BIOCONTROL POTENTIAL OF SOIL HYPHOMYCETES FOR ROOT-KNOT NEMATODES

Dissertation submitted for the Degree of

ifWaittr of ^l)ilo$(opI)p

\ ^ AGRICULTURE (PLANT PATHOLOGY) ^ ^

BY

MQflD. AY£1QB MA?4TQQ

INSTITUTE OF AGRICULTURE ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

1993

DS2146

He (A1I2*') tdught man th<a.t which he Knevif T»ot CThe Qurc»\96:5)

In memory

Of my Late

Grandmother

O/ io/. ,yfl. //aau/ '-yo/ic^'^^-I.Sc. (Ban.) Ph.D. (Alig.), F.L.S.. F.P.S.I. rijjtessor of Botany

n a m r a m w i w y v a"*^ ' •""» i»wiii"*^"»-

Laboratories

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY AUGARH-202002 INDIA Tel: (0571) 25676 (Office)

D a t e d . 1 1 . 1 2 . 9 3

C E R T I F I C f i T E

This is to csY'tify that Mr*. Mohd. Ayoob Mantoo has

prepav'ed this dissertation as required for M. Phil.

(Agricultuj^e) degree of the ftligarh Muslim University, ftligarh

in Plant Pathology, under rny supervision and guidance. He is

allowed to submit this dissertation in partial fulfilment of the

requirements for the degree of M. Phil, in Agriculture (Plant

Pathology).

(M. Wajid Khan)

Research Supervisor

flCKNOWLEDGEMENTS

From the taegmriing to the end of my M. Phil,

programme, suggestions, assistance and encouragement came from

several sources. Foremost among them was that from my supervisor

Prof- M. Wajid Khan, Depai-tment of Botany, A.M.U. , filigarh, who

went all out to help me. I am conscious of rny debb to hirn and

take this ooporbunity to opine my sense of reverence and

gratitude in all earnestness to him for his indefatigable

efforts, scient 11 lat m g suggestions, constant encouragement and

constructive criticism in the conduct of thus programme.

I owe a great deal of unquantiflable gratitude to

Prof. M. Shamim Jairajpuri, Director, Institute of ftgricultur-e

and Prof. Uazahat Husain, Chairman, Department of Botany,

A. M. U. , Oligarh, for providing necessary facilit3E>s.

It 15 a great pleasure to acknowledge the most

generous help from my seniors, Dr. N. A. Onsari, Dr. fl. ft. Khan,

Dr. M. R. Khan, Dr. K. S. Yadav, Messers S. T. Nabi, N. H. Shah,

F. ft. Lone and Z. ft. Makdoomi,

I obediently and dutifully offer my gratitude to

Messers M. H. Siddiqui, B. Khan and ft. H. Wani m particular <\rid

to Ms. L. Khalil, Mr. N. ft. Baba in general for their kind help.

Words are inadequate to expross rny gv^atitude towards

my parents, brothers and the sister Parveena Shaheen, for their

everlasting love and affection, who remained the source of

inspiration to my work.

l i

ftllah a l o n e i s t o be s o u g h t foi" h e l p and on Hirn

a l o n e we d e p e n d .

Mohd. ftyoob Mantoo I n s t i t u t e o f ftgricultur'B A.M. U. , flligarh.

C O N T E N T S

Page No.

1. INTRODUCTION 1

£. REVIEW OF LITERATURE e

3. MflTERIftLS fiND METHODS 43

1. Survey and collection 43

a. Isolation of fungi from soil samples 44

3. Isolation and identification of fungi

associabed with egg masses 44

4. In vitro inoculations ^5

5. In VIVO inoculations 47

(i) Nematode inoculation 47

(ii) Fungus inoculation 48

6. Root penetration 49

7. Soil drench and spray treatment 50

8. Recording of data 50

9. Root-knot disease parameters 51

10. Experimental design and statistical analysis 51

4. LITERATURE CITED 53

INTRODUCTION

Plant parasitic nematodes ar^e heterotrophic

organisms and occur worldwide in all the kinds of agricultural

soils. Depending upon their mode of parasitism and host-parasite

relationships, they are variously classified into different

groups like ecto-, semi-endo-, and endo-parasites. Endoparasitic

nematodes are more damaging and agriculturally important than

other groups. Omong the endo-parasities, root-knot nematodes

belonging to the genus Meloidogyne constitute the major group of

plant-pav^asit ic nematodes of outstanding economic importance.

The root-knot nematodes, known for more than a century have

attracted the attention of most nematologists and plant

pathologists all over the world, fi vast amount of literature is

testimony of their importance as parasites of crop. They at^e

ubiquitous and most widespread causing losses of many crops.

Though they attack almost every type of crop, causing

considerable losses of yield or affecting the quality of the

produce, vegetable crops mostly suffer greatest damage (Sasser,

1980).

The losses caused by nematodes both in quatity of

the yield and quality of the produce are enormous, fl loss of

$£50 million was estimated by Hutchinson e^ al. (1961) to be

due to nematodes. United States Department of Agriculture

(USDfi) estimated an annual crop loss of 37£,335000 dollars to 16

crops (Taylor ig&7). The society of Nernatolog ists (SON)

committee on crop losses led by Dr. Feldrnesser, estimated anriual

losses in U. S. Pi. in the fov m values of the order of

$1,036,374,300 in field crops; $££5,£45900 in fruit crops;

$£66,989, 100 in vegetable crops and *59,ai7,&34 in ornamental

crops due to nematodes <ftnon,, 1971). finnual-crop losses due to

nematodes on worldwide basis was estimated to the tune of *100

billion (Sasser and Frechman, 1987). The crop losses caused by

nematodes has given the grim picture of agricultural economy in

the United States (finon. , 1987).

There ar^e also some reports of crop losses from

India due to plant-parasitic nematodes like ftnquina t r i t i c i. the

seed gall nematode causing a loss of *10 million to wheat crop;

Heterodera a.ver\ae, cereal cyst nematode causing $8 million to

wheat and barley in Rajasthan State alone and Pratylenchus

coffeae. root lesion nematode causing *3 million to coffee.

However, (^&r\ Berkum and Seshadri, 1970). However, above figures

are not adequate to highlight the crop losses caused by

nematodes as only little information is available about crop

losses in the developing countries.,) The situation warrants

application of management measures in order to minimize the crop

losses. Various rneasuv-es that have been in use for management of

nematodes are classified as physical, chemical, cultural,

regulatory, use of host resistance and biological methods.

Hot watev^ trearnent, radiation treatmerit, ultrasonic,

washing processes, seed cleaning, heat treatment viz. steam

sterilization, solarisation and pasturization of soil etc.

<3outhey, 1965, 1978a; Mass, 1987) are physical measures applied

for management of certain categories of plant -parasitic

nematodes. Soil solarization is a recent technique to kill

nematodes but it has certain lirnitst ions and can be effective in

summer season in tropical and subtropical regions of the world

only.

Several reviews have provided detailed and valuable

information on the efficiency of chemical measures for

management of plant parasitci nematodes. (Van Gundy and Mc

Kinny, 1977; Whitehead, 1978; Winfield, 1978; Haq et. al.. , 1983).

fi number of chemicals both soil fumigants and

systemic were successfully used for management of plant-

parasitic nematodes including root—knot nematodes. But some

problems are associated with their application, ft number of them

have been banned, therefore, only a few are marketed. These

chemicals also create pollution causing toxic effects on

beneficial flora and fauna and on men involved in their

manufacturing and use. Their influence or\ the non-target

organisms has caused ecological imbalances which are sometimes

difficult to be restored. Furthermore, the nematode population

increases several months after the use of chemical. The

persistence in the soil, and contamination of ground water are

their otlTev characteristics that reject their usefulness.

Some cultural practices like fallowing, flooding,

ploughing, crop rotation etc. (Br-own, 197S; Noe, ISSG) are also

used for management of plant—parasitic nematodes. Fallowing of

the field results in economic loss. Flooding causes adverse

effect on soil structure and monetary losses to the farmers.

Cj- op rotation though effective in controlling nematodes, yet it

sometimes increases the population of other plant-par^asit ic

species. fls majov" species of root-knot nematodes are

polyphagous, it is difficult to design effective rotation

schemes for their management.

Regulatory methods aim at checking of

dissemination of nematodes (Southey, ig7Qb; Maas, 1987). The

method involves passage and enforcement of quarantine laws

desinged to prevent the spread of particular nematodes into

areas Known to be free. It is essentially not an approach to

manage the nematodes in infested fields. Successful employment

of nematodes resistance requires the manipulation of genetic

systems to transfer resistant genes form a resistant plant to

susceptible and acceptable type. Use of host resistance can be

most simple, practical and economical method for management of

planty parasitic nematodes but development of r^esistant

cultivars is itself a very time consuming and costly affair.

Some cultivates found resistant become susceptible subsequently

due to continuous variatioon in pathogens caused by host

selection pressure and erivirorirnental fluctuations. Bornetirnes,

new races of the nematodes may evolve due to selection pressures

caused by rnono-culturing of the resistant varieties in a

particular area (Jalala, 1986). Though some plants and

cultivars were claimed to be resistant to root-knot nematodes

(Fassuliotis, 1979), yet use of host resistance will require

still more efforts and is still a far dream for plant breeders.

Currently biocontrol measures have become most

significant methods for the management of plant-parasitic

nematodes. The biocontrol of pathogens have become a fascinating

dream and ultimate goal of many concerned with the health of

plants. In the present time due to greater awareness of the

essentility of pollution free environment, biocontrol seems to

be the most relevant and practically demanding approach. (Khan,

1990).

Biological control means different things to

different people. In the broadest sense, it is the natural

phenomenon of population regulation created by the interaction

of the biotic components of an ecosystem. The biological control

encompasses all biologically causes of mortality, including

competition between individuals of the same and the different

species, the effect of host defenses and host resistance, and

the direct or indirect results of attcaks by organisms belonging

to higher trophic levels. More applied biological control

involves the manipulation, conservation and augmentation to

sroecific kinds of orgariisms 3 n order to regulate populations of

undesirable species and thus to prevent or reduce their negative

impacts on human well-being.

Biocontrol is considered skilful rnani Dulat ion of the

biosphere against nematode pests for achieving maximum benefits

but m some cases, it has many difficulties m practical

applications. Other drawback of biocontrol is impatience of

scientists as well as farmer who alkways desire for instant

results.

Nematode egg-parasitizmg fungi have been reviewed

by Stirling et. a_l.. <197a), Nigh et. a^, (1979), Jatala (1986),

Cabanillas et. al.. (1388,1989), Khan (1990), fll-Ha = mi and Raz:ik

(1991), Marban et_ B1^. (199S). Some of these fungi were reported

to attack more than one develeomenta1 stages of nematodes.

The literature cited showed that much attention has

been given to the biocontrol of plant-parasitic nematodes

particularly root-knot nematodes. In biocontrol studies m

India, mdeginous soil hyphomycetes have not been evaluated for

their efficacy as biocontrol agents of root-knot nematodes and

other endoparasitic nematodes. Therefore in the proposed study

for Ph.D., investigations on biocontrol potential of soil

hyphomycetes for root-knot nematodes will be undertaken. It is

planned to conduct in the following experiments.

1. Sui- vey and collection of soil and root samples from fields

infested wibh root—knot nematodes from different parts of the

country.

2. Isolation, identification and multiplication of fungi

parasitic to root—knot nematodes from the collected samoles^.

3. In vitro studies of selected (identified) fungi

(hyphornycetes) for their efficacy as biocontrol agents

under:—

a) Effect of culture filtrates of selected fungi on

hatchability of juveniles of root-knot nematodes.

b) Larvicidal effect of culture filtrates of selected fungi C'rt

root-knot nematode juveniles.

c) Efficacv of the selected fungi as parasites of eggs/egg

masses of root-knot nematodes,

4. Effect of the selected fungi ori the development of root-knot

nematodes and root-galling in cowpea and tomato.

5. Effect of the selected fungi and root-knot nemabode

Meloidogyne .lavanica alone and in combination on plant

growth characters of cowpea and tomato.

6. Effect of the selected fungi (biocontrol agents) on

Meloidogyne .i avanica juvenile's penetration m the host roots.

7. Management trials of selected fungi as biocontrol arients

through soil drenching and spv^aying experiments in pots or

microplots.

REVIEW OF LITERftTURE

Root—l^not nematodes, Meloidogyne spp. , constitute a

major group pf plant-parasitic nematodes affecting crop

production. Thoy are world- wide in distribution and show

extensive host range. Their association with other plant

pathogenic organisms causes disease complexes m a number of

plants. They account for significant losses in yields of food,

feed and fibre? crops. The most frequent species encountered

world- wide Ar^e Me loodogyne incognita, M. .lavanica, M. arenaria

and M. hapla (Sasser, 1977). Pi recently concluded International

Meloidogyne Project (IMP) showed that these four species Br-e

major species of Meloidogyne. fibout 95/. of the populations

identified, through the efforts of IMP were represented by the

aboye mentioned four species of Meloidogyne (Taylor et_ al. ,

1982). The frequency of the species encountered was as follows:

M. inconnita 5i2/4; M. j ay an ica 3 IS; M. hapla BY- and M. arenaria

7%. Four races were differentiated in M. incognita. Race 1

comprised of 7c:"/.; Race £, 13%; Race 3, 13"/. and Race 4, cl'A of 472

populations studied. Within the species M. arenaria, two races

were identified and designated as Race 1 and Race £ (Sasser,

igS£).

Root—knot disease was first recorded by Berkeley

(1355) on glasshouse cucumbers i»i England who named the nematode

= s Vibrios. The prc- sent day name Meloidogyne was giyen by Goeldi

(1887, 189a) and Chitrwood (1949) confirmed it. On the basis of

morphological d i f f ey^ences particularly in the? perineal patterns

of adult females, Chitwood (1949) described four species vis.

Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 19A9;

M. .lavanica (Treub, 1885) Chitwood, 1949; M, arenaria (Neal,

1889) Chitwood, 1949; M. hapla Chitwood, 1949 and orxB sub­

species M. incognita acrita. Since then fv orn tirne to time new

species were discovered, described and added to the species list

of the genus Meloidoqyne. Till 1990, there Are as many as 73

species and L2 subspecies under the genus Meloidoqyne (Eisenback

et al., 1985; Hirschmann, 1986; Golden and Kaplan, 1986; ftbdel

Rahman and Maggenti, 1987; Rammah and Hirschmann, 1988; Golden,

1989; Rarnmah and Hirschmann, 1990).

Sasser (1977) summarized the distribution of

Meloidoqyne species in different parts of the world. Occurrence

of IS species in the United States, 11 species in fifrica, 11

species in Europe and the Mediterranean region, 10 species in

India and Sri Lanka, 9 species in Central and South America, 5

species in South East fisia, Australia and Fiji Islands and 3

species in Canada was included in the summary. In India, root-

knot nematodes were first reported by Barber (1901) or\ tea in

Kerala and named them as Heterodera rad icicola. Out of Ic!

species so far reported in India, Meloidogyne incoqnita and M.

.lavanica are the most widely distributed and attack a wide range

of crops (Khan and Khan, 1985; Haider and Khan, 1988; Khan,

1988).

10

Biocontrol of Nematodes

Plant-parasit ic riernatodes including root-knot

nematodes are managed by different methods which ar-e categorized

as chemical, cultural, biological etc. Biological or biocontrol

in the present time, seems to be practically demanding and the

most relevant approach due to greater awareness of the

essentiality of pollution-free environment (Khan, 1990).

Biocontrol is a natural phenomenon. Plant—parasitic nematodes

share the habitat with other organisms in the soil. The

relationship that is developed m common habitat is a continuous

process and maintains a natural balance m soil biotic

cornmi. nity. However, the efficiency of the organisms varies under

different ecological conditions or as such in the soil (Khan,

1990). Biocontrol has been defined in various ways by a number

of workers. Almost any process occuring naturally or done

artificially by man, which affects the relationship between

organisms in such a way that the natural biological balance is

restored, can be regarded as biocontrol. Recording to DeBach

(1964), biocontrol is defined as "any condition under which or

practice whereby survival or activity of pathogen is reduced

through the agency of any other living organism (except man

himself), with the result that there is a reduction m incidence

of the disease caused by the pathogen. Sewell (1965) defined

biocontrol as "natural or induced, direct or indirect,

limitations of a harmful organism or its effects by another

11

organism or a group of orgarnsrns"-

Swarup and Gokte (1986) defined biocontrol "as the

exploitation of a living organism for reducing the pest

population, with essential approach of trying to restore that

balance of nature that has been upset to the advantage of some

destructive organisms". Barker and Cook (1974) defined

biological control as "the reduction of inoculum density or

disease producing activities of a pathogen or parasite m its

active or doi-mant state, by oriB or more organisms, accomplished

naturally or through manipulation of the environment, host or

antagonist, or by mass introduction of one or more antagonists".

On average fertile field soil contains approximately

10 bacteria, 10 -10 act inomycet es, 10 -10 fungi and 10"^

proto::oa reproductive units per gram. In addition a large number

of fungivorouB nematodes, tardigrades, collembola, mites and

other assorted micro- and meiofauna are also found. Biological

antagonisms are common in soil, and biocontrol has often

occurred under natural conditions (Baker and Cook, 1974). Thus

in general, biocontrol with reference to plant - parasitic

nematodes is a means of promoting organisms that are harmless to

the plants but attack and destroy those ov^ganisms which are

harmful to the plants.

The possibilty of biocontrol of nematodes was first

suggested by Lohde (1874). This study generated interest in

12

many eav^ly scieritists (Kuhn, 1677; Zopf, 1888; Thome, 1927;

Linford, 1937; Linford and Oliveira, 1938; Daddington et_ al. ,

1956; Loewenbev'g et_ al_. , 1959), but organized and detailed

studies on this subject commenced in the I960's only. Now

biocontrol of nematodes has been the subject of several reviews

(Sayre, 1971, 1980; Mankau, 1980; Kerry, 1980; Jatala, 1986;

Morgan-Jones and Rodrigues-Kabana, 1987; Nordbring-Hertz, 1988;

Khan, 1990; Sundei-land, 1990; Galper et. a_l.. , 1991; Leij et. al. ,

1992, Stirling, 1992; Zaki and Maqbool, 199£).

Plant-parasit ic nematodes including i-oot-knot

nematodes a\^B controlled by a number of different kinds of

enemies. These enemies include predacious fungi, predacious

nematodes, and predacious animals; parasites like viruses,

bacteria, r'lckettsias, and fungi (Sayre, 1971; Manaku, 1980).

Organic amendments, use of trap crops and antagonistic plants

also reduce parasitic nematodes biologically.

The literature ctn predacious fungi, endozoic fungi

and fungi parasitic on eggs/cysts is reviewed.

Predacious Fungi

Predacious fungi are also called as nematophagous or

nematode trapping fungi. More than 100 such species have been

reported now (Mankau, 1980; Jatala, 1986). Nematode trapping

fungi were considered as potential biocontrol agents for the

control of nematodes (Duddington, 1957). Nematode trapping habit

15 found in several fungi belonging to order Zoopagales and

13

Moniliales of the class Zygomycetes arid Deuterornycetes

respectively. Predacious fungi have developed various dt^vices to

capture their prey. These devices are grouped into sticky traps

and mechanical traps. The sticky traps are of three types:

(i) Sticky branches e.g. Pact ylel la lobat a

(ii) Sticky rret works e.g. Pirthrobotrys ol i qospora

(ill) Sticky knobs e.g. Pactylella ellipsospora

The mechanical traps B.re of two kinds:

(i) Non-constricting rings e.g. Dactylaria Candida

(ii) Constricting rings e.g. Dactvlel la bernbicodes

(Duddmgton, 1960)

The mechanical traps were found to be more efficient

in reducing nematodes than sticky traps (Cooke, 196c:). Based on

germination of conidia m 16 fungi, predacious fungi were

divided into two ecological groups by Cooke (196A). ft sensitive

group in which conidia quickly give rise to traps, and

insensitive group in which scant trap formation occurs.

Sensitive group are efficient predators and poor saprophytes but

insensitive group are better saprophytes and poor predators.

The existence of fungi that trap and prey on

nematodes was first reported by Zopf (1888). Nematode trapping

fungi were the first entities with which efforts were made in

the beginning to control plant-parasitic nematodes (Drechsler,

1937). He pointed out that some hyphomycetes prey on free living

terricolous nematodes. When nematodes pass through the trapping

14

rings, are captured by the sticky hyphal networks, killed by the

constrict ion of globose structures and body content consumed by

the hyphae originating from these globose stv^uctures. The root-

knot nematode, Meloidoqyne sp. was claimed to be controlled by

adding chopped pineapple tops to the infested soil (Linford,

1937) but disappointing results were obtained when soil was

inoculated with predacious fungi to control Meloidoqyne sp. in

pineapple (Linford and Yap, 1939). Dactylella bembicodes and

flrthrobotrys oliqospora protected the begonias from infection of

root-knot nematodes (Deschiens et_ al. , 1943).

Experiments conducted under indoor conditions by

the application of predatory fungi to control root-knot nematode

Meloidoqyne marioni demonstrated the possibiity of reducing

nematode infection of plants. The increase in the dosage of the

fungi resulted in greater control of the nematodes (Gorlenko,

1956). Various kinds of fungi were reported to infect free

living nematodes isolated directly from forest litter. The

predacious fungi were found to be active in the natural

population of nematodes from a soil habitat (Capstick et_ al • ,

1957). They reported that some nematodes have been trapped by

hyphal snares of the constricting ring type, and some were

infected by spore forming fungi including Harposporium

oxycoracum but more infections were by hyphae or by small

spherical bodies, often associated with hyphae.

15

The constricting ring mechanism of predacious

hyphomycetes that trap nematodes was studied by Muller (1958),

but the chesrnical factors that induce the formation of traps were

demonstrated by Pramer and Stoll (1959) and Feder et_ al. (1963).

The use of nematode—trapping fungi to control root-knot nematode

diseases of tomato and okra was studied by Mankau (1961). He

pointed out that the immediate protection of crops highly

susceptible to root-knot nematodes by the application of

predacious fungi does not seem possible.

The ecology of nematode trapping fungi in the soil

was studied by Cooke (19G£). He found that the decomposition of

sucrose in the soil stimulates AVI increase in both the

population of free living nematodes and the actvity of

indigenous nematode trapping fungi. He further reported that the

fungi were incapable of v^emaining in a predaciously active state

in the absence of 3.n organic energy source other than the

nematodes. Predacious actvity of different species of nematode

trapping fungi in soil has been found to vary in both intensity

and duv^ation, and that constricting ring type of traps are more

efficient in reducing soil nematode populations than fungi

forming adhesive v^eticulate traps (Cooke, 1963). The

significance of ecological characteristics of nematode-trapping

hyphomycetes in relation to biocontrol of nematodes was

discussed by Cooke (1964).

16

Activity of nematode trappers is greatly influenced

by soil conditions such as pH, availablity of nutrients,

temperature and moisture (Mankau, 1968). The morphology of traps

of about 100 species of nematode trapping fungi used for

capturing nematodes have been studied. They vary from simple,

undifferentiated hyphae to -very highly specialized devices

(Sayre, 1971).

Pactylei la ov i paras i t i ca was found naturally

controlling root—knot nematodes even in the presence of a highly

susceptible variety "Lovell" of peaches in Califor-^riia. (Stirling

and Mankau, 1977). Even though the fungus was able to survive

without the riernatode, yet it control led the Meloidoqvne species

(Stirling et_ al. , 1979). Dactylel la arcuata. a nematophagous

hyaline hyphomycete, forms simple, one-celled, lateral, globose

sticky knobs at first but later on multicellular arches, rings

and networks are formed by branching and anastomosis, and then

trap the nematodes (Scheuer and Webstev^, 1990). The biology of

D. meqalospora was studied by Esser et_ al. (1991). The fungus

trapped and assimilated 18 genera of plant-parasitic nematodes;

9 non-parasitic nematodes were also trapped and assimilated by

the fungus (Esser et_ al. , 1991). Two predacious fungi, an

isolate named "Royal 300", of ftrthrobotrys robusta for the

control of Pitylenchus myeeliophaqus on mushrooms (Cayrol et

al.. 1978) and another isolate of flrthrobotrys named "Royal 350"

for the control of Weloidoqyne on tomato (Cayrol and Frankawski,

17

1379) are marketed for commercial use iri France.

In a study on attraction, induction of trap

formation and capturing of plant parasitic- nematodes by 13

nernatophagous fungi, it was observed that the ability of

different fungi to attract tended to increase with increasing

dependence of the fungi on nematodes for nutrition. Induction of

trap formation depended on their motility and on the

concentration of nutrients in the culture medium, fill nematodes

were rapidly captured when traps were present (Jansson and

Nordbring-Hertz, 1980).

The diversity, adaptations and distribution of

nematode destroying fungi, and taxonomic problems encountered in

their study are reviewed by Mankau (1980). He suggested that

fungi may be utilized as alternatives to chemical control after

a more thorough and expanded study of their biology and ecology.

In microplot and greenhouse experiments, it was

found that the reproduction of Meloidoqyne incognita and the

incidence of root galling in corn, were reduced by the addition

of flrthrobotrys conoides and/or green alfalfa. No interaction

was found between the fungus and green alfalfa in the reduction

of the nematode population <fll-Hazmi et_ al. , 198£). Further, the

effect of Q. conoides on M. incognita population densities as

affected by soil ten^peratut^e, inoculum density and green alfalfa

was determined in greehouse experiments. Q. conoides was most

effective in the nematode control, when the fungus was

18

iritroduced into the soil £ weeks before the nematode inoculation

and planting of c o m (Pll-Haarni et_ al. , 19a£) . Culture filtrates

of Rrt hrobot rys oli qospora, Dactylaria brochopaqa and Curvulan a

pallescence inhibited juvenile hatching of Weloidogyne incognita

and Hetev'odera zeae (Walia et_ al. , 1985).

The efficiency of nernatophagous fungus P)rt hr obot rys

dasquptae attacking a range of plant-parasitic nematodes was

studied by Bong et al. (1988). Predatory fungus ftrthrobotrys

irregularis protected against light and moderate infestation of

Meloidoqyne spp. (Cayrol, 1988). Use of nematode-trapping

fungus, ftrthrobotrys tortor against Meloidoqyne spp. on

different substrates in greenhouses was reported by Jawich and

Bochow (1989).

Entraping and assimilation of nematodes by fungi

like flrthrobotrys, Monacrospori um and Dactylaria spp. by

employing constricting rings was briefly reviewed by Sunderland

(1990). The nature of their nematode entrapment and their

biological control potential were also studied (Esser and

Shubert, 1991). The formation of three dimensional sticky

netwoks in flrthrobotrys oli qospora and Monacrospori um cionopaqum

and the capture of nematodes in soil was observed by using

fluorescence microscopy (Jensen and Lysek, 1991). In vitro

studies were carried by Nakasono and Gaspard (1991) to determine

the effectiveness of two nernatophagous fungi, ftrthrobotrys

dactyloides and Dactylei la haptotyle for Meloidoqyne incognita

19

and free living nematodes in soil.

In samples collected from forest, annual and

perennial crops in Minas Gerais (Brazil), the occut^r^Bnce^,

predatory capacity and growth of flrthrobotrys musiformis and 0.

conoides was studied by Naves and Campos (1991). Pria et. al.

(1991) also isolated and identified the predacious fungi from

soil samples collected from various regions of Brazil. In India,

Saxena and Mukerji (1991) surveyed 165 soil 3.rtd vegetation

samples from various sites of Varanasi and found that

nematophagous fungi were distributed in a variety of habitats.

ftrthrobotrvs oliqospora produce extra-cellular

proteases when grown in a liquid culture. The involvement of

protease in the infection and immobilation of nematodes by the

nematophagous fungus was investigated (Tunlid and Jansson,

1991). fl lectin- carbohydrate interaction leads to adhesion of

nematodes to Q. olioospora, which is the first step in the

infection of the nematodes (Tunlid, 199£).

In a study on the formation of conidial traps in

flrthrobotrys oligospora. it was found that the fungus developed

the traps in response to diffusing substances from cow faeces

rather than in response to living nematodes (Dackman and

Nordbring-Hertz, 199£). Trap productoon by nematophagous fungi

(0. dactyloides. ft. olioospora, Monacrospori urn ellipsosporium

and M. cionopaqum) growing from parasitized nematodes was

investigated by Jaffee et. al. (199S).

20

Endozoic -Fungi

In addition to nematode trappers, some endozoic

fungi s^re natural enemies of nematodes. fin endozoic fungus,

Harposporium anqui1lulae was first to be found effective in

checking nematode populations (Lohde, 1874). About 50 endozoic

fungi mostly belonging to Phycomycetes and Deuteromycetes srs

now known to be parasitic on nematodes. Ingestion of spores of

H. anqui11ulae prior to their germination, by plant-parasitic

nematodes was found to be effective for their control (Aschner

and Kohn, 1958).H. arthrosporum was reported to parasitize

nematodes only when its arthroconidla become lodged in the

nematode's oesophagous (Barron, 1979). H. arcuatum was observed

attacking nematodes in farmyard soils collected in Ontario,

Canada. The conidia were readily ingested by the nematodes and

conidiophores emerged through the nematode cuticle at the tail

end (Barron, 1980). H. oxycoraeum, H. cycloides and H. sicyodes

isolated from soil in Varanasi, India were found to be

parasitizing the plant -parasitic nematodes (Srivastava and

Dayal, 1983). The electron microscope study of initiation of

infection by conidia of H. subuliforme was carried out by

Saikawa and Morikawa (1985).

M e n a coniospora, Ar\ endoparasit ic nematophagous

fungus infects nematodes specially at the chemosensory organs

(Jansson and Nordbring-Hert z, 1933). Jansson et. §2,. (1984) gave

a additional information on M. coniospora at different stages of

21

nematode infection using LSM and TEM. M. coniospora conidia

showed diffE?rent patterns of adhesion to the cuticles of

nematodes (Jansson et_ al. . 1985).

Jansson et_ al • (1985) evaluated potential of M e n a

coniospora in reducing root galling caused by root-knot nematode

iri tomato. Experiments conducted to determine whether M.

coniospora, ftrthrobotrys oligospora and ft. flaqrans control the

root-knot nematode, Meloidogyne hapla on alfalfa and tomato,

showed that there was reduction in root galling without any

improvement in plant growth (Townshend et. al. . 1989).

Hirsutella rhossi1lensis produces spores that adhere

to and penetrate the nematode cuticle and assimilate the body

content prior to its emergence and sporulation (Jaffee and Zehr,

1985). The adhesion and infection of Drechmerla coniospora to

the plant-parasitic nematodes was studied and protein was found

to be involved in the adhesion process (Jansson et. al . , 1987).

Mycelial growth and rate of sporulation of Dj;_ coniospora.

Vert 1c1111um balanoides and Harpospori um angui1lulae were tested

in different liquid cultures. Conidia produced in liquid culture

retained their parasitic capabilities (Lohmann and Sikora,

1989). The ultrastructural study of adhesion and initial stages

of infection of nematodes by conidia of D. coniospora was

reviewed by Dijksterhuis et_ al. (1990). The colonization and

digestion of nematodes by D. coniospora was investigated by

Dijksterhuis et. al.. (1991).

22

Catenaria aux i laris arid Nernatophthor£^ qynoph i la are

two important zoosponc fungi involved in biocontrol of plarit-

parasitic nematodes. C. auxi 1 l a n s was observed on females of

Heterodera schachtii (Kuhn, 1877). It also parasitizes females

of H. avenae and Globodera rostochiensis (Kerry, 1S75; Kerry et

al_. , 1976). N. qynoph ilia parasitises H. avenae, H. trif ol ii, \±.

cruci ferae. H. gott mqiana, H. schacht 11 but not G.

rostoch1ensIS (Kerry and Crump, 1977). The ability of N.

qynophila to infect nematodes is dependent upon adequate soil

moisture (Kerry et_ al. . 1980). Roy (1984) reviewed the effects

of nitrogenous soil amendments on the parasitic actvity of C.

anqui 1 lulae c<ri root-knot nematodes. Jaffee (1986) examined the

parasitism of Xi phinema riven and X.. amer icanum by C.

anqui 11 ulae^, Laqenid lum caudaturw. ftphanomyces and Leptol eqnia.

Both nematodes were infected by all fungi. Parasitism by

flphancmyces and Leptoleqnia was imcreased when nematodes were

incubated m BY- soil extract for 4 days before exposure to

fungi.

Voss and Wyss (1990) determined the potential of

Catenaria anqui11ulae as a control agent against several plant-

parasitic nematodes. Variation between strains of the fungus and

its attack to Heterodera schachtii was also studied (Voss et

al. , 199i2). Two of the three isolates (C^, C J Q ) reduced

significantly the number of cysts produced while one isolate

(C^) showed no effect on H. schacht 11 population. In vitro

23

observation on the infection of Meloidoqyne incognita eggs by

the zoosponc fungus C. anqui 1 lulae was reported by Wyss et_ al_.

il33S.) . The embryo was killed withen a few rninutes following

mass aggregation and encystment of the zoospores. Jansson and

Thiman (139£) studied chemotxis of zoospores of C. angui1lulae.

The characterization, entrapment, habitat, hosts,

life cycle and biological control potential of fungi that

utilize zoospores to parasitize the nematodes were briefly

outlined by Esser and Schubert (1963). Three species of

Myzocytiurn viz. M. papi1latum, M. q1utinosporum and M. anomalum

isolated from Ontario, Canada produce zoospores which encysted

directly on the host cuticle before penetration and produce

zoospores (Barron, 1976a), whereas M. hurnicola produced

zoospores that did not attack the nematodes directly but

encysted and produced adhesive buds that atacked and penetrated

nematodes (Barron and Percy, 1975). In Myzocytlum intermedium,

the encysted zoospores germinated by germtubes which penetrated

through the body orifices of the nematodes (Barron, 1976b).

Fungal parasites of eggs/cysts

Several fungal species have been found to be

parasitic on eggs and/or cysts of plant parasitic- nematodes.

Vertici11lum chlamydosporlum, V. falcatum, Cy1indrocarpon

destructans. Humicola qrisea, Fusariurn oxysporum and F. so1ani

were parasitic on cysts of Heterodera schachtii (Vinduska, 1979;

Heijbroek, 1983). Chaetomiurn cochllodes. Exophiala pisciphi la.

Fusarium oxysporum. F. solani, Phytophthora cinnamomi. Pythium

sp. and Trichosporon beiqelii were isolated from young cysts and

24

newly exposed females of Heterodera glycines from soybean fields

in Alabama, U.S.ft- (Ownley, 1383),

fl filamentous, non-sporulating fungus, designated as

Arkansas Fungus 18 <fiRF 18), was isolated from Heterodera

q 1 ycines in Arkansas, U.S.A. It parasitised 89^- of H. glycines

eggs in =cysts when tested in Petri dishes. The fungus also

infected eggs of Meloidoqyne incognita and eggs in the cysts of

H. qraminophila. H. lespedezae. H. leuceilyma, H. schachti i, H.

trifoli i and Cactodera betulae (Kim and Riggs, 1991).

Eggs of root-knot and cyst nematodes Bf^e found to be

parasitised by certain fungi. Destruction of nematode eggs by

Fusarium and Cephalospori urn species was first reported by Lysek

(1963). Later on Vert ici11ium chlamydospori um, V. bulbillosum,

Paecilomyces 1ilacinus, Acremonium bacilosporum etc. were found

to perforate egg shells and enter eggs of nematodes (Lysek,

1966).

The eggs of Heterodera schacht i i were found to be

attacked by flcremoni um strict urn and Fusarium oxysporum in

California sugarbeet fields (Nigh, 1979). Of 14 fungal isolates

evaluated as biocontrol agents isolated from the Meloidoqyne

incognita egg masses, Paecilomyces 1ilac inus was most effective

(Villanueva and Davide, 1984). The infection by F. so1ani on and

within the females, eggs and juveniles of Meloidogyne incognita

was observed by Khan and Hussain (1986). Infected juveniles and

adults were defov^med and thickly covered with fungal mycelium

25

arid chlarnydospores. F. solarn as natuY-^ally present m seal and

may be an impov^tant factor m natural biocontrol of root-knot

nematodes M. .lavanica and M. incognita as it infects the eggs

and larvae of these nematodes.

Vertici11lum ch1amydosporium has been reported not

only infecting Meloidogyne species but developing females of

Heterodera avenae prior to their production stages also (Kerry,

19S0). The A species of Vertici11ium and 2 species of

Paecilomvces have been found as parasites of cysts and eggs of

Heterodera glycines^ Meloidogyne arenaria and M. incoqnita in

Alabama U. S.0. glasshouse studies (Morgan-Jones and Rodriguez—

Kabana, 1984). Out of 15 different fungi isolated from the

different stages of H. a\/eriS(B life cycle, the egg parasite V.

chlamydospor1um was common in young cysts (Dackman and

Nordbring-Hertz, 1985).

The infection of Heterodera avenae and H. schachtii

eggs by six strains of Vertici111um chlamydosporium on water

agar was studied. Dead and immature eggs were most readily

colonised by all strains of the fungus. Eggs of H. avenae were

more susceptible to parasitism than those of H. schachtii

(Irumg and Kevn-y, 1986). The average growth rate, optimum

temperature for growth and production of chlamydospores varied

m these strains. They also varied significantly in their

pathogenecity to H. avenae eggs (Kerry et. al. 1986). However,

ore pathogenic strains of V. ch lamydospor i um are beirig m

26

developed at Rotharnsted Ex per-" i mental Station, U.K., to control

cyst and root-knob nematodes (Carlisle, 1387). Two methods seed

inoculation and paper pot, were used by Coosemaus (1991) for

introducing V. ch lamydospori um into the soil to conty'^ol H.

schacht11. The entomopathogenic fungus V. lecani i has been

tested for utilizing as an biocontrol agent against H. schacht i i

cysts (Hanssler and Hermanns, 1981) and against Globodera

pal 1 ida under field conditions (flvia and Sil^ora, 199£; Crump and

Irving, 199£; Meyer, 199£; Uziel and Sikora, 1992).

Some factors in the development of Vert ici11ium

chlamydospori um and Pasteuria penetrans as biocontrol agents of

cysts and root-knot nematodes were studied by Kerry (1988). Leij

et al. (199S) tested potential control of Meloidoqyne incognita

by V. ch 1 amydospi-irium and P. penetrans, alone and in combination

in a pot experiment. V. chlamydospori um was most effective.

Majority of egg masses were colonized by £. penetrans and both

resulted 9£"/4 population control.

The biocontrol potential of the Vert ici11i um

chlamydospori um for root-knot nematodes has been reviewed by

Cabrera et. at.. (1987) and Leij and Kerry (1991). The effect of

temperature on interactions between V. chlamydospori um and

Meloidoqyne spp. , was demonstrated by Leij et. at. (1995). V.

chlamydospori um required some external nutrients for its

establishment in soil. The colonization of nematode eggs and egg

masses depended on fungal inoculum and on galling caused by

27

nematodes <Leij et_ al. , 19S£)-

Vert ici 11 i urn ch 1 amydospori um parasitises females of

Meloidogyne arenaria (Morgsn-Jones et_ al. 1961), and cysts of

Heterodera glycines (Ginitis et. al. , 1983). It reduces the

population of M. arenaria. by adversely effecting its egg

hatching and inducing juvenile mortality. Both egg shell and

larval cu-ticle aY"-B disv^upted by the fv^r^gus. Ul-trastructural

studies have shown that chitin and lipid layers of egg shell and

basal layer of larval cuticle are disorganised (Morgan-Jones et_

al., 1983).

Strains of Cv1indrocarpon radicicola, Monotorpora

deleae, Catenar ia anqui 11 ulae, Fusari um oxysporum s/av^. lonqius

were tested in laboratory and greenhouse experiments for the

control of Heterodera rostoch iens is. Cylindrocarpon radicicola

1320 and Catenaria anqui11ulae £436 gave the best control of the

nematode (Kondakova, 1973).

For assessing the ability of a soil to suppress

multiplication of the sugar beet cyst nematode and for isolating

the fungus, Cylindrocarpen destructans from the infected

females, was devised by Crump (1987). Surveys of tropical

regions indicated that C. destructans and Ulccladium atrum were

the most commom species associated with root-knot and cyst

nematodes. It has been suggested that multi- cropping systems

may be designed to increase the frequency of occurrence of

microbial species antagonistic to phytonematodes in most

28

tropical regions (Rodr iguez-Kabana and Morgc^n-Jones, 1988).

Dadri and Saleh (1990) tested the impact of sevriral

nernatophagous fungi on Heterodena schacht 11 and Meloj doqyne

lavanica by inhibition of the fungi in field soil and by their

addition to nematode cultures on culture media and m pots.

flcremonium sclerotiqnum, Preussia sp., Vertici11lum

ch I anrydospor i urn and Fusari um so I a m inflected 8B-7c:'/- i±. schacht 11

eggs. The latter two fungi similarly infected the eggs of M..

.lavanj ca. Pis per pi 1 lus versicolor, ft. f urn i gat us, Cyl indrocarpon

ol id 1 urn. Fusarium oxysporum, F. equiseti also infected 25-46"/.

eggs of H. schacht 11 and/or M. .lavanica. Out of six fungal

species tested as biocontrol agents, only two species flcremonium

persicinum and ftsperqi11 us ochraceous were found most effective

against M. .lavan ica (fil- Ha=mi and Razik, 1991). Culture

filtrates of ftcremonium strictum, ftcrophia1ophora fusispora,

fllternaria alternata. ftsperai11 us flavus, ft. niqer and

Penici11lum spinulosuw were found to be inhibitory against M.

incognita (Shabana and Khan, 199£) .

Pythium monospermum^ P. aphanidermatum and

Phvtophthora palmivora were found to be capable of destroying

some non-stylet bearing nematodes through endozoic parasitism by

hyphae from ingested zoospores (Tzean and Estey, 1981). Pythium

tracheiphilum. reduced the population of Meloidoqyne hapla and

Pratylenchus penetrans on lettuce (Gracia, 1991).

29

The perforation in nematode egg shell by

Paeci lornyces 1 i lac in us was first reported by Lysek ( t966) .

Later, infection of eggs, and females of Meloidoqyne incognita

acrita by the fungus was recorded by Jatala et. aj^. (1979). P.

1i1acinus produces the antibiotic P-168 which has a wide

antimicrobial activity not only against fungi, yeast and

gram positive bacteria but also on the eggs, juveniles and

females of nematodes (Isogai et. al. , 1380). The efficiency oF P.

1i1acinus in controlling M. incognita and Globodera pal 1ida was

demonstrated by Franco et. al. (1981) and Jatala et_ al. (1981).

Paecilornyces 1i1acinus was found associated with

developmental stages of Heterodera glycines in soybean field in

Alabama (Gintis et. al. 1983). The parasitism of Meloidoqyne

arenaria eggs and juveniles by P. 1i1acinus was studied by

Morgan-Jones et. al. (1984). They found two species of

Paeci1omyces as parasites of cysts and eggs of H. glycines. M.

arenaria and M. incognita.'The eggs of M. incognita are deformed

by the fungus with the help of diffusable toxic metabolites

(Jatala et. aj.. , 1985; Jatala, 1986).^ Effectiveness of £.

1 i 1 acinus against M. .lavanica on tomato was studied by Croshier

et al. (1985). Davide and Zorilla (1985) reported that P.

1ilacinus significantly reduced the M. incognita population when

applied as soil drench or mixed with substrates such as rice

hulls and rice bran. They also found that nematicide treatment

using isozafos at 3Kg a. i./ha gave about 10"/- higher control of

30

the nematode in the soil than the fungus tr'eat rnenb, Roman and

Podi-^iguez-Marcano (1985) examined the effect of £, 11 I acinus on

the larval populations and root-knot formation of M. incognita

in tomato. The fungus controlled the nematodes and reduced root-

knot formation. Significantly fewer larvae were found m roots

and soil of plants inoculated with the fungus 5 days prior to

nematode inoculation.

Paec11omyces 1i1acinus has demonstrated tremendous

potential as biocontrol agent of nematodes. In case of

Meloidoqyne, Tylenchulus and Nacobbus. the fungal hyphae first

grow m the gelatinous matrix, then form a network around the

eggs and finally penetrate them (Jatala, 19S6). In Panama, £.

111acinus protected tomatoes and potatoes in field conditions

from infection of M. incognita. The fungus treated plots had

higher yield than did the control plots and those treated with

carbofuran (Jatala, 1986). Penici11lum anatolicum a related

fungus, also produces noxious compounds which interfere in the

development of Globodera pal1ida and G. rostochiensis without

parasitizing the females (Jatala, 1988).

The possibility of using Paeci1omyces 1ilacinus

along with the bacterial parasite Pasteuria penetrans for

controlling Meloidoqyne mcopnita in field microplots was

investigated. Nematode control was more effectively obtained

when both organisms were applied together (Dube and Smart,

1987). Similar results were obtained m case of M. .lavanica on

31

tomato by Maheshwari and Mani (1986). The effects of application

of P.. li lacrinus on the development and population of Meloidogyne

5pp., on tomato plants was tested by Rohana et. aj... (1987). The

fungus inhibited the population development of the nematode arid

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

soil. Shahsad and Ghaffar (1987) reported that P. lilacinus

alone and combined with carbofuran reduced root-knot disease of

okra and mung.

Cabanillas et. al. (1988) studied the histology of

the interactions of Paecilomyces 1i1 acinus with Meloidogyne

incognita on tomato. Root galling and giant cell formation were

absent in tomato roots inoculated with nematode eggs infected by

P. 1 i 1 acinus. Hewlett et_ al.. (1988) evaluated the efficacy of P.

1i1acinus alone and in combination with phenamiphos and

ethoprop, for controlling M. .lavanica on tomato. The fungus did

not control the nematode. Plants with M. ASiva.r^ica alone or in

combination with £. 1i1acinus had galling indices of 5.0. The

latter produced lower yields than all other treatments.

Jimenez and Gallo (1988) found that £. 1i1acinus

under glasshouse conditions infected eggs and sometimes females

of M. incognita, M. lavanica and M. arevaria.

Microplot experiments conducted to evaluate the

effects of inoculum level and time of application of

Paecilomyces 1i1 acinus on Meloidogyne incognita to protect

tomato (Cabanillas and Barker, 1939) and to determine the

32

irifluerice of carrier and storage oF £. 1 i ] acinus on tornat-o

against M. incorjriita (Cabanillas et_ al.. , 1989), showed that best

protection against M. incognita was attained with 10 and £0 g of

fungus infested wheat kernels and fungus alone delivered into

soil 10 days before planting, and that greabest suppression of

egg. development occured in plots treated with £. 11 lacmus jn

pellets, wheat gv ain and granules. The effects oF temperatuY^e

on growth of 13 isolates of £. 1i1acinus and on their efficacy

in controlling M. incognita were investigated by Cabanillas et

al. (1989). MaKimurn fungal growth occoured from 54 to 30"C and

least growth was found at 12 and 36 C. fis soil temperature

increased from 16—28 C, both root—knot damage caused by M.

incognita and percentage of egg masses infected by P. 111 acinus

increased. The culture filtrates of £. 1i1acinus were inhibitory

to juvenile hatching of M. incognita (Khan et_ al. 1988) and

showed nematicidal actvity mostly against Heteroderidae

(Meloidogyne and Heterodera) (Cayrol et_ al. 1989) .

The genus Paecilomyces was first described by Bainer

(1907) as a close relative of Penici11lum but differing in the

absence of green coloured colonies and by short cylindrical

phial ides which taper into long neck. Samson (1975) placed

Penici11lum 1ilacmum and some other fungi m the genus

Paecilomyces and proposed Paecilomyces 1ilacinus.

Paeci lomyces 1<»lacinus has been the subject of many

recent researches for evaluating its biocontrol potential

33

against nernat odes. In Philippines, it is being produced

commercially under the trade name of "BIOCON" (Jatala, 1986).

Khan and Esfahani (1990) showed the efficacy of

Paeci lomuces' 1 i 1 acinus for controlling Meloidoqyne .i av an ica on

tomato in greenhouse. Root galling and egg mass production were

greatly reduced. The fungus was severely effective when both

organisms were inoculated simultaneously or the fungus preceded

the nematode in sequential inoculation . ft high percentage of

eggs were found to be infected. £. 1i1acinus also reduced damage

to cowpea caused by M. incognita and Rotylenchulus reni formi s

(Khan and Hussain, 1990). Combination of £. 1i1acinus and castor

leaves was found to be better for reduction in gall index,

second stage juveniles, eggs/egg masses, egg destruction and egg

mass infection of M. .lavanica than either alone (Zaki and

Bhatti, 1990).

Two applications of isolates of Paecilomyces

marquandi i from suppressive Chinampa soils or P_. 1 i lacinus from

Peru, was reported to give better control of tomato root-knot

due to Meloidoqyne incognita than did a single application

(Marban et_ al. , 199£). Siddiqui and Mahmood (1992) found that

out of P. 1ilacinus. ftcroph ialophora fusisphora. Baci1lus

1icheniformis and ftlcaliqenes faecal is used for the control of

M. incognita race 3 and Macrophomina phased ina on chickpea, the

last one was leess effective and that first one was most

34

effect ive.

Organisms with inhibitory metabolites

Besides predators and parasites of plant-parasitic

nematodes, some other organisms have also the capacity of

biochemically harming them by the way of producing toxicants

which are mostly by products of metabolism. Ori the hand, some

microorganisms act as decomposers of organic residues and

release substances deleterious to nematodes. These metabolites

and decomposition products s^r^e quite comparable to nematicides

and thus their producers may be considered as biocontrol agents.

Fungi are known to produce metabolites with

nematicidal, antibiotic or fungistatic potentialites.

Penicil1ium anatolicuw produces a series of compounds that can

alter the permeablity of the egg shell to cause free movement of

noxious compounds into the eggs, causing abortive embryonic

development and complete vacuolation of eggs (Jatala et. al. ,

1985). The inhibitory effects of Gliocladium spp., Trichocladium

spp., Trichurus spp., UIocladium spp. and Drechsiera spp. were

manifested as embryonic obliteration, alteration in the egg

shell makeup and reduced hatching which were attributed to the

actvities of their enzymatic and exopathic diffusible toxic

metabolites (Jatala et. al. , 1985). The toxic substances produced

by Paecilomyces 1ilacinus, also cause above mentioned effects

when come in contact with eggs of nematodes (Jatala et. al.. ,

1985). fill concentrations of culture filtrates of P. 1i1 acinus

35

were found effective m inhibiting hatching of Me] oidogyi'ie

incognita juveniles (Khan et_ al. , 1988). The nematicidal

efficiency of metabolites from 16 strains of fungi and their

specificity in controlling nematodes viz. Mtiloidogyne incogni ta,

Globodera pal 1 ida and Nacobbus aberrans were tested by Jatala et_

al. (1990).

The current knowledge on the metabolites (toxins)

produced by fungi is summerized under two main headings (Cayr-'ol ,

1989).

1. Toxins produced in cultures ori liquid media

£. Toxins produced in cultures on solid gel media

Withm the first category, metabolites active

against larvae and adults include species of Fusariurn,

Tr ichoderma and Pis per gi 11 us ni ger (effective against

Meloidoqyne and Heterodera) . Filtrates of 0. niqer and P..

11lacmus were also active against Meloidoqyne eggs (Cayrol,

1989).

The effect of culture filtrates of Rhizoctonia

solam and Sclerot i um rolfsii on hatching and mortality of

Meloidoqyne .lavanica was reported by flli (1989). The culture

filtrates of flspergill us niqer and Rhiroctonia solam reduced

larval penetration, suppressed nematode reproduction and gall

formation on tomato roots. The culture filtrates of former were

more effective than latter (Khan et. aj_. , 1984; Mukhta et_ al.. ,

1991). The culture filtrates of fl. mqer. ft. fumiqatus, ft.

36

ochr-aceus, Peru ci 111 urn isc''la>"'dicurn. P.. no bat urn and P. utriae

were found responsible for considerable reduction m root and

soil populations, maturatic^n and reproduction of Meloidoqyne

incognita and Roty lenchul us reniforrnis (01 i, 1990), Psperai 11 us

ochraceous culture filtrates were tested against M. incoqniba,

Roty lenchul us reniforrnis and He 1 i cot y 1 ench us spp. , in the

laboratory. The nematodes showed iOO'/- mortality withen two

minutes <fimeen, 1991). The structure and confirmation of

ophiobolin K and 6 epiophiobolin K from Q. ustus as a

nematicidal agent was reported by Singh et. ai.. (1990).

Some characteristics of culture filtrates of

Fusari urn so1ani toxic to Meloidoqyne incognita were reported by

Mani and Sethi, (19a4a). They also reported (in 1984b) that

culture filtrates of F. oxyspo^'i urn f. sp. c i cer i and F. so 1 ar, i

have a profound effect on the hatching and mortality of

Neloidoqyne incognita.

Pimong bacteria, Clostridium butyricum produces a

mixture of formic, acetic, propionic and butyric acids in the

culture filtrates that can be toxic to Tylenchorhynchus martini

(Johnston, 1959) and Desulfo'>^ibrio desulfurirans releases H^S to

control nematodes in flooded rice fields (Rodriguez-Kabana et.

ai. , 1965).

Roots of tomato ^nd cucumber plants were inoculated

with 3£6 bacterial isolates and £8 actinomycetes and then

infected with Meloidogyne incognita. Serrat i a marcescens was

37

found to produce volatile metabolite that wa<3 nenictt ox ic. firnmorna

was found to be the m a m metabolic product involved m nematode

inactivation in in vitno conditions (Zavaleta-Mejla, 1985;.

Influence of organic additives on soil microorganisms

Several kinds of organic additives have been found

effective in suppressing plant-parasitic nematodes (Sayre, 1971;

Khan et_ al. , 1974; Rodr i guez-Kabana e; aJL- 1983;'Haq et_ al. .

1986; fibu-Laban and Saleh, ISSZ; Borah and Phukan, 1992).

Initially it was the finding of Lmford (1937) who claimed a

degree of control of Meloidoqyne by adding chopped pineapple

tops to the infested soil. The multifold increase in the

rhizosphere population of saprophytic fungi with application of

oil-cakes in soil prior to transpi atat ion was studied by Khan et

al. (1974) and Khan et_ al. (1976). Soil treatment with organic

matter not only increased microbial actvities but also caused

enhanced enzymatic actvities (Rodriguez-Kabana et_ a_l_. , 1983).

Soil treatment with ground oil-cakes, DD, DBCP, phorate,

fensulfothion, aldicarb or carbofuran reduced soil population of

plant parasitic nematodes in presence or absence of tomatoes

(Haq et. al. , 1986). Fresh and composted rice straw, cacawate

leaves, saw dust and chicken dung were found to reduce nematode

populations of root-knot and reniform initially but results were

not lasting long (Duhaylongsod, 1988). Effects of organic

amendments for controlling root-knot nematodes were also studied

by Darekar et. al, (1980) and Saifullah and Gul (1990).

38

fi col lagenolyt ic fungus C urm i n q h ame 11a eleqari'5

reduced root galling, egg hatching and immobilized second stage

juveniles of Meloidoovne ,iavanica in tomato plants when collagen

was used as soil amendment <Galper et_ al. , 1991). Four oil-cakes

VIZ. castor, groundnut, honge and neem, in combination wibh

carbofuran gave least root galling and highest yield of okra

(Reddy and Khan, 1991). Omong different organic amendments,

karang, neem and mahuva cakes supported growth and sporulation

of Paecilomyces 1ilacinus. Pressmud and farm yard manure were

least effective while piludi cake was toxic to fungus growth

(Patel et. al, , 1991).

Different animal manures were evaluated and compared

with wheat g r a m medium for mass production, storage and

application of some nematode egg parasitic fungi (fibu-Laban and

Saleh, 199E:). Mycelial growth indices of flcremonium

sclerot iqnum, Fusari urn solani, F. oxysporum, Microascus

triqanosporus. Paecilomyces 1i1acinus and Phoma level 1 lei on

animal manures were similar o^'^ higher than on wheat grains. The

fungi were effective in reducing root galling on glasshouse

tomato plants caused by Meloidonvne .lavanica. The organic

amendments vin. poultry manure, mustard cake, neem cake, proved

to be effective in reducing galls and egg masses in roots of

green gram (Borah and Phukan, 199£).

The role of microbes associated with chicken litter

in the suppression of Moloidoqyne arenaria in amended soil was

39

investigated. Microbial degradation of the egg shell and deaxtli

of Snd stage juveniles was apparent (Kaplan et_ aj^. , 199S).

Bacteiria

Bacteria attacking the nematodes are pathogenic or

saprophytic is not yet distinct, because in most cases the

bacteria have been observed within the body cavity, gut and

gonads (Dollfus, 1346). Sayre (1971) stated that tv-ue nature of

bacterial parasitism on nematodes could be resolved only after

application of Koch's postulates.

Pasteuria penetrans; a bacterium attacking nematodes

has been effectively used as biocontrol agent against root-knot

nematodes. Biocontrol efficiency of this bacterium has been

observed to be varied under greenhouse and field conditions

(Mankau, 1975; Sayre, 1980; Stirling, 1984).

The spore-forming bacterium Pasteuria penetrans,

was originally described as a protozoan Duboscqia penetrans by

Thorne (1940). Then it was renamed as Baci1lus penetrans by

Mankau (1975) when its prokaryotic nature was established by

electron microscopy. Sayre and Starr (1985) later placed it in

the genus Pasteuria.

Pasteuria penetrans has shown excellent

synchronisation with the devlopmental stages of root-knot

nematode and that is why it has proved very effective. Baci11 us

penetrans inhibited the penetration by Meloidoqyne incognita 2nd

stage juveniles into tomato roots (Brown et_ al. 1985).

40

Population of M. incognita were lower in the presence oF B.

penetrans in tabacco at 59 days after planting and were higher

after 91 days in tobacco and at sowing with soybeans. The

pathogenic effect of Baci11 us penetrans on Meloidoqyne incognita

was investigated in concrete bordered plots of tobacco,

soyabeans and Vicia y i 1 losa (Brown et_ al. , 1985). Treatment of

M. .lavanica infested soil with Pasteuria penetrans spores as

well as aldicarb or carbofuran, reduced root galling of tomato

seedlings (Brown and Nordrneyer, 1985). The bacterium Pseudornonas

denitrificans has been found to play an important role in

reducing the population of X i phinema americanum (fldams and

Eichenmuller, 19&3). The effect of Ozotobacter chroococcum VP—5

on the hatching of egg masses and eggs of M. incognita was

investigated. Eggs were more susceptible as compared to egg

masses (Chahal and Chahal, 1986). In in vivo and in vitro tests,

0. chroococcum inhibited the hatching of egg masses of M.

incognita and did not allow the larvae to penetrate into the

roots of host to form galls (Chahal and Chahal, 1988).

Preliminary studies on the potential of Pasteuria

penetrans to control Meloidoqyne spp., were done by Channer and

Gowen (1988), Jaya Raj and Mani (1988), Maheswari and Mani

(1988) and Daudi et_ al. (1990). During interaction between

bacteria and nematodes in the soil, the residual products

released are toxic, antibiotics or inhibitory to plant nematodes

(Sayre, 1988). £. penetrans parasitized larvae of Heterodera

41

species also. Maxirmjrii reductions in the cys:,t population of H.

averiAB and H. zeae were qibtained when spore infested soil was

incubated at 30'"'c before application (Bhattacharya and Swarup,

19S9).

Of five bacterial isolates tested against larvae of

Meloidoqyne incognita, Hfeterodera ca.iani, H. zeae, H. a venae,

the most effective were Baci 11 us subt 11 is and B. purni lis. B.

cerus and two Pseudornonas species were also larvicidal (Gokte . r-

and Swarup, 1989).

The biology, specificity and invasion to nematodes,

survival and movement of spores in a soil of Pasteuria spp. is

discussed by Dickson and Oostendon (1990) and Gowen and flhmed

(1990). £. penetrans with Paecjlomyces 1i1acinus gave a greatest

suppression of Meloidoqyne .lavanica ori mung bean (Shahzad et

al. , 1990). The attachment of P,. penetrans spores tq the root-

knot nematode M. .lavanica m soil and its effects on infect ivity

was demonstrated by Stirling et. al. (1990).

Out of four biocontrol agents including bacteria

VIZ. Baci 1 lus 11 cheh i form is. Pseudornonas m m d o c m a ,

Rcrophialophora fusispora and ftsperqi11 us flavus, of root-knot

nematodes, individually B. 1icheniformis was found to be the

best (Siddiqui and Hussain, 1991). The effect of temperature on

attachment, development and interactions of £. penetrans on

Meloidoqyne ay^eris^r i a. was traced out by Hatz and Dickson (199c:).

The greatest attachment rate of endospores of £. penetrans

42

occurv^ed on Snd fc>baQe juveniles at 50"C.

The attachment of g. penetran'3. on Me I oi dopvne larvae

was found to vary with the concentration of spores in the

suspension (Zaki and Maqbool, 1992). The undiluted concen-crat ion

was most effective and reduced 50% root—knot ii'ifection.

Significant reduction of root-knot nematodes on brinjal and niung

beans and increased plant ^ro^^th were obtained when P. penetrans

was combined with Paecilomyces 111 acinus (Zaki and Maqbool,

133£).

The literature r&viewed shows that in recent years

more attention has been given to corlt 'o endopaf-asi t ic nematodes

particularly root-knot nem^E^todes bv usmq opportunistic soil

hyphornycetes, especially P. 11 1 acinus and V. ch 1 amyd'"'soon ura.

Some success has been achieved. Efforts are m progress to sbudy

various aspects of their occurrance, growth, culturing,

adaptablity and efficiency under various agro-climatic

conditions. Some studies in this direction have been made m

India as well. However, in most studies made in the country,

imported strains of these fungi particularly P. 1ilacinus have

been used. Evaluation of Iindian soil fungi (hyphornycetes) has

not been done for their efficacy as biocontrol agents of root-

knot nematodes and other er^doparasites.

MftTERIfiLS AND METHODS

Materials to be used and methods to be employed to

carry out the proposed stilidy are generalized as follows:

1. Survey and collection

Survey will b^ conducted to collect and soil samples

from fields infested with the root-knot nematodes and grown

preferrably with vegetable crops, in different parts of the

country. For collection of the root samples, top soil will be

removed at the base of a plant showing poor growth and .roots

will be examined for root-knots. Five to ten root samples will

be collected at random from each locality of the area undev-

survey, in polythene bags. Samples will be properly labelled and

brought to laboratoty for-^ further examination.

Soil samples will be collected from the vicinity of

the root system having root-knots, fi number of sub-samples from

a particular field will be mixed together and from the bulk

about £50 g of soil will be taken in polythene bags. Samples

will be properly labelled and brought to laboratory for further

examinat ion.

Root samples will be throughly washed and examined

for the presence of galls and egg masses. Root-knot nematode

species will be identified by using perineal pattern

characteristics (Eisenbabk et. al_. 1981) and conducting North

Carolina host differential test (Taylor and Sasser, 1973).

44

Meloidoqyrie .lavardca (Treub) Chit wood will be selected for the

proposed study. Single egg mass culture (Taylor and Sasser^,

1378) of the species will be raised and population will be

multiplied and maintainedion tomato in greenhouse for use in the

experiments.

2- Isolation of fungi from 5oil samples

Soil samples collected from different parts of the

country from root-knot infested fields will be used for

isolation of fungi. Warcup method will be employed for isolation

of fungi from the samples, ft small amount of soil will be added

to sterlized petriplates with the help of spatula and cooled

potato dextrose agar (PDA) or Mav^tin's rose bengal agar will be

poured m the plates. The plates will be shaken gently in order

to attain uniform distribution of soil particles in the plates.

The petriplates will then be incubated at c:5"C in 3.ri incubator.

Soil hyphomycetes present in the samples growing in culture

plates will be identified, isolated and stocked on PDfi slants

with proper markings for future use in experiments. Sub-

culturing will be done periodically.

3. Isolation and identification of fungi associated with

egg masses

0 few egg masses selected randomly from the roots of

the infected host plants collected during the survey will be

washed throughly and plated on PDA contained in sterli::ed petri

dishes. Potato dextrose agar (PDO) will contain the follov«jing

45

consb ituents:

Peeled and sliced potato - £00 g

Dextrose - £0 \;i

agar - £0 g

Distilled Water - 1000 ml

The medium will be prepared in the laborator^y and £0

ml of it (heated and cooled at 45 C) will be taken m each

sterlized petv^iplate. The egg masses will be plated under

aseptic conditions on a laminar flow bench. Before it, a small

amount of streptocycline ^ill also be added to each petriplate.

The petriplates will be then incubated at £5~C for a week in an

incubator and fungal colpnies devloping around egg masses will

be examined and identified. The fungi apparently penetrating the

eggs and egg masses will be then isolated, multiplied and

maintained on PDR slants for future studies.

4. In vitro inoculations

To study the effect on hatchability of juveniles of

the nematode Meloidoqyne .lavanica. 5 egg masses of the nematodes

will be placed in 5 ml of different concentrations of culture

selected filtrates of fungi contained in sterlized petriplates

(3 cm dia. >. The plates' will be examined after £4, 43 and 7£

hours for hatching of eggs. The number of juveniles hatched will

be counted.

To study the larvicidal effect of culture filtrate

of selected fungi, 100 second stage juveniles (Jg) of root-knot

46

nematode, Meloidoqyrig JLiv aniES. will be placed in 5 ml of

different concentrations,of culture filtrate of selected fungi

contained in sterlized petriplates. The plates will be examined

after £4, 48 and 7£ hours. The number of dead juveniles will be

counted.

To study thi efficacy of some selected fungi as

biocontrol agent, egg miisses of Meloidoqyne .lavanica will be

taken and treated with mei"curic chloride (0.01"/.) for 1-3 minutes

and washed in disti1 led,water repeatedly to remove mercuric

chloride. The surface sterlized egg masses will be then placed

on PDO in petriplates. The fungus to be tested will be

inoculated over these egg masses. The whole procedure will be

carried out at 1 ami nor -flow bench. In some petriplates, only

surface sterlized egg masses will be plated and rio fungal

inoculum will be added. Tt;ie5e petriplates will serve as control.

The petriplates with egg masses, inoculated or uninoculated with

i-i

the fungus will be then i lcubated at £5 C for a week.

fit the end o f the incubation period, the egg masses

will be taken out from the petriplates and examined for the

penetration of the fungus into the egg masses, subsequently

infecting the eggs. For ^determining the percentage of infected

eggs in each egg mass, the egg masses obtained from both

treatments (inoculated or control), will be stained with cotton

blue in lactophenol and each egg mass will be gently pressed

o\er a glass slide to sbperate the eggs. The number of eggs

47

irjfocted by thf? test fungus will be counted under the

microscopic fields and percentage oF infected eggs will be

calculated. The deForrnity and abnormal development of root-knot

nematode juveniles if afiy in the inoculated egg masses, will

also be examined and noted.

5. In vivo inoculations

For m vivo studies, root—knot nematode, f. .lavanica

and some selected fungi kill serve as test pathogens. Cow pea,

Vi nna sinensis L- and tomato, Lycopersican eoculent um Mill.,

will serve as test plants.

(i) Nematode inoculation

For inoculation, nematode inoculum will be obtained

by either of the following two methods:

(a) A la>-"ge number of egg masses of M_. lavanica

collected from the roots maintaining single egg mass culture of

the species will be kept on a double layer of facial tissue

paper supported by a coarse sieve. It will be placed over a

petriplate (9 cm dia.) having sufficient water to touch the

bottom of the support, ft small amount of water will also be

poured over the egg masses. PIfter £4 h, the hatched juveniles

will be collected from the petriplate and used for inoculation

of the seedlings in the experiments. Their number m measured

quantity of the suspension will be determined.

(b) The roots maintaining single egg mass culture of M_.

.Tavanica will be cut into pieces (£-4 cm) after thoroughly

48

washing with i tap water. The pieces will be placed m a 1000 ml

container with £00 rnl faf 0.5"/. sodium hypochlorite (NaOCl )

solution. The tightly capj^ed container will be shaken vigorously

for three minutes. Shkking will partially dissolve the

gelabmous matrix, thus freeing eggs from the egg masses. The

liquid suspension of eg^s will be poured through a .SOO-mesh

sieve, nested upon a 50O-rnesh sieve- Eggs suspended in the

agitated solution will pass through the £00-mesh sieve and will

be collected on the 500-mesh sieve. Eggs will be washed free of

residual NaOCl solution under a slow stream of tap water. The

concentration of eggs pfer milliliter will be standardised by

counting the eggs from ten, 1 ml samples and the average number

will be used to represent the number of eggs per ml.

For inoculation, depending upon the inoculum

density, volume of the suspension containing eggs or second

stage juveniles will be taken in pipette. Roots of seedlings (3-

4 week old) will be partially exposed carefully removing the top

layer of soil. The suspension will be poured uniformly on the

exposed roots. Then, the roots will be covered with same soil

and light watering will be done.

(ii) Fungus inoculation

Pure cultures of the selected fungi will be

maintained in the culture tubes, containing PDA. The fungi will

be grown on Czapek's liquid medium in Erlenmeyer flasks. The

sterilized medium in the flasks will be inoculated V'jith the

49

desired fungus and the? flasks will be incubated in an incubator

at £5 C for a week.

After the intubation period, the mycelial mat will

be removed and washed in distilled water to remove the traces of

the medium. Then it will be gently pressed between sterile

blotting papers to remove the excess amount of water. Inoculum

will be prepared by shaking 10 g fungal mycelium in 100 ml of

sterilized distilled water and blending it for 30 sec. in a

waring blender (Stemerding, 1963). In this way each 10 ml of

this homogenate will coV-itain 1 g of the fungus. The desired

amount of the suspensibn will be added to roots exposed by

removing the top layer of the soil. The roots will be covered

with the soil again. Cane will be taken to keep the inoculated

pots moist for 3-4 days for the stabilization of the fungus.

6. Root penetration

For studying the effect of the selected fungi on

root penetration by juveniles of M. .lavanica in their presence,

the experiment will be set up by adding suspension of the

biocontrol agents (selected fungi) and M. .lavanica as given

above. One replicate from each treatment will be uprooted after

every four days of inoculation. Th^ roots will be washed,

labelled and taken to laboratory for examination. The roots will

be stained with acid fuchsin in the laboratory and cleaned in

lactophenol. The number of penetrated juveniles or the other

development stages of the? nematode in the roots will be counted

50

under the microscope.

7. Soil dr-ench and spray treatment

Efficacy of the selected soil fungi found to be

effective as biobontrol agents in earlier experiments will be

tested for their application as soil drench and spray

treatments. The fungus inoculum will be applied on the soil

surface in quantities sufficient to wet 10-15 cm of the soil in

the microplots or £'4" clay pots. This treatment will be applied

before or after sowing of cowpea seeds and tomato seedlings. The

nematode suspension will be added in the soil at the primary

seedling stage. Uninoculated plants will serve as control.

To study the efficacy of biocontrol agents by

spraying the fungus, the inoculum will be sprayed on the host

plants in the microplots or £4" clay pots. The nematode

suspension will be added in the soil after one week of spraying.

Uninoculated plants will feerve as control.

8. Recording of data

fit the end of the experiments, plant growth

parameters and parameters related to root-knot disease on the

host will be considered. Plants will be uprooted after 60 days

of inoculation and the roots will be thoroughly and gently

washed. The length (in cm) and fresh and dry weights <in g) of

shoot and root will be determined separately. Before weighing

the plants for weight, excess amount of water will be removed by

putting shoot and root between blotting sheets. For dry weight,

51

o shoot and root will be dried in 3.ri oven at 60 C and weighed,

3. Root—knot disease parameters

Before drying the plants, roots of plants from the

treatments will be examined. The number of galls and egg masses

present will be counted. Root—gall iridex (GI) and egg mass index

(EMI) will be rated on 0-5 scale of Taylor and Sasser (1976) as

given below:

1_ = O No galls, no egg mass

£ = 1-10 galls/egg masses

3 = 11-30 galls/egg masses

4 = 31-100 galls/egg masses

5 = Pibove 100 galls/egg masses

The percentage of egg masses infected with the

fungus added as biocontrol agents will be determined. Similarly,

percentage of eggs in each egg mass infected by the fungus will

also be determined by exafnining the eggs under the microscope.

Root population and soil population of the nematode

will also be determined by the standard methods.

10. Experimental design and statistical analysis

Pots or pet^'i plates in the experiments will be

arranged according to the complete randomized block design

(CRBD). The data will be subjected to Analysis of Variance

(RNOVft) and L.S.D. will be calculated to determine the

significance between the treatments.

52

The materials and methods described above will be suitably modified or changed during the course of investigations, whenever felt necessary.

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Brown, B. M. and D. Nordrneyer (1985). Synergist jc reducb K-ICI in root galling by Me] oidogyne .lavanica with PastRKrj a penotraris and nematicides. Revue ds- Nernatologie 6(3):£85-£a6.

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Cabrera, R.I., J.P. Latge, D. Dominguez and Y.G. Gonzalez (1987). The fungus Vertici111um chlamydospora um m biological control of the phytonematodes of the genus Meloidogyne in guava orchards, Psid ium qua.iava. Ciencia Teenica el la Ogricultura, citricos y Otros Erutales 10(4):79-87.

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