BIOLOGICAL CONTROL OF PLANT-PARASITIC NEMATODES

Jf y - '- ' DISSERTATION SUBMITTED TO THE

/ ALIGARH MUSLIM UNIVERSITY, ALIGARH ~ IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

jUaiter of pi)iIogopI)p IN

^ % n BOTANY

v,>^ A V

MOHAMMAD ARIF

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH ( INDIA) 1995

DS2939

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CiiECKED-2002 S i \ : t \ 0 . ^

2 A OCT 199''

^\ft aiigarl) iWuglim ffinibersfitg^JlUgarlj

Ph.D. ,O.Sc. ,PGNomatol . .FPSl , FBS, FBRS

Professor of Plant Nematology Deputy Director, Institute of Agriculture

Editor : Afro-Asian Journal of Nematology Afro-Asian Nematology Network

PHONE: (0571) 401016 (Bot ) (0571 ) 400516 (Agr ) (0571) 400030 (Res )

(0571)400920-23 Ext. 31 4 (Bot.) TELEX : 564230 AMU IN FAX •. ^0571 \ 4,00 52S, 400105

Department of Botany Aiigarh Muslim University Aligarh-202002 (India)

Dated 0 5 . 0 2 . 1 9 9 6

CERTIFICATE

Tills is to certify that the dissertation '

entitled, "Biological Control of Plant Parasitic

Nematodes", is a faithful record of the bonafide

research (Jork carried ovkt by Wx. Mohawsaad Axif vmder Trty

guidance and supervision. This work is up-to-date and

original. He is allowed to submit the dissertation to

the Aiigarh Muslim University, Aiigarh for consideration

of the award of the degree of Master of Philosophy in

Botany.

(M. MASHKOOR ALAM)

ACKNOWLEDGEMENT

First, I offer my thanks to the Almighty Allah, who

bestowed upon me the capabilities and inspiration to

achieve this goal.

I deem utmost pleasure in expressing profound

gratitude and indebtedness to my supervisor.

Prof. M. Mashkoor Alam, Department of Botany, Aligarh

Muslim University, Aligarh for his unrelenting and

unstinted help extended to me during the course of

completion of this dissertation.

I also submit my sincere thanks to Prof. Wazahat

Hussain, Chairman, Deptt. of Botany, Aligarh Muslim

University, Aligarh for generously providing me all the

facilities.

I am also thankful to all my colleagues and friends

who assited me, in one way or the other during the course

of present study.

(MOHAMMAD ARIF)

CONTENTS

PAGE NO.

INTRODUCTION 01-08

REVIEW OF LITERATURE 02-29

MATERIALS & METHODS 30-42

REFERENCES 43-49

mzKCDuezjoj^

1. INTRODUCTION

Nematodes constitute the largest and most ubiquitous

group of the animal kingdom, comprising 80-90 per cent of

all the multicellular animals. Basically, the nematodes are

aquatic animals thriving best in water but they have

adapted to terrestrial habitats. They are mostly found in

soil in almost all regions, from snowy mountains to

deserts, in oceans and lakes. In soil, they are the most

common of all the soil fauna, numbering from 1.8 to 120

million per square meter of soil in some situations (Kevan,

1965). Only a fraction of their number have the ability to

parasitize plants and animals.

According to Sasser & Freckman (1987) the average

loss of crops due to nematodes was about 12.3 per cent,

more being in developing countries. They have also

estimated the losses in potato, tomato eggplant, okra and

pepper which were 12.2, 20.6, 16.19, 20.4 and 12.2 per cent

respectively. Even in a developed country like U.S.A. the

losses caused by nematodes are not so low. In the

mid-sixties about 7.2 per cent of the annual crop value was

lost; at 1967-68 prices this amounted to $1.6 billion. In

field crops 6 per cent ($110 million), fruits and nuts 12

per cent ($ 225 million) and vegetables 11 per cent ($267

million) were estimated to be lost. In U.K. the potato cyst

nematode caused an annual loss of £20 million (Southey &

Samuel, 1954). In India alone about 10 per cent crop losses

due to nematodes occurred annualy; this included loss of

Rs. 30 million in barley and Rs. 40 million in wheat due to

"molya disease" caused by Heterodera avenae (Seshadri,

1970).

As soon as soil-inhabiting nematodes were recognised

as pathogens, attempts were made to control them, the

earliest records are the observations of Lohde in 1874,

Kuhn in 1877 and zopf in 1888 regarding the fungi

destroying the nematodes. The discovery of the nematicidal

properties of D D (1,3-dichloropropene +

1,2-dichloropropane) and related C3 hydrocarbons, EDB

(ethylene dibromide) and DBCP (1,2-dibromo-3 chloropropane)

during the 1940s and early 1950s heralded a new era in

plant nematology. Since most nematicides are closely

related to the insecticides they also show problems such as

pesticidal resistance, resurgence of pest populations

following their use, accumulation of residues. Thus many of

these have been found environmentally unsafe and have

created many problems of pollution.

As we approach the end of the twentieth century

there has ever been a greater need to find alternative

methods of nematode control. In some countries, the

furaigants DBCP and EDB which have formed the basis of

nematode control programmes on many crops since the end of

World War II, have been removed from the market because of

health and environmental considerations. With the

increasing cost of testing and registering pesticides, the

development of new nematicides has almost come to a halt,

necessitating nonchemical means of nematode control to be

found. In the recent years, biological control of plant

parasitic nematodes has emerged as one of the prospective

alternatives to the traditional control means especially

with nematicides.

The importance of selecting biological control

agents that are able to compete with endemic

micro-organisms is particularly apparent when it is

considered that they must act against nematodes in the

rhizosphere, the moslt microbially active zone in soil. The

biological complexity of the soil and the rhizosphere

environment ensure that any introducd organism, including

an antagonist of nematodes, faces competition from the

endemic components of the microflora. The chances of

selecting effective antagonists for field use may thereofre

be improved by isolating them from this environment, as

such organisms are likely to have evolved the capacity to

maintain themselves in the vicinity of roots.

The relatively stable behaviour of animal

populations in natural environments should serve as a

constant reminder that in nature, all organisms are

subjected to a constant series of checks and balances.

Populations of individual species do not increase

indefinitely but tend to be constrained by the physical

environment and by the community of organisms with which

they co-exist. Cyclic changes in populations will occur but

provided there is no major change in the physical or biotic

environment; populations fluctuate between certain upper

and lower limits. This phenomenon is commonly referred to

as 'biological balance' or the 'balance of nature".

Plant parasitic nematodes spend at least some part

of their lives in soil, one of the most complex of

environments. Their activities are not only influenced by

variations in soil physical factors such as temperature,

moisture and aeration but also by a vast array of living

organisms, including other nematodes, bacteria, fungi,

algae, protozoans, insects, mites and other soil animals.

These biological components of the soil ecosystem are

particularly important in limiting and more or less

stabilizing nematode populations. All organisms are

competitors for resources such as space and oxygen;

root-feeding insects and plant pathogens compete with plant

parasitic nematodes for the same food sources, some

bacteria and fungi produce metabolic byproducts which

interfere with nematode behaviour and many soil organisms

parasitize or prey on nematodes. The action of such

organisms in maintaining nematode population densities at a

lower average level than would occur in their absence is

generally termed as "biological control".

It is generally accepted that biological control is

a broad concept which encompasses a range of control

strategies including cultural practices, host plant

resistance, and the introduction or encouragement of

antagonistic organisms. Cook & Baker tl984) gave definition

of biological control of plant parasitic nematodes as

"reduction in nematode damage by organisms antagonistic to

nematodes through the regulation of nematode populations

and/or a reduction in the capacity of nematodes to cause

damage, which occurs naturally or is accomplished through

the manipulation of the environment or by the mass

introduction of antagonists". Such a definition recognizes

that reduction in nematode damage is the primary aim of any

control measure and that it need not necessarily be

accomplished by reducing nematode numbers. It embrances

situations, for example, where control is achieved with

antagonists which restrict the movement or invasion of

nematodes rather than killing them.

Nematologists have been interested in using natural

enemies to control nematodes since the 1920s, when Cobb

showed an interest in the predatory nematodes (Cobb, 1920),

and Linford and Yap (1939) conducted experiments with the

nematode-traping fungi. Despite a considerable amount of

interest and many optimistic pronouncements during the last

50 years, only a few examples of natural biological control

of nematodes have been reported, and that there are no

examples of the widespread commercial use of introduced

biological control agents. This situation is in marked

contrast to other fields such as entomology and weed

science where there are many well documented examples of

biological control. Even in the related fields of plant

pathology, where interest in biocontrol quickened during

the last decade or so, biological control now offers

answers to many serious disease problems in modern

agriculture. The relatively recent recognition of the

importance of plant parasitic nematodes, the need to

develop basic information on their taxonomy, physiology,

biology and ecology, the availability of effective

nematicides, and the complexity of the soil environment are

perhaps some of the reasons as to why development of

biological control of nematodes has legged behind one other

fields.

Fungi that are antagonistic to nematodes play a

great role in keeping the long coevolution of nematodes and

fungi, which obviously occurned in the close confines of the

soil habitat, it is not surprising that a great variety of

interrelationships have developed between the two groups.

Fungi possesing the capacity to destroy or deleteriously

affect nematodes vary considerably in both their biology

and taxonomic relationships. They range from obligate,

endoparasitic forms, many of which are zoosporic, to

predacious trap forming species and opportunistic fungi

that colonize reproductive structures, such as cysts and

eggs.

The discovery of opportunistics fungi capable of

colonizing nematode reproductive structures and

deleteriously affecting them, has been very recent.

Nematodes belonging to such genera as Globodera, Heterodera

and Meloidogyne at sedentary stages of their life cycles,

are vulnerable to attack by fungi either within host plant

roots or when exposed on the root surface or in the soil.

When Globodera and Heterodera, as obese females, break in

through the root cortex and mature into cysts, they become

increasingly susceptible to fungal colonization, as do

emerging eggmasses of Meloidogyne.

Fungi that parasitize eggs, prefer eggs of

heteroderid nematodes and those which deposit them in

gelatinous matrix. The oviposition nature (both in the cyst

and eggmass) of these nematodes makes them most vulnerable

to the attack by these fungi. In some cases there is

enzymatic disruption of nematode structural elements such

as egg shells and larval cuticle or physiological

disturbances due to biosynthesis of diffusable toxic

metabilities.

The genus Paecilomyces / a close relative of

Penicillium has been one of the principal genera of

interest in biological control studies in recent years.

Paeciloymces lilacinus has been the subject of many recent

investigations due to its many desirable qualities. It was

found parasitizing eggs of M_ incognita in Peru (Jatala et

al., 1979), and has since been associated with root-knot

and cyst nematodes in many geographical locations.

Paecilomyces has given many successful results in the

Philippines and this is the ma3or reason for its commercial

production under the trade name of "BIOCON" in that country

(Davide, 1990). Due to its mode of nutrition (i.e.

saprophytic and/or parasitic) it is becoming increasingly

apparent that P_ lilacinus may act in a more complex manner

than simply as an egg parasite and this may help explain

the variable results that have sometimes been obtained with

this fungus. An overview on this aspect is given in the

next chapter.

KSVjeW 07 aZSKAZUKS

2. REVIEW OF LITERATURE

The progress made in the control of plant parasitic

nematodes with biological agents in laboratory and green

house conditions has been quite impressive. However, it is

a costly affair to prepare large quantities of inocula of

biocontrol agents for use on field scale. In biological

control the most desirable characters of natural enemies

are: (i) mobility and the ability to search out its prey

(ii)adaptability to the environment, (iii) host specificity

(iv) synchronization with the host and (v) ability to

survive host free periods. Due to paucity of information on

these aspects the biocontrol agents have not been used

properly on large scale. Moreover, this kind of control

measure requires a thorough knowledge of biology and

ecology of the biocontrol agents. Recently, a parasitic

fungus, Paecilomyces lilacinus has aroused interest of

scientists for its use in the biocontrol of plant parasitic

nematodes mainly because of its opportunistic behaviour. A

review on this aspect is given below.

Jatala (1986) and Alam (1990) have thoroughly

reviewed the work on Paecilomyces lilacinus. The most

important quality of the fungus P_ lilacinus is its ability

to rapidly colonize nematode reproductive structures in

short span of time and to survive host-free periods by its

10

capacity to grow axenically. It is a good competitor to

other soil microorganisms. Moreover, P_ lilacinus has a

quite long lasting residual effect in field condition. The

fungus is highly effective in controlling plant parasitic

nematodes particularly root-knot and cyst nematodes on

various plants.

Paecilomyces lilacinus was isolated from eggmass of

Meloidogyne incognita actrita,' picked from infected potato

roots in central Peruvian highland in the Huanuco Valley

(Anon, 1979; Strttner, 1979; Jatala et. al_. , 1979; Jatala,

1982). Silva et aJ^. (1992) noted P_ lilacinus infesting

eggs of M^ incognita in the northwest of Parana State. The

fungus when inoculated was able to infest eggs and eggmass

of Mj_ incognita race 2. The density of P_ lilacinus was 680

propagules/g of soil obtained from a mulberry (Morus alba)

infested with M_^ incognita.

Jatala (1985) reviewed the use of biological control

agents in reducing numbers of Meloidogyne spp. with

particular reference to the use of Paecilomyces lilacinus

in controlling Meloidogyne incognita on potatoes. It was

found that the fungus significantly reduced the tuber- and

root-galling due to the nematodes.

Roman & Marcano (1985) conducted two experiments in

the green house to determine the effect of P_ lilancinus in

11

controlling the larvae and root-knot or root gall formation

of M^ incognita in tomato cv. Florodel. The plants were

inoculated with the fungus alone, the nematode alone or

both simultaneously at different time intervals and

combinations. Observations, made 60 days after the

inoculations, showed that the fungus controlled the

nematodes and reduced root-knot formation. In the first

experiment, significantly fewer larvae were found in root

and soil of plants inoculated with the fungus three days

prior to nematode inoculation. A higher root-knot index in

plants inoculated with nematode alonge, with fungus and

nematodes simultaneously, or with the fungus alone three

days after nematode inoculation, was also obtained. In the

second experiemnt, a tendency for a reduced number of

nematodes in soil and root gall indices was observed as the

time of inoculation of the fungus before the nematode

inoculation was increased. Plants inoculated with the

fungus four and six days before nematode inoculation and

significantly lower gall indices than the other plants

inoculated with the fungus, the nematode or both.

Rohana et al. (1987) conducted experiments to

investigate the effects of application of P_ lilacinus on

population levels of root-knot nematode (Meloidogyne spp.)

and tomato plant growth. P.lilacinus was found to inhibit

the population development of Meloidogyne spp. and

12

maintained growth of tomato at 0.5 and 1.0 g inoculum/750 g

soil.

Cabanillas et al. (1988) examined the excised tomato

roots histologically for interactions of the fungus, P.

lilacinus and M_ incognita race 1. Root galling and giant

cell formation were absent in tomato roots inoculated with

nematode eggs infected with P_ lilacinus. Little or no

giant cell formation was found in roots dippped in a spore

suspension of P_ lilacinus and inoculated with M.

incognita.Numerous large galls and giant cells were present

in roots inoculated only with M_ incognita. P. lilacinus

colonized the surface of epidermal cells as well as the

internal cells of epidermis and cortex.

Cabanillas & Barker (1989) also conducted microplot

experiments to evaluate the effects of inoculum level and

time of application of P_ lilacinus on the protection of

tomato against M_ incognita. Greatest protection against

this pathogen was obtained when P_; lilacinus was delivered

into soil ten days before planting and again at planting.

Yield increased two fold compared with yield in plots with

nematodes alone or with M^ incognita + fungus. Percentage

of Pj^ lilacinus infected eggmasses were greatest in plots

treated at mid-season or at mid season with early

application compared with plots treated with the fungus ten

days before planting and/or at planting time.

13

cabanillas et al . (1989a) conducted laboratory and

microplot experiments to determine the influence of storage

of P_ lilancins on M_ incognita on tomato. Application of

P. lilacinus in field microplots resulted in about 25%

increase in tomato yield and 25% galls suppression,

compared with nematode alone. Greatest suppression of egg

development occurred in plots treated with P_ lilacinus on

pellets of wheat grains and granules. Cabanillas ejt al.

(1989b) investigated the potential of 13 isolates of

P. lilacinus from various geographical regions as

biocontrol agents against M^ incognita, the effects of

temperature on their growth and characterization of the

impact of soil temperature on the efficacy for controlling

this nematode. Maximum fungal growth, as determined by dry

weight of the inoculum, occurred from 24 to 30 C; least

growth was at 12 and 36°C. The best control of M^ incognita

was provided by an isolate from Peru or mixture of isolates

of P_ lilacinus. As soil temperature increased from 16 to

28°C, both root damage caused by M^ incognita and

percentage of eggmasses infected with P_ lilacinus

increased. The greatest residual activity of P_ lilacinus

on M.incognita was attained with a mixture of fungal

isolates. These isolates affected lower root galling and

necrosis, egg development and enhanced shoot growth

compared with plants inoculated with M^ incognita alone.

14

Sharma et al . (1980) observed the reduction in M.

incognita population by using fungus P_ lilacinus grown on

different rice levels. Maximum control was found with

Rice(R) + Nematode (N) + Fungus (F) treatment having 40 g

rice level. P_ lilacinus surrounded the eggmasses and the

mycelium penetrated the eggs deeply.

Sharma & Trivedi (1989) used four inoculum levels of

P. lilacinus (1,5,15 and 75 g fungus infested rice) for the

control of M_ incognita on aubergine. The number of

eggmasses and number of eggs per eggmass were much lower in

plants treated with the fungus compared to controls. There

was 34, 75, 87 and 91% reduction in nematode in plants

treated with 1,5,15 and 75 g doses of the fungus-infested

rice respectively. Sharma & trivedi (1989) also conducted

an experiment to control M_ incognita infecting Trigonella

foenum-graecum P. lilacinus raised on goat dung or sesame

oil cake as substrates. Relatively better reduction in the

nematode population was observed on the substrate + fungus

+ nematode treatment compared with substrate + nematode

alone. Root-knot index was lower in the sesame oil cake +

fungus + nematode combination than in the one with dung +

fungus + nematode. The fungus penetrated the eggs and fed

upon their contents leaving emptyshells. Invaded eggs were

swollen in comparison with uncolonized ones.

15

Fernandez £t aj . (1989) evaluated the effectiveness

5 9 of 10 -10 conidia/ml concentrations of the Peruvian strain

of P^ lilacinus in the biological control of M^ incognita

7 in vitro. A concentration of 10 conidia/ml and higher

levels resulted in 100% colonization of the nematode eggs

at 12 days of exposure with a reduction of 65% in their

hatch.

Davide (1990) reported results of studies on the

biological control of plant parasitic nematodes in the

Philipines which had been going on for the past 16 years.

Since 1981 most of the studies were focused on the use of P.

lilacinus. The best isolate (Philipine strain No.l) is now

commercially mass produced and marked as "BIOCON" BY

Asiatic Technologies Inc. in Manila. A review of the

nematodes attacking economic crops and their control by P.

lilacinus has been given. Integration of the fungus with

other control measures has also been discussed (Davide,

1990) .

Tandingan & Asunction (1990) applied P_ lilacinus

with or without organic substrate or fenamiphos at 100 ppm.

All the treatments reduced populations of M^ incognita in

the soil and number of galls and eggmasses on Boehmeria

musae roots 3 months after treatment. Soil populations

decreased by 54.85% with P_ lilacinus treated plots showing

16

the greatest reduction. Eggmasses decreased by 80 and 82%

in plots with P^ lilacinus as soil mix and soil drench

respectively.

Trivedi (1990) evaluated the fungus P_ lilacinus for

the biological control of root-knot nematode, M^ incognita

on Solanum melogena. P. lilacinus was used to control M.

incognita under Indian agroclimatic conditions. The fungus

was screened on sixteen different culture substrates.

Maximum spore load per gram was found on rice grains.

Better plant reductions in galling, final soil nematode

population, number of eggmasses and number of eggs per

eggmass were noted in fungus treated aubergine plants.

Khan et al . (1992) investigated the importance of P.

lilacinus for seed dressing of Luf fa aegyptica for the

control of M^ incognita acrita during seed germination.

Spores of P^ lilacinus in polyvinyl alcohol and gelatine

with vegetable charcoal mixtures as vehicles were used

separately for seed dressing of Luffa aegyptica in pot

experiments. The medium and high doses of P_ lilacinus (50

and 75 mg spores respectively) significantly reduced

root-galling by M^ incognita acrita.

Pandey & Trivedi (1992) applied £^ lilacinus to

Capsicum annuum seedlings infested with M^ incognita in a

17

pot experiment. In treated pots there was a decrease in the

number of galls, eggs per eggmass, final soil population

and hatching of M^ incognita eggs.

Croshier et aJ. (1985) obseved the effectiveness of

p. lilacinus in the control of the root-knot nematode

Meloidogyne javanica on tomato. High percentage of egg

infection was found.

Carneiro & Cayrol (1991) evaluated the relationship

between inoculum density of the nematophagous fungus P.

lilacinus and control of Meloidogyne arenaria on tomato.

Five doses (0.01, 0.1, 1.0, 10 and 100 g/m^) of a

commercial product of P_ lilacinus isolated from eggs of M.

incognita were applied in a powder formulation (10

spores/g of the product) in a glass house pot experiment

against large infestations of M_ arenaria. The trial was

conducted over eleven months on three successive tomato cv.

Saint Pierre crops. Results showed that the number of the

fungal propagules in the soil was correlated to the initial

dose applied and decreased progressively through the time

with increased dose. Populations of M_ arenaria were

significantly reduced by the fungus at 10 and 100 g of

2 spores/m in the second and third nematode generations. The

number of colonized eggmasses and the number of non-viable

eggs increased with fungal inoculum and the fungus was most

18

effective at a density of 10 spores/g of soil. In the

highest level of control (100% colonized eggmasses) only

50% of the eggs were parasitized. Twenty three percent of

the larvae remained which constituted an important residual

inoculum potential.

Jimnez & Gallo (1988) evaluated the efficiency of P.

lilacinus as a biological control agent of Meloidoqyne

spp. , viz. M^ javanica, M. arenaria and M^ incognita under

green house conditions. The fungus infected eggs and

occasionally females.

Zaki & Maqbool (1991) investigated the possible

control of Meloidoqyne javanica by P_ lilacinus on

chickpea. Chickpea seedlings were grown in pots inoculated

with M_ javinica alone, simultaneous inoculation of M.

javanica and P_ lilacinus, inoculation of M^ javanica

followed by P_ lilacinus after 1 wk. Root-knot indices

were greatly reduced by P_ lilacinus after 60 days, the

fungus being most effective at 2 g/pot, 1 week before

nematode inoculation.

Franco £t a^. (1981) evaluated the efficiency of P.

lilacinus as a biocontrol agent of Globodra pallida. One

year old cysts of G^ palida were surface sterilized with

10% clorax and sprinkled in Petri plates containing PDA, V8

19

or water agar medium recently inoculated with P^ lilacinus

spores. The plates were maintained at 20 C for 5, 10, 15,

20, 25 and 30 days. At the end of leach period, cysts were

recovered from plates, washed and divided in two groups.

Infected eggs were counted in one group while the second

group of cysts were exposed to potato root exudates to

determine the extent of hatching. The percentage of

infected eggs increased as the time of exposure to fungus

increased, but there was no difference in egg infection in

different media. Results of the hatching tests indicated a

stimulatory effect of media upto 25 days, thereafter,

hatching decreased significantly. The decrease in hatching

was correlated with the increase in infection of eggs by P.

lilacinus.

Jatala et al . (1985) reported hatching stimulation

and inhibition of Globodera pallida eggs by enzymatic and

exopathic toxic compounds of some biocontrol agents

particularly P_ lilacinus. Laboratory and green house

studies on the effect of P_ lilacinus on G_ pallida

indicated up to 30 per cent egg damage without direct

penetration while the hatching was stimulated prematurely

in the eggs containing second stage juveniles.

Saifullah & Gul (1991) tested the efficacy of P.

lilacinus against Globodera rostochiensis in a pot

experiment using two methods of inoculation : direct

pouring of fungus-infected wheat grains 7g (A) and 14g (B)

20

to the soil and coating potato tubers with fungus. Final

nematode populations were 70, 70 and 75 cysts/50 cm soil,

2.75, 11.25 and 12.25 cysts/tuber and 5.0, 5.5 and 7.75

cysts/5cm roots were recorded due to fungus coated tubers,

dose B and dose A, respectively. Yield and plant height

were greatest with tuber coating while fresh shoot weight

was greatest with dose.

Paecilomyces lilacinus in association with

Verticillium chlamydosporium have also been found effective

against root-knot nematodes. Population densities of M.

incognita and the nematophagous fungi, P_ lilacinus and V.

chlamydosporium were determined by Gaspard et_ a]^. (1990b)

in 20 Northern California tomato fields over two growing

seasons. All p_ lilacinus and V_ chylamydosporium field

isolates parasitized M_^ incognita eggs iji vitro. In green

house study, number of V_ chlamydosporium and Pj_ lilacinus

increased more in soil with M_ in cogn it a-infected tomato

plants than in soil v/ith uninfected tomato plants. After 10

weeks, the pf/pi of second stage 3uveniles in soil infested

with P_ lilacinus, V. chlamydosporium and M^ incognita was

47.1 to 295.6. The results suggested that V.

chlamydosporium and P_ lilacinus were not effectively

suppressing population of M^ incognita in California tomato

fields. The survival of M_ incognita in soil infested with

P. lilacinus and V_ chlamydosporium was studied by Gaspard

21

et al. (1990a). Meloidoqyne incognita infected tomato

seddlings were transplated into sterilized soil or

unsterilized soil collected from 20 California tomato

fields to measures suppression caused by P_ lilacinus, V.

chlamydosporium and other naturally occurring antagonists.

Unsterilized soils contained 35, 39 and 55% fewer M.

incognita second stage juveniles (J2) than did sterilized

soil 1 month after infected tomato seedlings were

transplanted to these soils and placed in a green house.

Three months after infected seedlings were transplanted,

unsterilized soils had 97, 62 and 86% fewer J2 than the

corresponding sterilized soils. Unsterilized soils of M.

incognita infected seedlings that were maintained one month

in green house followed by 1 or 2 month of post-harvest

inoculation contained J2 numbers equal to or greater than,

numbers in the corresponding sterilized soil. The most

suppressive of the unsterilized soils, K and Q were not

infested with V_ chlamydosporium and P_^ lilacinus in colony

forming units in unsterilized soil of all bioassays, but

they were not associated with lower numbers of J2.

Stirling & West (1991) evaluated the fungal

parasites of root-knot nematode eggs from tropical and

subtropical regions of Australia. A survey of 48 Queensland

soils for nematophagous fungi revealed the presence of V.

chlamydosporium and P_ lilacinus. To identify the isolates

22

with the biocontrol potential, 26 isolates of P_ lilacinus

and 13 isolates of V_ chlamydosporium were screened for

activity against M ^ javanica eggs using 3 different tests.

Two isolates of V_ chlamydosporium and one isolate of P.

lilacinus were found to be highly parasitic in all 3 tests.

There are also reports regarding the possibility of

using P_ lilacinus alongwith a bacterial parasite of

nematodes, Pasteuria penetrans for controlling root-knot

nematode on various plants. Dube & smart (1987) reported

the possibility of using P_ lilacinus alongwith P.

penetrans for controlling M_j_ incognita on tomato, tobacco,

soybeans, Vicia villosa and Capsicum annuum. They noted

greater nematode control when P_ lilacinus and P_; penetrans

were applied together. Similar results were obtained by

Maheswari & Mani (1988) in case of M^ javanica on tomato

and Zaki & Maqbool (1992) in case of M_ javanica on brinjal

and mung.

Several workers made comparative study about the

efficacy of P_ lilacinus with different nematicides against

plant parasitic nematodes on various plants. Jatala et al.

(1980) compared the efficacy of P_ lilacinus with

nematicides and organic matter against M_ incognita on

potato cv. Cuzco. The root galling was significantly lower

in plants grown in plots applied with the fungus than those

23

from plots applied with Temik 10% G at 25 kg/ha, Nemacur 5%

G at 50 kg/ha, furadan 5% Gat 50 kg/ha and organic matter

at 10 tons/ha.

Davide & Zorilla (1988, 1986) found a nematicide

isozofos (Miral 10 G) more effective than P_ lilacinus in

controlling M^ incognita on okra. Novavetti et_ a]^. (1986)

and Shahzad & Ghaffar (1987) have used P_ lilacinus and

furadan for nematode control, while Khan (1988) compared

the efficacy of P_ lilacinus with aldicarb for controlling

Globodera rostochiensis on potato.

Diskson & Mitchell (1985) evaluated P_^ lilacinus

alone and in combination with ethoprop and phenomiphos for

management of M^ javanica on tobacco in microplots. The

yield of crop was maximum in the treatment where fungus was

present.

Hewlett et al . (1988) also evaluated P_ lilacinus as

a biocontrol agent of Mj_ javanica on tobacco. The efficacy

of P_ lilacinus alone and in combination with fenamiphos,

and ethoprops for controlling M^ javanica on tobacco cv.

NC 2326 and the ability of te fngus to colonize in soil

under field conditions were evaluated for 2 years in

microplots. Nematicides showed higher field in both years

while reverse was true with P_ lilacinus. But the fungal

population decreased two folds.

24

Reddy & Khan (1989) investigated the effect of P.

lilacinus alone and in combination with carbofuran on the

management of reniform nematode, Rotylenchulus reniformis

infecting brinjal. Inoculation of plants with £_ lilacinus

grown on 4g sterilized rice was found to be effective in

increasing plant height and root weight and reducing the

nematode population both in soil and roots. The fungus gave

least reproduction factor and increased the percentage of

males. P_ lilacinus infected both eggs and mature females

of reniform nematode.

Vicente et al. (1989) evaluated the effect of

nematicides and P_ lilacinus for nematode control in water

melon. In a field trial the following treatments were

compared: (a) £_ lilacinus applied to soil 2 week before

planting water melons, (b) P_ lilacinus incorporated at

planting, (c) 2.24 and 4.48 kg carbofuran lOG/ha, (d) 6.73

and 13.4 kg fenamiphos 15G/ha; (c) and (d) were

incorporated at planting. All treatments showed substantial

reduction in nematode populations 90 days after planting.

Yields from the carbofuran treatments were significantly

higher than those from the fenamiphos treatments and from

plots to which P^ lilacinus was added at planting. No

significant yield differences were found between the 2

rates of the 2 nematicides.

25

Vicente et_ a]^. (1991) showed the effect of granular

nematicides and the fungus P_ lilacinus in nematode control

in water melon. P\_ lilacinus was added to soil 2 week prior

to planting or carbofuran at 2.24 kg/ha and fenamiphos at

6.73 or 13.46 kg/ha applied to water melon in the field in

Puerto Rico for control of M^ incognita and Rotylenchulus

reniformis. All treatments effectively controlled

nematodes, numbers being lowest in treatments with P.

lilacinus.

Walia et a_] . (1991) conducted a pot experiment in

which P_ lilacinus cultured on wheat bran (WB) was applied

either as a soil treatment 10 days before or after sowing

8 7

at 1 g/kg soil (5x10 spores), or as seed treatment (3x10

spores/seed), or in different combinations, to sandy soil

infected with M_ javanica. Fungus application in general,

resulted in better top growth of okra. The methods

(soil/seed treatment) or time (pre/post sowing) of fungus

applications were found to be equally effective. WB alone

also promoted shoot length and fresh shoot weight to a

lesser extent. Root galling was significantly reduced in

fungus treatments. Application of WB alone failed to reduce

root galling. The time and method of fungus application

were at par, as reduction was concerned. WB proved better

than carbofuran (1kg a.i./ha) in gall suppression.

26

Abid et a3_. (1992) investigated the effects of

oilcakes, Bradyrhizobium sp. , P_ lilacinus and Furadan on

root nodulation and root-knot nematode in mungbean. In a

pot experiment complete control of iM_ incognita infection

in mungbean (Vigna radiata) was observed in plants grown in

3% w/w cotton cake mixed 25 ml Bradyrhizobium sp. , P. q

lilacinus (3x10 conidia/ml) or furadan (carbofuran) at 55

kg/ha with cotton and neem (Azadirachta indica) cakes was

more effective than individual applications. P_ lilacinus

stimulated nodule formation by Bradyrhizobium sp. Maximum

fresh shoot weight was rerorded in 3% cotton cake mixed

with P_ lilacinus followed by 3% cotton cake with

Bradyrhizobium sp. Of the oilcakes, mustard cake showed

inhibitory effect on seed germination.

Vicente & Acosta (1992) evaluated the biological and

chemical control of nematodes in Capsicum annuum L. In a

field experiment, the fungus P_^ lilacinus (added 1 week

before planting or at planting) and carbofuran (IX or 2X)

were added to see the effect on yield of pepper and on

population level of M_ incognita and Rotylenchulus

reniformis. Significantly more and heavier fruits were

obtained from fungus (1 week before planting) and

carbof uran 2X treated plots than from the check. A similar

trend was observed in the nematode population dynamics;

although the percentage of nematode reduction was high in

27

all treated plots; it was higher in those treated with the

2X fungus 1 week before planting and with carbofuran

Besides Pj lilacinus ohter species of Paecilomyces,

viz. P_ marquandii has also been observed by Marbon-Mendoza

et al. (1992) as one of the soil organism that contributes

to nematode suppression in the Chinampa agricultural soil.

Two applications of isolates of P_ marquandii from

suppressive Chinama soil or P_ lilacinus from Peru,

generally gave better control of tomato root-knot due to M.

incognita than did a single application. The effects of

root galling by each of the Paecilomyces isolates varied

between experiments, however, the ovicidal potential of the

three isolated did not differ significantly.

FUTURE PLAN OF WORK;

The nematode control strategies including the

biocontrol agents have gained momentum these days.

Paecilomyces lilacinus, a nematode biocontrol fungus has

been reported to grow on organic matter in absence of its

living substrate, i.e. nematodes. However, the studies on

this aspect have been mostly confined to other countries.

Therefore, it is considered worthwhile to systematically

study this problem, the details of which are as follows:

28

1. To isolate and identify nematode antagonistic fungi from field, and to determine correlation between their population and those of root-knot and reniform nematodes.

2. To study the suitability of different growth media for culturing Paecilomyces lilacinus.

3. To study the effect of culture filtrates of Paecilomyces lilacinus on mortality of second stage juveniles (J2) of root-knot nematode, Meloidoqyne incognita in vitro.

4. To study the effect of culture filtrates of Paecilomyces lilacinue on mortality of reniform nematode, Rotylenchulus reniformis in vitro.

5. To study the effect of culture filtrates of Paecilomyces lilacinus on hatching of second stage juveniles (J2) of root-knot nematode, Meloidoqyne incognita in vitro.

6. To study the effect of culture filtrates of Paecilomyces lilacinus on penetration of second stage juveniles (J2) of root-knot nematode, Meloidoqyne incognita into the roots of tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

7. To study the effect of root exudates of antagonistic plants on mycelial growth and sporulation of Paecilomyces lilacinus in vitro.

8. To study the effect of different concentrations of a nematicide carbofuran on mycelial growth and sporulation of Paecilomyces lilacinus in vitro.

9. To study the efficacy of Paecilomyces lilacinus against root-knot nematode, Meloidogyne incognita in presence/absence of organic amendments on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

10. To study the efficacy of Paecilomyces lilacinus against reniform nematode, Rotylenchulus reniformis in presence/ absence of organic amendments on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

29

11. To study the efficacy of Paecilomyces lilacinus in combination with antagonistic plants against root-knot nematode, Meloidogyne incognita on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

12. To study the efficacy of Paecilomyces lilacinus in combination with antagonistic plants against reniform nematode, Rotylenchulus reniformis on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

13. To study the efficacy of Paecilomyces lilacinus in combination with carbofuran on root-knot nematode, Meloidogyne incognita on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

14. To study the efficacy of Paecilomyces lilacinus in combination with carbofuran against reniform nematode, Rotylenchulus reniformis on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

15. To study the efficacy of seed soaking with culture filtrates of Paecilomyces lilacinus against root-knot nematode, Meloidogyne incognita on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

16. To study the efficacy of seed soaking with culture filtrates of Paecilomyces lilacinus against reniform nematode, Rotylenchulus reniformis on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

17. to study the efficacy of bare-root-dip treatment with culture filtrates of Paecilomyces lilacinus against root-knot nematode, Meloidogyne incognita on tomato (Lycopersicon esculetum) and eggplant (Solanum melongena).

18. To study the efficacy of bare-root-dip treatment with culture filtrates of Paecilomyces lilacinus against reniform nematode, Rotylenchulus reniformis on tomato (Lycopersicon esculentum) and eggplant (Solanum melongena).

MAZSKJAJCS i MezMom

3. MATERIALS AND METHODS

3.1 Selection of test materials

Two nematode species, viz. the root-knot nematode,

Meloidogyne incognita (Kofoid & White) Chitwood and the

reniform nematode, Rotylenchulus reniformis Linford &

Olivera, and a fungus, Paecilomyces lilacinus (Thorn.)

Samson will be selected for the present study. Tomato

(Lycopersicon esculentum Mill.) and eggplant (Solanum

melongena Linn.) will be taken as host plant. Organic

amendments will include chopped leaves, stem, etc, and

oil-seed cakes of neem (Azadirachta indica A. Juss. ) ,

castor (Ricinus communis Linn.) and rice polish.

Antagonistic plants and a nematicide, carbofuran will also

be used for nematode control in the presence of the

antagonistic fungus.

3.2 Preparation of nematode inoculum

Separate cultures of the root-knot and reniform

nematodes will be maintained on tomato in concrete

microplots.

In case of root-knot nematode, a single eggmass will

be taken out from the infected tomato roots with the help

of a sterilised forcep and will be placed on a small coarse

sieve (1 mm pore size) lined with moist tissue paper which

31

will then be placed in a Petri dish (10 cm diameter)

containing distilled water. Second Stage juveniles (J2),

which will be hatched out, will be collected alongwith the

water from Petri dishes after every 24 h. Fresh water will

be added to the Petri dishes after each collection. This

process will continue for 5-7 days. These second stage

juveniles (J2) will serve as inoculum of the root-knot

nematode, Meloidogyne incognita for raising culture on

tomato.

For the extraction of the reniform nematode, soil

will be collected from around the roots of infected castor

plants. The soil will be processed by using Cobb's sieving

and decanting method alongwith modified Bearman's funnel

technique (Southey, 1986). The nematode suspension thus

collected/obtained, will serve as the inoculum of the

reniform nematode, Rotylenchulus reniformis for raising

culture on tomato.

Separate suspensions of the test nematodes, will be

obtained as above and will be gently stirred for making

homogenous distribution of the nematodes and then 5 ml

suspension will be transferred to the counting dish

(Southey, 1985) and the number of second stage juveniles

(J2) in case of Meloidogyne incognita and immature females

in case of Rotylenchulus reniformis in each sample will be

32

counted under stereoscopic microscope. An average of five

counts will be made in each case to determine the density

of nematodes per unit volume of these suspensions.

3.3 Preparation of inoculum of the fungus

Pure culture of the fungus, Paecilomyces lilacinus

will be maintained on Potato-Dextrose-Agar (PDA), which,

will be prepared by using following ingredients:

Potato 250.0 g Dextrose 17.0 g Agar 20.0 g Distilled water 1000.0 ml

First of all, the pealed pieces of the potato will

be boiled in distilled water and then the extract so

obtained (after removing the potato pieces) will be mixed

with dextrose and agar, and boiled again. The medium in

liquid condition (when still hot) will be transferred to

culture tubes which will be plugged with cotton. These

tubes will then be autoclaved and after cooling the fungus

will be transferred to the culture tubes in an aseptic

chamber taking all the precautions prescribed for such

operations.

The inoculum of the fungus, Paecilomyces lilacinus

will be raised on Richard's liquid medium (Riker & Riker,

19 36) having the following composition:

33

Potassium nitrate 10.00 g Potassium dihydrogen phosphate 5.00 g Magnesium sulphate 2.50 g Ferric chloride 50.00 g Sucrose 50.00 g Distilled water 1000.00 ml

One hundred ml of the above medium will be

transferred to 250 ml Erlenmeyer flasks and will be plugged

with cotton covered with butter paper. After that these

flasks will be autoclaved. Small bits of the test fungus

will be transferred to these conical flasks in an aseptic

chamber taking all the precautions prescribed for such

operations. The fungus will then be incubated for 15 days

in B.O.D. running at 25+l°C temperature.

After the incubation period, the mycelial mats will

be removed and then gently pressed between sterile blotting

sheets to remove the excess amount of liquid. This liquid

will later be used as culture filtrate of the test fungus.

The inoculum will be prepared by mixing 10 g fungal

mycelium in 100 ml of sterilised distilled water in a

waring blender for few seconds (Stemerding, 1964). In this

way each 10 ml of the homogenate will contain 1 g of fungal

mycelium.

3.4 Effect of culture filtrates of Paecilomyces lilacinus on Juvenile hatching of Meloidogyne incognita in vitro;

Five freshly picked eggmasses of Meloidogyne

incognita of average size will be transferred to Petri

34

dishes (40 mm diameter) containing 5 ml culture filtrates

of Paecilomyces lilacinus. These will be replicated five

times including distilled water control. The Petri dishes

will be incubated at 25 C and the total number of hatched

juveniles will be counted after 5 days with the help of

Doncaster's dish (Southey, 1986).

3.5 Effect of culture filtrate of Paecilomyces lilacinus on mortality of second stage juveniles (J2) of Meloidogyne incognita and immature females of Rotylenchulus reniformis in vitro.

About 100 freshly hatched second stage juveniles

(J2) of Meloidogyne incognita and same number of immature

females of Rotylenchulus reniformis will be transferred to

Petri dishes containing 5 ml culture filtrates of P.

lilacinus following the procedure described by Alam (1985).

Each treatment will be replicated five times including

distilleld water control. Number of immobile

juveniles/immature females, will be counted after

12,24,48,72,96 hours. Death of nematodes will be

ascertained by transferring them into plain water and then

per cent mortality will be determined.

3.6 To study the effect of culture filtrates of Paecilomyces lilacinus on penetration of second stage juveniles (J2) of Meloidogyne incognita into the roots of tomato, Lycopersicon esculentum cv. Pusa Ruby and eggplant, Solanum melongena;

The experiment will be conducted as per procedure

35

described by Ismail & Alam (1975). The seedlings of the

test plants will be raised in 25 cm diameter clay pots

containing autoclaved soil-manure mixture. Seedlings when 3

week old, will be taken out and roots will be washed

thoroughly and gently to remove soil particles adhering to

it. The seedlings will be then separately placed in beakers

containing cultural filtrates of fungus for different

durations, i.e. 12, 24 and 48h. The seedlings will be

placed in such a way that their root systems remain dipped

in the cultural filtrate. After that, the seedlings will be

taken out, their roots will be washed thoroughly and gently

and then will be transferred to thermocole cups (5 cm

diameter) containing acid-leached and thoroughly washed

river sand. Each treatment, including un-dipped control,

will be replicated five times. The seedlings will be

inoculated with 1000 freshly hatched second stage 3uveniles

(J2) of Meloidogne incognita. Care will be taken to keep

the sand moist for 5 days by gently adding water with the

help of pipette along the rim of the cup. After 5 days, the

cup will be placed in a bucket and will be washed gently to

take out the seedlings. The roots as well as cups will be

washed properly in bucket and then will be taken out from

the bucket. From these buckets the juveniles will be

extracted following Cobb's sieving and decanting technique.

Number of juveniles will be counted in each sample and

deducted from the number of initial inoculum, i.e. 1000.

36

Thus the number of juveniles penetrated into the roots will

be determined.

3.7 To study the efficacy of Paecilomyces lilacinus against the root-knot nematode, Meloidogyne incognita and reniform nematode, Rotylenchulus reniformis on tomato and eggplant in presence/absence of organic amendments:

Fifteen cm clay pots will be filled with soil. The

soil will be amended with oil-sed cakes and chopped leaves,

etc. at the rate of Ig N/kg soil. Untreated pots will serve

as control. Watering will be done daily for a period of ten

days to ensure proper decomposition of the additives.

Thereafter, three week old seedlings of test plants will be

transplanted into these pots. When three week old, these

plants will be inoculated with 5000 juveniles of M.

incognita or immature females of R^ reniformis separately.

With the nematode inoculation, fungus, P_^ lilacinus will

also be inoculated simultaneously at the rate of 10

mycelial suspension per plant. Uninoculated plants will be

used as control. The experiment will be terminated after 90

days of seedling transplantation and the growth parameters

of the plants will be determined as described below.

Recording of data:

The experiment will be terminated after 90 days of

transplanting of the seedlings and plant growth parameters

37

(length, fresh weight, dry weight, and number of fruits,

etc.) will be determined.

Chlorophyll content of leaf will be estimated by the

method of Hiscox & Israelstam (1979). One hundred milligram

of leaf pieces will be placed in a vial containing 7 ml

DMSO (Dimethyl feulphoxide) and the chlorophyll will be

extracted into the fluid at 65 C by incubating it for 60

minutes. The extract will be transferred to a graduated

tube and made up to 10 ml with DMSO and will be assayed

immediately. A sample of 3.0 ml chlorophyll extract will be

transferred in cuvette and the OD (optical density) values

at 645 and 663 nm will be read in spectronic-1001

spectrophotometer against a DMSO blank. chlorophyll

contents will be calculated by the following formulae:

Chi.a = [12.7 (663)]-L2.69( 645)] x V/1000 x W

Chl.b = [22.9 (645)j-L4.68( 663)j x V/1000 x W

Where,

V = Volume of the extract in ml

W = Fresh weight of the sample in g

Number of root-galls per plant will be determined in

case of plants inoculated with the root-knot nematode.

While in case of reniform nematode, the pot soil from each

treatment will be separately processed after the

38

termination of the experiment according to Cobb's sieving

and decanting method followed by the modified Bearmann's

funnel technique (Southey, 1986). Nematode population in

roots will be determined by macerating the root-pieces in a

waring blender for few seconds and then counting their

numbers. Reproduction factor (R) of the nematode will be

calculated following the method of Oostenbrink (1966) by

dividing the final population (Pf) with initial population

(Pi).

R = Pf/Pi

In the experiment separate sets of 5 plants for each

treatment will also be kept for studying the effect on

pollen fertility. Pollen fertility (%age) at flowering

stage will be estimated by the method of Brown (1949) using

stainability of pollen grains. The pollen grains which took

up stain and have regular outline will be considered

fertile while those which will be empty without stain and

have irregular shape will be considered sterile.

Water absorption capability of plant roots will be

assesed prior to the recording of plant weights by using

the method of Alam et al_. (1974). Erlenmeyer flasks of 250

ml capacity will be filled with water and weighed. Plants

from different treatments will be placed in each flasks

39

singly. Flasks containing water without seedlings will be

kept as control. After 24h the plants will be taken out

from the flasks and the amount of water loss will be

determined. Amount of water lost from the control flasks

will be deducted from the amount of water lost from the

flasks having plants. The difference will give the actual

amount of water absorbed by the roots.

3.8 To study the effect of different concentrations of nematicide, carbofuran on mycelial growth and sporulation of Paecilomyces lilacinus in vitro;

Different standard concentrations of the nematicide,

carbofuran will be prepared. One ml of these solutions will

be poured into the Erlenmeyer flasks containing 99 ml of

Richard's liquid medium. In these flasks the test fungus,

Paecilomyces lilacinus will be inoculated as per procedure

described earlier and the data will be observed

accordingly.

3.9 To study the efficacy of Paecilomyces lilacinus in combination with antagonisti plants against root-knot and reniform nematodes on tomato, Lycopersicon esculentum and eggplant, Solanum melongena;

Fifteen cm diameter clay pots will be filled with

soil manure mixture, these pots will be autoclaved at 15

2 lb/in . Three-week-old seedling of the test plant will be

transplanted singly to these pots in the centre alongwith 5

40

seedlings of antagonistic plants in the periphery. One

week later the plants will be inoculated with 5000 J2 of

the root-knot or 5000 immature females of the reniform

nematodes as the case may be. Simultaneously with the

inoculation of nematodes, the fungus Paecilomyces lilacinus

will be inoculated in the pots. Uninoculated pots will

serve as control. After 90 days the experiment will be

terminated and the plant growth parameters etc. will be

determined as described earlier.

3.10 To study the efficacy of Paecilomyces lilacinus in combination with carbofuran on root-knot and reniform nematodes on tomato, Lycopersicon esculentum and eggplant, Solanum melongena;

Fifteen cm clay pots will be filled with soil-manure

mixture as described earlier. In these pots different

concentrations of the nematicide carbofuran will be added,

and watering will be done. After one week, 3 week old

seedling of the test plant will be transplanted. One week

after transplanting, the plants will be inoculated with

5000 J2 of Mj^ incognita or immature females of R.

reniformis seperately. Simultaneously with the inoculation

of nematodes, 10 ml of mycelial suspension will also be

inoculated into the pots. The uninoculated pots will serve

as control. Each treatment will be replicated five time.

After care will be given as and when required. The

41

experiment will be terminated after 90 days of

transplantation. The plant growth parameters, etc. will be

determined on the same lines as described earlier.

3.11 To study the effect of bare-root-dip treatment with culture filtrate of Paecilomyces 111acinus against root-knot and reniform nematode on tomato, Lycopersicon esculentum and eggplant, Solanum melongena;

Thoroughly washed roots of three-week old seelings

of the test plants will be dipped in different

concentration of culture filtrate of P_ lilacinus. Root-dip

treatment in distilled water will serve as control. Later

on the seedlings will be transplanted in clay pots. Two

days after the transplantation these will be inoculated

with 5000 J2 of M^ incognita or immature females of R.

reniformis separately. Unioculated plants will serve as

control. There will be five replicates in each treatment.

Aftercare of plants will be done as and when required.

3.12 To study the effect of seed soaking with culture filtrate of Paecilomyces lilacinus against root-knot and reniform nematode on tomato, Lycopersicon esculentum and eggplant, Solanum melongena;

Seeds of the test plants will be soaked in different

concentration of culture filtrate of P_ lilacinus. Seeds

soaked in distilled water will serve as control. Four

treated seeds will be sown in each pot. After germination

42

thinning will be done and only one plant per pot will be

allowed to grow. Three-week old plants will be inoculated

with 5000 J2 or immature females of M^ incognita and R.

reniformis respectively. Uninoculated plants will serve as

control. Each treatment will be replicated five times.

Aftercare of the plants will be done as and when required

and the plant growth parameters, etc. will be determined as

described earlier.

nsjSKSj^ees

4. REFERENCES

Abid, M.; Haque, S.E.; Maqbool, M.A. & Ghaffar, A. (1992). Effects of Oilcakes, Bradyrhizobium spp., Paecilomyces lilacinus and Furadon on root nodulation and root-knot nematode in raungbean. Pak. J. Nematol. 10(2): 145-150.

Alam, M.M. (1985). A simple method in. vitro screening of chemicals for nematoxicity. Int. Nematol. Net. Newsl.2(l):6.

Alam, M.M. (1990). Paecilomyces lilacinus a nematode biocontrol agent. In Nematode bio-control (aspects and prospects) eds. M.S. Jairajpuri, M.M. Alam and I. Ahmad: 71-82.

Alam, M.M.; Hasan, N. & Saxena, S.K. (1974). Effect of root-knot nematode, Meloidogyne incognita on water absorption capability of tomato roots. Indian J. Nematol. 4(2): 244-246.

Anon (1979) Control of important nematode and insect pests of potatoes. CIP Ann.Lima, Peru, pp 35-47.

Brown, G.T.(1949). Pollen Slide Studies. Charles C. Thomas Publication, spring Field Illinois.

Cabanillas, E. & Barker, K.R. (1989). Impact of Paecilomyces lilacinus inoculum level and application time on control of Meloidogyne incognita on tomato. J. Nematol. 21(1): 115-120.

Cabanillas, E.; Barker, K.R. & Daykin, M.E. (1988). Histology of the interactions of Paecilomyces lilacinus with Meloidogyne incognita on tomato. J. Nematol. 20(3): 362-365.

Cabanillas, E.; Barker, K.R. & Nelson, L.A. (1989a). Survival of Paecilomyces lilacinus in selected carriers and related effects on Meloidogyne incognita on tomato. J^ Nematol. 21(2): 164-172.

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44

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