice husk, mungbean husk, wood charcoal, saw dust were used in this screening. Rice husk emerged as best substrate for growth of V. chlamydosporium

139 as It showed maximum spore load on this waste organic material Rice husk was also good for A stnctum and F oxyspurum But better growth and highest spore load of these two fungi was found on gram husk The vialbility of the fungal spores of all the three fungi were checked after their growth for different duration (i e

2, 4, 6 and 8 weeks) on the agricultural waste materials of plant origin Spores of all the three fungi were viable at each interval of observation The spore count of

V c hlamydusponum peaked after 6 weeks In case oiA stnctum and F oxysporum, highest spore count was found after 4 weeks

When fungus colonized organic materials of agricultural origin were applied in soil and tomato plants were grown in it, rice husk colonized with V chlamydosporium was most effective in reducing the root-knot disease A stnctum and F oxysporum, which were applied on gram husk, also reduced the disease severity Root galling egg mass production and soil and root populations of the nematode were reduced Consequently plant showed improved growth In sequential inoculations, prior application of the agricultural waste colonized with either fungus was more effective than the application made after the nematode inoculation Simultaneous application was, however, also effective

The study presented in the second part of the thesis deals with the potential of a predatory nematode, Mononchoides longicaudatus, as biocontrl agent for root-knot nematode For mass production of M longicaudatus soil, agar and farmyard manure were used in different combinations and it was found that farmyard manure and soil agar medium were suitable culture media where the populations of the predatory nematode was higher than other two combinations of

140 culture media Prey catching and feeding, pattern of feeding the predatory nematode was studied. The nematode was predatory on second stage juveniles (J2) of the root-knot nematode The effect of some factore on predation were also studied.

In VIVO experiment, it was found that when the higher numbers of M longicaudatus were inoculated along with M. incognita, the root galling and egg mass production decreased The population of nematode also declined. As a result tomato plants showed better growth.

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Zaki, F.A., Bhatti, D.S. (1991). Effect of culture media on sporulation of Paecilomyces lilacinus and its efficacy against Meloidogyne javanica in tomato. Nematologia Mediterranea, 19 : 211-212.

Zaki, M.J., Maqbool, M.A. (1996). Effect of Paecilomyces lilacinus and Vetncillium chlamydosporium on root-knot disease of chickpea. International Chickpea and Pigeonpea Newsletter, 3 : 38-39.

Zopf, W. (1888). Zur kenntris der Infektions Krankeiten Niederer Thiere and Pflanzen. Nova Acta Leopold, Carol 52 : 314-376.

167 ANOVA TABLE SECTION!

Table 1 : Juvenile mortality of Meloidogyne incognita in different concentrations of culture filtrates of some soil fungi

Table la : Juvenile mortality in culture filtrate of V. chlamydosporium af­

ter 24 hours

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 0.3281 0.0820 0.3116

Treatment 5 70966.5703 14193.3145 53909.3242

Error 20 5.2656 0.2633

Table lb : Mortality of juveniles in the culture filtrate of V.

chlamydosporium after 48 hours

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.4688 0.1172 1.0067

Treatment 5 69680.1719 13936.0342 119718.9531

Error 20 2.3281 0.1164

Table Ic : Mortality of juveniles in the culture filtrate of V.

chlamydosporium after 72 hours

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 7.5313 1.8828 0.7016

Treatment 5 66686.6641 13337.3330 4969.9521

Error 20 53.6719 2.6836 Table Id : Mortality of juveniles in the culture filtrate of Acremonium strictum after 24 hours SOURCE D.F. s.s. M.S.S. F. VAL. Replicate 4 2.8672 0.7168 0.6092 Treatment 5 58725.4688 11745.0938 9982.5498 Error 20 23.5313 1.1766

Table le : Mortality of juveniles in the culture filtrate oi Acremonium strictum after 48 hours SOURCE D.E S.S. M.S.S. F. VAL. Replicate 4 3.4688 0.8672 1.2272 Treatment 5 53911.3672 10782.2734 15258.4971 Error 20 14.1328 0.7066

Table If: Mortality of juveniles in the culture filtrate of Acremonium strictum after 72 hours SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 3.1328 0.7832 0.5351 Treatment 5 59739.8906 11947.9785 8163.0166 Error 20 29.2734 1.4637

Table Ig : Mortality of juveniles in the culture filtrate of Fusarium oxysporum after 24 hours SOURCE D.E S.S. M.S.S. F. VAL. Replicate 4 4.5332 1.1333 0.8500 Treatment 5 23540.6680 4708.1338 3531.1865 Error 20 26.6660 1.3333 Table Ih : Mortality of juveniles in the culture filtrate of Fusarium

oxysporum after 48 hours

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 0.8672 0.2168 0.6848

Treatment 5 39676.1680 7935.2334 25063.7832

Error 20 6.3320 0.3166

Table li : Mortality of juveniles in the culture filtrate of Fusarium

oxysporum after 72 hours

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 1.5332 0.3833 0.7615

Treatment 5 60041.7656 12008.3535 23858.2715

Error 20 10.0664 0.5033 Table 2 : Juvenile hatehing of Meloidogyne incognita in different concentrations of culture filtrates of some soil fungi

Table 2a: Juvenile hatching of M. incognita in culture filtrate of V.

chlamydosporium after 24 hours

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 85.5000 21.3750 1.8874

Treatment 5 1644940.5000 328988.0938 29049.7227

Error 20 226.5000 11.3250

Table 2b: Juvenile hatching of M. incognita in culture filtrate of V.

chlamydosporium after 48 hours

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 29.000 7.2500 1.2393

Treatment 5 3930211.0000 786042.1875 134366.1875

Error 20 117.0000 5.8500

Table 2c : Juvenile hatching of M. incognita in culture filtrate of K

chlamydosporium after 72 hours

SOURCE D.F. S.S. IVi.S.S. F. VAL.

Replicate 4 340.0000 85.0000 0.9487

Treatment 5 6452254.0000 1290450.7500 14402.3525

Error 20 1792.0000 89.6000 Table 2d : Juvenile hatching of M. incognita in culture filtrate of A. strictum after 24 hours SOURCE D.F. s.s. M.S.S. F. VAL. Replicate 4 5.0000 1.2500 0.1838 Treatment 5 1667901.0000 333580.1875 49055.9102 Error 20 136.0000 6.8000

Table 2e : Juvenile hatching of M. incognita in culture filtrate of A. strictum after 48 hours SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 29.0000 7.2500 1.6667 Treatment 5 3612808.0000 722561.6250 166106.1250 Error 20 87.0000 4.3500

Table 2f: Juvenile hatching of M. incognita in culture filtrate of ^4. strictum after 72 hours SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 1070.0000 267.5000 2.1213 Treatment 5 6842444.0000 1368488.7500 10852.4092 Error 20 2522.0000 126.1000

Table 2g : Juvenile hatching of M. incognita in culture filtrate of F. oxysporum after 24 hours SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 71.0000 17.7500 2.7953 Treatment 5 1278048.0000 255609.5938 40253.4805 Error 20 127.0000 6.3500 Table 2h: Juvenile hatching of M. incognita in culture filtrate of 7^ oxysporum after 48 hours SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 40.0000 10.0000 2.0833 Treatment 5 2480708.0000 496141.5938 103362.8281 Error 20 96.0000 4.8000

Table 2i : Juvenile hatching of M. incognita in culture filtrate of F. oxysporum after 72 hours SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 1204.0000 301.0000 1.0013 Treatment 5 5256868.0000 1051373.6250 3497.5835 Error 20 6012.0000 300.6000 Table 4 : Effect of soil application of Verticillium chlamydosporium on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 4a : Fresh weight of shoot

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 0.0576 0.0144 0.5598

Treatment 5 573.9292 114.7858 4460.7476

Error 20 0.5146 0.0257

Table 4b : Fresh weight of root

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0598 0.0150 1.0996

Treatment 5 149.2325 29.8465 2194.8169

Error 20 0.2720 0.0136

Table 4c : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0602 0.0151 1.5597

Treatment 5 41.4478 3.2896 858.9160

Error 20 0.1930 0.0097

Table 4d : Dry weight of root

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0068 0.0017 0.9424

Treatment 5 1.8063 0.3613 199.8850 Error 20 0.0361 0.0018 Table 4e : Length of shoot

SOURCE D.F. s.s. M.S.S. F.VAL.

Replicate 4 2.1211 0.5303 1.3988

Treatment 5 1464.9043 292.9809 772.8295

Error 20 7.5820 0.3791

Table 4f: Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.4639 0.1160 0.8966

Treatment 5 1231.6689 246.3338 1904.4606

Error 20 2.5869 0.1293

Table 4g :GI

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 0.1280 0.0320 0.7693

Treatment 5 81.9147 16.3829 393.8274

Error 20 0.8320 0.0416

Table 4h : EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0100 0.0025 0.1699

Treatment 5 65.8377 13.1675 895.8011

Error 20 0.2940 0.0147 Table 4i : Egg/Egg mass

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 4.9375 1.2344 0.4484

Treatment 5 508748.1250 101749.6250 36957.8672

Error 20 55.0625 2.7531

Table 4j : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0977 0.0244 0.1111

Treatment 5 45191.2500 9038.2500 41134.0781

Error 16

Table 4k : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 41.0000 10.2500 2.5000

Treatment 5 19839686.0000 3967937.2500 967789.6250

Error 20 82.0000 4.1000

Table 41 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 5.5332 1.3833 1.0949

Treatment 5 17385.0664 3477.0132 2752.1541

Error 20 25.2676 1.2634 Table 4in : Population of female

SOURCE D.F. s.s. M.S.S. F.VAL.

Replicate 4 18.0000 4.5000 5.8065

Treatment 5 2137771.2500 427554.2500 551682.9375

Error 20 15.5000 0.7750

Table 4n : Population of male

SOURCE D.F. S.S. iVl.O.^. F. VAL.

Replicate 1.4667 0.3667 2.2000

Treatment 14.1667 2.8333 17.0000

Error 20 3.3333 0.1667

Table 4o : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 60.0000 15.0000 4.4118

Treatment 5 35154712.0000 7030942.5000 2067924.2500

Error 20 68.0000 3.4000

Table 4p :R; f

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 1.2879

Treatment 5 8.7745 1.7549 278808.6250

Error 20 0.0001 0.0000 Table 5 : Effect of soil application of Acremonium sirictum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 5a : Fresh weight of shoot SOURCE D.E s.s. M.S.S. E VAL. Replicate 4 0.0400 0.0100 0.4280

Treatment 5 386.4678 77.2936 3304.7432

Error 20 0.4678 0.0234

Table 5b : Fresh weight of root

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0248 0.0062 0.1962

Treatment 5 69.9786 13.9957 443.2747

Error 20 0.6315 0.0316

Table 5c : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.1646 0.0412 2.0012

Treatment 5 21.0386 4.2077 204.5983

Error 20 0.4113 0.0206

Table 5d : Dry weight of root

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 0.0023 0.0006 0.9267 Treatment 5 1.0189 0.2038 335.4778

Error 20 0.0121 0.0006 Table 5e : Length of shoot

SOURCE D.F. s.s. M.S.S. F.VAL.

Replicate 4 0.1406 0.0352 0.5936

Treatment 5 779.6055 155.9211 2632.5342

Error 20 1.1846 0.0592

Table 5f : Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 2.7715 0.6929 1.1412

Treatment 5 559.5342 111.9068 184.3214

Error 20 12.1426 0.6071

Table 5g :GI

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0053 0.0013 0.1333

Treatment 5 88.1475 17.6295 1780.4996

Error 20 0.1980 0.0099

Table 5h : EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0052 0.0013 0.2638

Treatment 5 26.6175 5.3235 1071.1312 Error 20 0.0994 0.0050 Table 5i : Egg/Egg mass

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 5.7500 1.4375 0.6745

Treatment 5 577175.0625 115435.0156 54163.586

Error 20 42.6250 2.1312

Table 5j : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.3535 0.0884 0.4756

Treatment 5 30110.5781 6022.1157 32404.8672

Error 20 3.7168 0.1858

Table 5k : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 26.0000 6.5000 1.7568

Treatment 5 19953244.0000 3990648.7500 1078553.7500

Error 20 74.0000 3.7000

Table 51 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 1.8672 0.4668 0.3934 Treatment 5 13857.7656 2771.5532 2335.4761 Error 20 23.7344 1.1867 Table 5m : Population of female

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 1.7500 0.4375 0.3333

Treatment 5 2083983.5000 416796.6875 317559.3750

Error 20 26.2500 1.3125

Table 5n : Population of male

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0087 0.0022 1.2258

Treatment 5 19.2097 3.8419 2174.5432

Error 20 0.0353 0.0018

Table So : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 356.0000 89.0000 1.0137

Treatment 5 33537352.0000 6707470.5000 76394.8750

Error 20 1756.0000 87.8000

Table 5p ;; Rf

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0000 0.0000 -1.0000 Treatment 5 8.3384 1.6677 -6994772.0000 Error 20 0.0000 0.0000 Table 6 : Effect of soil application of Fusarium oxysporum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 6a : Fresh weight of shoot

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0117 0.0029 0.1583

Treatment 5 268.8816 53.7763 2905.9075

Error 20 0.3701 0.0185

Table 6b : Fresh weight of root

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 0.1053 0.0263 1.2114

Treatment 5 134.7992 26.9598 1240.0618

Error 20 0.4348 0.0217

Table 6c : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0006 0.0002 0.0148

Treatment 5 36.4846 7.2969 706.9952 Error 20 0,2064 0.0103

Table 6d : Dry weight of root SOURCE D.F. S.S. M.S.S. E VAL. Replicate 4 0.0134 0.0034 0.8317 Treatment 5 6.8499 1.3700 339.9890 Error 20 0.0806 0.0040 Table 6e : Length of shoot

SOURCE D.F. s.s. M.S.S. F. VAL,

Replicate 4 0.2998 0.0750 0.5732

Treatment 5 1082.4971 216.4994 1655.6790

Error 20 2.6152 0.1308

Table 6f: Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0479 0.0120 0.3635

Treatment 5 538.1387 107.6277 3270.3501

Error 20 0.6582 0.0329

Table 6g :GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0287 0.0072 1.4441

Treatment 5 91.4307 18.2861 3683.9849 Error 20 0.0993 0.0050

Table 6h : EMI SOURCE D.F. S.S. M.S.S. F. VAL, Replicate 4 0.0500 0.0125 1.7120 Treatment 5 68.7507 13.7501 1883.6302 Error 20 0.1460 0.0073 Table 6i : Egg/Egg mass

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 6.1250 1.5313 0.7313

Treatment 5 562101.4375 112420.2891 53693.2734

Error 20 41.8750 2.0938

Table 6j : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.4316 0.1079 2.4610

Treatment 5 27227.6934 5445.5386 124192.2344

Error 20 0.8770 0.0438

Table 6k : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 32.0000 8.0000 2.5000

Treatment 5 19741482.0000 3948296.5000 1233842.6250

Error 20 64.0000 3.2000

Table 61 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 16.4648 4.1162 1.7465 Treatment 5 25395.8633 5079.1729 2155.0813 Error 20 47.1367 2.3568 Table 6m : Population of female

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 17.5000 4.3750 2.9661

Treatment 5 2833506.0000 566701.1875 384204.1875

Error 20 29.5000 1.4750

Table 6n : Population of male

SOURCE D.F. S.S. iVl.S.S. F. VAL.

Replicate 4 1.5333 0.3833 2.6744

Treatment 5 10.8000 2.1600 15.0698

Error 20 2.8667 0.1433

Table 6o : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 164.0000 41.0000 4.1837

Treatment 5 38219292.0000 7643858.5000 779985.5625

Error 20 196.0000 9.8000

Table 6p :; Rf

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0001 0.0000 2.5301 Treatment 5 9.5151 1.9030 240415.8750 Error 20 0.0002 0.0000 Table 10 : Efficacy of some organic materials of agricultural origin for soil application of V. chlamydosporium for the management of M. incognita on tomato

Table 10a : Fresh weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1338 0.0334 0.9958

Treatment 6 449.9194 74.9866 2232.4290

Error 24 0.8062 0.336

Table 10b : Fresh weight of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1606 0.0402 1.5565

Treatment 6 150.1335 25.0223 969.7567

Error 24 0.6193 0.0258

TabI e 10c : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1212 0.0303 1.1808

Treatment 6 32.4933 5.4156 211.1325 Error 24 0.6156 0.0257

TabI e lOd : Dry weight of root

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0090 0.0022 0.2208 Treatment 6 4.6910 0.7818 76.8350 Error 24 0.2442 0.0102 Table lOe : Length of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 4.0801 1 0200 1.6242

Treatment 6 3029.1152 504.8525 803.8912

Error 24 15.0723 0.6280

Table lOf : Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 2.8174 0.7043 0.9831

Treatment 6 1252.5479 208.7580 291.3862

Error 24 17.1943 0.7164

Table lOg : GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0154 0.0039 0.1626

Treatment 6 82.9429 13.8238 583.5160

Error 24 0.5686 0.0237

Table 1 Oh: EMI

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.1417 0.0354 1.2613 Treatment 6 57.7000 9.6167 342.3115 Error 24 0.6742 0.0281 Table lOi : Egg/Egg mass

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 23 7500 5 9375 1 7273

Treatment 6 600541 0000 100090 1641 29117 1387

Error 24 82 5000 3 4375

Table lOj : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 1 0078 0 2520 0 9591

Treatment 6 44181 3906 7363 5649 28030 8203

Error 24 6 3047 0 2627

Table 10k : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 6 0000 1 5000 0 3273

Treatment 6 24709010 0000 4118168 2500 898509 3750

Error 24 110 0000 4 5833

Table 101 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 4 8438 1 2109 0 4572

Treatment 6 72510 1563 12085 0264 4563 0776

Error 24 63 5625 2 6484 Table 10m : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 38.0000 9.5000 3.0000

Treatment 6 2040026.0000 340004.3438 107369.7891

Error 24 76.0000 3.1667

Table lOn : Population of male

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 2.4571 0.6143 1.1569

Treatment 32.9714 5.4952 10.3498

Error 24 12.7429 0.5310

Table lOO : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 60.0000 15.0000 1.3846

Treatment 6 42287796.0000 7047966.0000 650581.5000

Error 24 260,0000 10.8333

Table lOp :Rf

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0000 0.0000 1.4328 Treatment 6 10.5472 1.7579 330134.1250 Error 24 0.0001 0.0000 Table 11 : Efficacy of some organic materials of agricultural origin for soil application of V. chlamydosporium for the management of A/, incognita on tomato

Table 11a : Fresh weight of shoot SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.2566 0.0641 1.8069

Treatment 6 303.2195 50.5366 1423.4807

Error 24 0.8521 0.0355

Tabl€; lib : Fresh weight of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1765 0.0441 1.7345

Treatment 6 116.3665 19.3944 762.3143

Error 24 0.6106 0.0254

Tabl e lie : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0670 0.0167 1.2473

Treatment 6 27.2738 4.5456 338.5582

Error 24 0.3222 0.0134

Table lid : Dry weight of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0316 0.0079 0.8494 Treatment 6 3.7673 0.6279 67.5344 Error 24 0.2231 0.0093 Table lie : Length of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1758 0 0439 0,2731

Treatment 6 2052.4980 342.0830 2126.2095

Error 24 3.8613 0.1609

Table llf : Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 141887.1094 35471.7773 1.0030

Treatment 6 203706.0938 33951.0156 0.9600

Error 24 848795.3125 35366.4727

Table llg :: GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0886 0.0221 1.1876

Treatment 6 80.5468 13.4245 720.1034

Error 24 0.4474 0.0186

Table llh: EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1943 0.0486 0.9932

Treatment 6 67.3720 11.2287 229.5954

Error 24 1.1738 0.0489 Table Hi : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 19.6016 4.9004 4.1759

Treatment 6 39051.1016 6508.5171 5546.2314

Error 24 28.1641 1.1735

Table llj : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 8.0000 2.0000 0.5714

Treatment 6 23237838.0000 3872973.0000 1106563.7500

Error 24 84.0000 3.5000

Table Ilk : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 24.4375 6.1094 1.7547

Treatment 58805.5938 9800.9326 2814.9275

Error 24 83.5625 3.4818

Table 111 : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 33.5000 8.3750 1.0836

Treatment 6 1213911.0000 202318.5000 26175.9785

Error 24 185.5000 7.7292 Table 11m : Population of male

SOURCE D.F. s.s. IVI.b.o. F. VAL.

Replicate 0 7429 0 1857 0 4815

Treatment 15 6000 2 6000 6 7407

Error 24 9 2571 0 3857

Table 11 n : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 108 0000 27 0000 1 6701

Treatment 6 35198388 0000 5866398 0000 362870 0000

Error 24 388 0000 16 1667

Table llo :Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0 0000 0 0000 1 2353

Treatment 6 8 7687 1 4615 540861 7500

Error 24 0 0001 0 0000 Table 12e ; Length of shoot

SOURCE D.F. s.s. Ivl.S.S. F. VAL.

Replicate 4 2.1289 0.5322 1.3838

Treatment 6 1874,1582 312.3597 812.1617

Error 24 9.2305 0.3846

Table 12f : Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1846 0.0461 0.3345

Treatment 6 845.8154 140.9692 1021.9646

Error 24 3.3105 0.1379

Table 1I2 g : GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0599 0.0150 0.7369

Treatment 6 71.0320 11.8387 582.2217

Error 24 0.4880 0.0203

Table 12h: EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1914 0.0478 1.7488

Treatment 6 61.1777 10.1963 372.7018

Error 24 0.6566 0.0274 Tabic 12i : Eggs/Egg mass

SOIRCE D.I. S.S. M.S.S. F. VAL.

Replicate 4 11 0000 2 7500 0 9362

Tieatmcnt 6 518687 2500 89781 2109 30563 8164

Error 24 70 5000 2 9375

Table 12j : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0 5156 0 1289 1 3289

Treatment 6 30957 0234 5159 5039 53187 9063

Error 24 2 3281 0 0970

Table 12k : Population of J2

SOURCE D.F. S.S. M.S.S. F. \AL.

Replicate 4 24 0000 6 0000 2 2500

Treatment 5 20853420 0000 3475570 0000 1303338 7500

Error 24 64 0000 2 6667

Table 121 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 19 1250 4 7813 2 1374

Treatment 68025 7500 11337 6250 5068 2744

Error 24 53 6875 2 2370 Table 12m : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 9 5000 2 3 750 0 3738

Treatment 6 1885582 5000 314263 7500 49457 9023

Error 24 152 5000 6 3542

Table 12n : Population of male

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 1 1703 0 2926 1 2229

Treatment 36 8069 6 1345 25 6417

Error 24 5 7417 0 2392

Table 12o : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 96 0000 24 0000 1 8462

Treatment 6 37298864 0000 62164-77 5000 478190 5625

Error 24 312 0000 13 0000

Table 12p : Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0 0000 0 0000 1 6000

Treatment 6 9 2946 1 5491 649736 6250

Error 24 0 0001 0 0000 Table 13 : Effect of soil application of rice husk colonized with V. chlamydosporium on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 13a : Fresh weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Rephcate 4 0 0640 0 0160 1.0315

Treatment 7 875 0728 125 0104 8063 6626

Error 28 0 4341 0 0155

Table 13b : Fresh weight of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1670 0 0417 2 8696

Treatment 7 167.3088 23 9013 1642.9055

Error 28 0 4073 0.0145

Table 13c : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0 0759 0 0190 1 5313

Treatment 7 31 3843 4 4835 361 6988

Error 28 0.3471 0 0124

Table 13d : Dry weight of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 1412 6567 353.1642 1 0016

Treatment 7 2506 4165 358 0595 1.0155 Error 28 9872 9033 352 6037 Table 13e : Length of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Rcplitatc 4 0 1641 0 0410 0 412"^

Treatment 7 6594 0195 942 0028 9470 21M

Error 28 2 7852 0 995

Table 13f: Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 1 2100 0 3025 2 2273

Treatment 7 938 2412 134 0345 986 9122

Error 28 3 8027 0 1358

Table 1 3g:GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0 1075 0 0269 1 5280

Treatment 7 146 7550 20 9650 1191 9735

Error 28 0 4925 0 0176

Table 13h : EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0 0396 0 0099 0 9641

Treatment 7 111 9915 15 9988 1559 0614

Error 28 0 2873 0 0103 Table J3i ; Egg/Egg mass

SOIRCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 4.7500 1 1875 0.4283

Treatment 7 850265,6250 121466.5156 43814.0078

Error 28 77.6250 2.7723

Table 13j : % of infected eggs

SOIRCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0234 0.0059 0.3333

Treatment 7 60203.1875 8600.4551 489270.3438

Error 28 0.4922 0.0176

Table 13k : Population of J2

SOIRCE D.F. S.S. M.S.S. F. VAL.

Replicate 10.0000 2.5000 0.7143

Treatment 30899556.0000 4414222.5000 1261206,3750

Error 28 98.0000 3.5000

Table 131 : Population of J3/J4

SOIRCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 29.0938 7.2734 2.3872

Treatment 7 204773.5625 29253.3652 9601.1045

Error 28 85,3125 3.0469 Table 13m : Population of female

SOURCE D.F. S.S. Ivl.S.S. F. VAL.

Replicate 4 14.5000 3.6250 0.6486

Treatment 7 4524765.0000 646395.0000 115648.9453

Error 28 156.5000 5.5893

Table 13n : Population of male

SOURCE D.F. S.S. Ivl.S.S. F. VAL.

Replicate 0.1500 0.0375 0.1795

Treatment 20.7750 2.9679 14.2051

Error 28 5.8500 0.2089

Table 13o : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 84.0000 21.0000 2.5789

Treatment 7 65054508.0000 9293501.0000 1141307.1250

Error 28 228.0000 8.1429

Table 13p : Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 0.7819

Treatment 7 16.2264 2.3181 181006.8281

Error 28 0.0004 0.0000 Tabic 14 : Effect of soil application of gram husk colonized with Acremonium strictum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 14a : Fresh weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0078 0.0020 0.1955

Treatment 7 524.3623 74.9089 7496.6421

Error 28 0.2798 0.0100

« Table 14b ; FresI• i weight of root SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 0.0178 0.0045 0.4301

Treatment 7 141.4126 20.2018 1950.2560

Error 28 0.2900 0.0104

Table 14c : Dry weight of shoot .

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0554 0.0138 1.4554

Treatment 7 25.5636 3.6519 384.0307 Error 28 0.2663 0.0095

Table 14d : Dry weight of root

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0071 0.0018 0.5064 Treatment 7 4.6807 0.6687 190.4927 Error 28 0.0983 0.0035 Table 14e : Length of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 1133 1719 283.2930 0.9909

Treatment 7 5600.2773 800.0396 2.7983

Error 28 8005.3711 285.9061

Table 14f : Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0313 0.0078 0.2427

Treatment 7 1595.9951 227.9993 7082.5522

Error 28 0.9014 0.0322

Table 14g : GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0500 0.0125 1.1908

Treatment 7 145.7998 20.8285 1984.1425

Error 28 0.2939 0.0105

Table 14h : EMI

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0315 0.0079 0.4302 Treatment 7 111.3038 15.9005 868.7951 Error 28 0.5125 0.0183 Table 14i : Eggs/Egg mass

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 6.1250 1.5313 0.9346

Treatment 7 885429.1250 126489.8750 77203.6320

Error 28 45.8750 1.6384

Table 14j : % of infected eggs

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 1.2539 0.3135 1.2738

Treatment 7 70316.4375 10045.2051 40818.6094

Error 28 6.8906 0.2461

Table 14k : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 12.0000 3.0000 1.3125

Treatment 7 32087244.0000 4583892.0000 2005452.6250

Error 28 64.0000 2.2857

Table 141 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 10.000 2.5000 0.8293 Treatment 7 181746.9688 25963.8535 8612.9629 Error 28 84.4063 3.0145 Table 14m : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 2.2500 0.5625 0.3387

Treatmenl 7 4869858.5000 695694.0625 418912.5625

Error 28 46.5000 1.6607

Table 14n : Population of male

SOURCE D.F. S.S. IVl.O.O. E VAL.

Replicate 0.0000 0.0000 0.000

Treatment 19.3750 2.7679 9.6875

Error 28 8.0000 0.2857

Table 14o : Total population

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 36.0000 9.0000 2.1724

Treatment 7 66896156.0000 9556594.0000 2306764.0000

Error 28 116.0000 4.1429

Table 14p :Rf

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.0000 0.0000 0.8140 Treatment 7 16.6462 2.3780 405926.7813 Error 28 0.0002 0.0000 Table 15 : Effect of soil application of gram husk colonized with Fusarium oxysporum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 15a : Fresh weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0869 0.0217 0.9644 Treatment 7 545.4277 77.9183 3458.3159

Error 28 0.6309 0.0225

Table 15b : Fresh weight of root

SOURCE D.F. S.S. M.S.S. ¥. VAL.

Replicate 4 0.0527 0.0132 1.1622

Treatment 7 110.5356 15.7908 1392.0184

Error 28 0.3176 0.0113

Tabl(e 15c : Dry weight of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0626 0.0156 1.3422

Treatment 7 22.6506 3.2358 277.5414

Error 28 0.3264 0.0117

TabI e 15d : Dry weight of root

SOURCE D.F. S.S. M.S.S. ¥. VAL.

Replicate 4 0.0012 0.0003 0.2273

Treatment 7 3.6033 0.5148 400.9657

Error 28 0.0359 0.0013 Table 15e : Length of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.3750 0.0938 0.9243

Treatment 7 4296.8711 613.8387 6052.2646

Error 28 2.8398 0.1014

Table 15f: Length of root

SOURCE D.F. S.S. M.S.S. E VAL.

Replicate 4 0.1592 0.0398 0.4407

Treatment 7 1200.8291 171.5470 1899.8053

Error 28 2.5283 0.0903

Table 15g : GI

SOURCE D.F. S.S. M.S.S. n ¥. VAL.

Replicate 4 0.0075 0.0019 0.2686

Treatment 7 150.9298 21.5614 3073.7520

Error 28 0.1964 0.0070

Table 15h: EMI

SOURCE D.F. S.S. M.S.S. R VAL.

Replicate 4 0.1535 0.0384 2.3031

Treatment 7 126.9297 18.1328 1088.3416

Error 28 0.4665 0.0167 Table 15i : Egg/Egg mass

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 3.6250 0.9063 0,3447

Treatment 7 951937.1250 135991.0156 51718.1445

Error 28 73.6250 2.6295

Table 15j : % of infected eggs

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 0.2773 0.0693 0.1700

Treatment 7 55814.8398 7973.5483 19553.3340

Error 28 11.4180 0.4078

Table 15k : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 12.0000 3.0000 0.9545

Treatment 7 30523012.0000 4360430.5000 1387409.7500

Error 28 88.0000 3.1429

Table 151 : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 6.1563 1.5391 0.9318

Treatment 7 206141.3750 29448.7676 17828.4434

Error 28 46.2500 1.6518 Table ISm : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 8.0000 2.0000 1.2584

Treatment 7 4150296.7500 592899.5625 373060.4063

Error 28 44.5000 1.5893

Table 15n : Population of male

SOURCE D.F. S.S. IVl.iS.iS. F. VAL.

Replicate 0.1500 0.0375 0.3043

Treatment 30.8000 4.4000 35.7101

Error 28 3.4500 0.1232

Table ISo : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 32.0000 8.0000 1.6471

Treatment 7 63151960.0000 9021709.0000 1857410.6250

Error 28 136.0000 4.8571

Table ISp :Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 1.1382

Treatment 7 15.7497 2.2500 268532.5313

Error 28 0.0002 0.0000 Table 16: Effect of seed soaking in different concentrations of culture filtrate of Verticillium chlamydosporium on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 16a : Fresh weight of shoot SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.4883 0.1221 0.1796 Treatment 5 1380.5527 276.1105 406.2899 Error 20 13.5918 0.6796

Table ! 16b : Fresh1 weighlt of root SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 23.0352 5.7588 0.7023 Treatment 5 297.9756 59.5951 7.2679 Error 20 163.9961 8.1998

TabI e 16c : Dry weight ([) f shoot SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.2628 0.0657 1.2350 Treatment 5 23.8104 4.7621 89.5287 Error 20 1.0638 0.0532

Tabll e 16d : Dry weight of root SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.0127 0.0032 0.6529 Treatment 5 5.5189 1.1038 226.3914 Error 20 0.0975 0.0049 Table 16e : Length of shoot

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 1.4609 0.3652 0.3015

Treatment 5 2284.0820 456.8164 377.1507

Error 20 24.2246 1.2112

Table 16f: Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.5000 0.1250 1.0535

Treatment 5 1198.3818 239.6764 2019.9884

Error 20 2.3730 0.1187

Table 16g :GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1100 0.0275 1.2008

Treatment 5 82.5270 16.5054 720.7355

Error 20 0.4580 0.0229

Table 16h : EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0820 0.0205 1.5893

Treatment 5 64.6187 12.9237 1001.9163

Error 20 0.2580 0.0129 Table 16i : Eggs/Egg mass

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 4.5000 1.1250 0.4500

Treatment 5 906451.0000 181290.2031 72516.0781

Error 20 50.0000 2.5000

Table 16j : Population of J2

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 1.0000 0.2500 0.1190

Treatment 5 29787224.0000 5957445.0000 2836878.7500

Error 20 42.0000 2.1000

Table 16k : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 4.8672 1.2168 1.0002

Treatment 5 18174.1680 3634.8335 2987.6941

Error 20 24.3320 1.2166

Table 161 : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 4.1250 1.0313 0.2437

Treatment 5 1271868.6250 254373.7188 60117.8672

Error 20 84.6250 4.2312 Table 16m : Population of male SOURCE D.F. s.s. IM.S.S. F. VAL. Replicate 0.5333 0.1333 0.3571

Treatment 18.7000 3.7400 10.0179

Error 20 7.4667 0.3733

Table 16n : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 20.0000 5.0000 0.3731

Treatment 5 44627428.0000 8925486.0000 666081.0625

Error 20 268.0000 13.4000

Table 16o :Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 0.7979

Treatment 5 11.1321 2.2264 331145.0938

Error 20 0.0001 0.0000 Table 17 : Effect of seed soaking in different concentrations of culture filtrate of Acremonium strictum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 17a : Fresh weight of shoot SOURCE D.E S.S. M.S.S. F. VAL. Replicate 4 4.2734 1.0684 2.6113 Treatment 5 1191.7051 238.3410 582.5544 Error 20 8.1826 0.4091

Table 17b : Fresh weight of root SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 0.2085 0.0521 1.2727 Treatment 5 219.2390 43.8478 1070.6444 Error 20 0.8191 0.0410

Table 17c : Dry weight of shoot SOURCE D.E S.S. M.S.S. R VAL. Replicate 4 0.1400 0.0350 1.7154 Treatment 5 21.5070 4.3014 210.8665 Error 20 0.4080 0.0204

Table 17d : Dry weight of root SOURCE D.E S.S. M.S.S. F. VAL. Replicate 4 0.0009 0.0002 1.3934 Treatment 5 1.9676 0.3935 2309.0476 Error 20 0.0034 0.0002 Table 17e : Length of shoot

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 1,5254 0.3813 0.8484

Treatment 5 1353.2715 270.6543 602.1074

Error 20 8.9902 0.4495

Table 17f : Length of root

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.4014 0.1003 2.2732

Treatment 5 500.1641 100.0328 2266.2300

Error 20 0.8828 0.0441

Table ]I7 g : GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1887 0.0472 4.1495

Treatment 5 101.2227 20.2445 1781.1061

Error 20 0.2273 0.0114

Table 17h: EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.2253 0.0563 1.3629

Treatment 5 76.0467 15.2093 367.9776

Error 20 0.8266 0.0413 Table 17i : Egg/Egg mass

SOURCE D.F. s.s. M.S.S. F. VAL.

Replicate 4 12.8750 3.2188 0.8864

Treatment 5 940957.8750 188191.5781 51825.5664

Error 20 72.6250 3.6312

Table 17j : Population of J2

SOURCE D.F. S.S. M.S.S. F. VAL,

Replicate 4 12.0000 3.0000 1.8750

Treatment 5 34580472.0000 6916094.5000 4322559.0000

Error 20 32.0000 1.6000

Table 17k : Population of J3/J4

SOURCE D.F. S.S. M.S.S*. F.VAL.

Replicate 4 2.5332 0.6333 0.4025

Treatment 5 17839.8672 3567.9734 2267.7703

Error 20 31.4668 1.5733

Table 171 : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 3.1250 0.7813 0.4647

Treatment 5 1172285.5000 234457.0938 139454.0313

Error 20 33.6250 1.6812 Table 17in : Population of male

SOURCE D.F. s.s. IM.a.o. F. VAL.

Replicate 0.1333 0.0333 0.1923

Treatment 33.3667 6.6733 38.5000

Error 20 3.4667 0.1733

Table 17n : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 12.0000 3.0000 0.5556

Treatment 5 49709736.0000 9941947.0000 1841101.2500

Error 20 108.0000 5.4000

Table 17o :Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 0.9122

Treatment 5 12.4156 2.4831 703714.4375

Error 20 0.0001 0.0000 Tabic 18 : Effect of seed soaking in different concentrations of culture filtrate of Fusarium oxysporum on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 18a : Fresh weight of shoot SOURCE D.F. S.S. M.S.S. E VAL. Replicate 4 1.3853 0.3463 1.7861 Treatment 5 626.4600 125.2920 646.1798 Error 20 3.8779 0.1939

Table 18b : Fresh1 weight of root SOURCE D.F. S.S. M.S.S. F.VAL. Replicate 4 0.0964 0.0241 0.9118 Treatment 5 157.9552 31.5910 1194.8005 Error 20 0.5288 0.0264

Table 18c : Dry weight (a f shoot SOURCE D.F. S.S. M.S.S. R VAL. Replicate 4 0.0314 0.0079 0.6061 Treatment 5 21.1717 4.2343 326.5884 Error 20 0.2593 0.0130

Table 18d : Dry weight of root SOURCE D.E S.S. M.S.S. E VAL. Replicate 4 16.6443 4.1611 1.0328 Treatment 5 28.9311 5.7862 1.4362 Error 20 80.5765 4.0288 Table 18m : Population of male

SOURCE D.F. s.s. 1V1.S.S. F. VAL.

Replicate 4 2.4667 0.6167 1.6372

Treatment 5 18.9667 3.7933 10.0708

Error 20 7.5333 0.3767

Table 18n : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 182.0000 45.5000 3.6694

Treatment 5 37190644.0000 7438129.0000 599849.1250

Error 20 248.0000 12.4000

Table 18o : Rf

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0001 0.0000 1.9577

Treatment 5 9.2887 1.8577 206136.4375

Error 20 0.0002 0.0000 ANOVA TABLE SECTION-II

Table 1 : Comparative suitability of some culture media for mass culturing of a predatory nematode, Mononchoides longicaudatus

Table la : Population after 14 days

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 14.3984 3.5996 0.9728 Treatment 2 28056.3984 14028.1992 3791.2051

Error 8 29.6016 3.7002

Table lb : Final population after 30 days SOURCE D.F. S.S. M.S.S. F. VAL. Replicate 4 13.7344 3.4336 0.9199

Treatment 2 76068.1406 38034.0703 10190.1855

Error 8 29.8594 3.7324 Table 2e : Length of shoot

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 65.0391 16 2598 2.9510

Treatment 4 785.0391 196.2598 35.6188

Error 16 88.1602 5.5100

Table 2f : Length of root

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 7.4399 1.8600 1.4762

Treatment 4 83.4399 20.8600 16.5554

Error 16 20.1602 1.2600

Table 2g : GI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.1063 0.0266 2.2427

Treatment 4 85.0263 21.2566 1793.4619

Error 16 0.1896 0.0119

Table 2h: EMI

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0456 0.0144 1.0220

Treatment 4 97.1696 24.2924 2178.8066

Error 16 0.1784 0.0111 Table 2 : Effect of a predatory nematode, Mononchoides longicaudatus on plant growth and development of root-knot nematode, Meloidogyne incognita on tomato

Table 2a : Fresh weight of shoot

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 6.2402 1.5601 0.2732 Treatment 4 274.6401 68.6600 12.0245 Error 16 91.3599 5.7100

Table 2b : Eresh weight of root

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 0.1036 0.0259 0.4076

Treatment 4 48.4286 12.1071 190.4823

Error 16 1.0170 0.0636

Table 2c : Dry weight of shoot

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 0.0216 0.0054 0.2146

Treatment 4 7.5176 1.8794 74.7267

Error 16 0.4024 0.0252

Table 2d : Dry weight of root

SOURCE D.E S.S. M.S.S. E VAL.

Replicate 4 0.0457 0.0114 0.6426

Treatment 4 1.3751 0.3438 19.3502 Error 16 0.2843 0.0178 Table 2i : Population of J2

SOURCE D.F. S.S. M.S.S. F.VAL.

Replicate 4 10.000 2.5000 1.9048

Treatment 4 2920165.5000 730041.3750 556222.0000

Error 16 21.0000 1.3125

Table 2j : Population of J3/J4

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 3.8398 0.9600 0.3714

Treatment 4 4361.0410 1090.2603 421.7705

Error 16 41.3594 2.5850

Table 2k : Population of female

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 8.2383 2.0596 0.7743

Treatment 5565.0430 3891.2607 1462.9283

Error 16 42.5586 2.6599

Table 21 : Population of male

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 0.0000

Treatment 4 20.8000 5.2000 26.0000

Error 16 3.2000 0.2000 Table 2m : Total population

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 7.0000 1 7500 0.1573

Treatment 4 3545885.0000 886471.2500 79682.8125 Error 16 178.0000 11.1250

Table 2n ;:R f

SOURCE D.F. S.S. M.S.S. F. VAL.

Replicate 4 0.0000 0.0000 0.0836

Treatment 4 3.5119 0.8780 51323.9023

Error 16 0.0003 0.0000