Biological control of plant parasitic nematodes by fungi: A review

Bioresource T~vhnologv 58 (1996) 229-239 Copyright © 1997 Elsevier Science Limited Printed in Great Britain. All rights reserved I)960-8524/96 $15.00 ELSEVIER

PII:S0960-8524(96)00122-8

BIOLOGICAL CONTROL OF PLANT PARASITIC NEMATODES BY FUNGI: A REVIEW Zaki A. Siddiqui* & Irshad Mahmood Department of Botany, Aligarh Muslim University,Aligarh 202002, India (Received 28 August 1996; accepted 10 September 1996) nematodes, insects, mites and some invertebrates, have been found to parasitize or prey on nematodes (Stirling, 1991). Of the microorganisms that parasitize or prey on nematodes, fungi hold an important position and some of them have shown great potential as biocontrol agents (Jatala, 1986; Stirling, 1991). Fungi may contribute up to 80% of the total microbial biomass in many soils (Clark & Paul, 1970; Shields et al., 1973). In nature, fungi continuously destroy nematodes in virtually all soils because of their constant association with nematodes in the rhizosphere. The fungal antagonism consists of a great variety of organisms which vary considerably in their biology and taxonomy and play a major role in recycling the carbon, nitrogen and other important elements from the rather substantial biomass of nematodes. Some 70 genera and 160 species of fungi have been found associated with nematodes (Qadri, 1989), but only a few of them are successful biocontrol agents. Jatala (1986) described 16 desirable attributes of a successful biocontrol agent, but according to Kim and Riggs (1992) the characteristics of good biological control agents are: (i) highly parasitic to nematodes, (ii) host range primarily nematode spp. but not pathogenic to crop plants or higher animals, (iii) growth at suitable pH and temperature ranges, (iv) growth on artificial media, (v) indentifiable and with mode of action known, (vi) can be formulated in usable form; competition with other soil microorganisms should be considered.

Abstract

Of the microorganisms that parasitize or prey on nematodes or reduce nematode populations by their antagonistic behaviour, fungi hold important positions and some of them have shown great potential as biocontrol agents. Fungi continuously destroy nematodes in virtually all soils because of their constant association with nematodes in the rhizosphere. A large number of fungi are known to trap or prey on nematodes but the most important genera include Paecilomyces, Verticillium, Hirsutella, Nematophthora, Arthrobotrys, Drechmeria, Fusarium and Monacrosporium. Application of some of these fungi has given very interesting results. There is an urgent need to develop some easy technologies for formulation and mass production of fungi at a commercial scale for fieM application. Some of these fungi may be used in integrated nematode management programmes despite some obstacles. Copyright © 1997 Elsevier Science Ltd. Key words: Biological control, fungi, plant parasitic nematodes, production and formulation of fungi. INTRODUCTION

De Bach (1964) defined biological control as "The action of parasites, predators or pathogens in maintaining another organism's population density at a lower average than would occur in their absence". However, according to Stirling (1991) biological control is "A reduction of nematode populations which is accomplished through the action of living organisms other than the nematode-resistant host plant, which occurs naturally or through the manipulation of the environment or the introduction of antagonists". Kfihn (1877) was the first to observe the parasitism of females of Heterodera schachtii by a fungus. Later, in 1881, he named this fungus Tarichum auxiliare (Ktihn, 1881). Since then a large number of organisms, such as fungi, bacteria, viruses, predatory

FUNGAL BIOCONTROL AGENTS OF NEMATODES

Some important genera of fungi that are known to reduce nematode multiplication by parasitism, predation or antagonism are Tarichum (Kuhn, 1877); Entomophthora, Isaria (Baunacke, 1922); Cylindrocarpon (Goffart, 1932); Aniriopsis. Colletotrichum, Margarinomyces, Monotospora, Penicillium, Phoma, Phialophora, Pseudeurotium (Van der Laan, 1953; 1956); Verticillium (Bursnali & Tribe, 1974); Cephalosporium (Willcox & Tribe, 1974); Catenaria

*Author to whom correspondence should be addressed. 229

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Z.A. Siddiqui, I. Mahmood

(Tribe, 1977); Paecilomyces (Jatala et al., 1979); Nematophthora (Kerry & Crump, 1980); Exophiala, Neocosrnospora (Morgan-Jones & RodriguezKabana, 1981); Chaetomium, Gliocladium, Stagnospora, Thielavia (Rodriguez-Kabana et al., 1981); Fusariurn, Pseudopopulospora (Godoy et al., 1983); Arthrobotrys (Villanueva & Davide, 1983); Chaetopsinea, Diheterospora, Monocillium, Olpidium, Rhizopus, Trichoderma (Grant & Elliott, 1984); Humicola (Vinduska, 1984); Aphanomyces, Lagenidium, Leptolegnia (Jaffee, 1986); Meristacrum, Myzocytium, Phytophthora, Trichosporan (Jatala, 1986); Monacrosporium (Mankau & Wu, 1985); Microdochium (Dackman & Nordbring-Hertz, 1985); Trichotheciurn (Swarup & Gokte, 1986); Dactylella, Harposporium, Metarhizium (Morgan-Jones & Rodriguez-Kabana, 1987); Cladosporium (Roessner, 1987); Ulocladium (Rodriguez-Kabana & MorganJones, 1988); Dactylaria (Jansson & NordbringHertz, 1990); Epicoccum, Scytalidium (Meyer et al., 1990); Acremonium, Preussia (Qadri & Saleh, 1990); Acrophialophora (Siddiqui & Husain, 1991); Allomyces, Coniothyrium, Cystopage, Macrobiotophthora, Rhopalomyces, Stylopage (Stirling, 1991); Mortierella, Trichocladium (Hay & Skipp, 1993); Botryotrichum (Dos Santos et al., 1993); Drechmeria, Hirsutella, Nernatoctonus (Timper & Brodie, 1993); Aspergillus (Zuckerman et al., 1994) and Pleurotus (Hibbet & Thorn, 1994). However, only a few have a significant role in nematode control. The main aim of biological control is to increase the natural enemies of nematodes in the soil so as to reduce nematode density. Microorganisms that can grow in the rhizosphere provide the front line defence for roots against pathogen attack and are ideal for use as biocontrol agents (Weller, 1988). Several comprehensive texts and reviews have appeared in quick succession on this aspect (Kerry, 1984; Swarup & Gokte, 1986; Jatala, 1986; MorganJones & Rodriguez-Kabana, 1987; RodriguezKabana, 1991; Stirling, 1991). The present review examines the role of fungi in nematode management, production and formulation of some of these fungi, development of practical control systems, conclusion and future prospects. Mycorrhizal fungi also have the ability to reduce nematode multiplication, but they will not be discussed here as they have been dealt with separately in another review (Siddiqui & Mahmood, 1995a). The fungi involved in the biological control of nematodes are broadly of three types; i.e. predator, parasite and antagonist. These fungi can be grouped into (i) endoparasitic fungi, (ii) predacious fungi, (iii) opportunistic fungi.

Endoparasitic fungi The endoparasitic fungi are often obligate parasites and have a limited saprophytic phase. They produce almost no mycelium in soil and complete their lifecycle within the body of their hosts. The

endoparasites of nematodes show considerable diversity. The endoparasite with encysting zoospores belong to the Chytridiomycetes and Oomycetes and their infective propagule is a flagellated zoospore. Endoparasites with adhesive spores are unique because the infection process is initiated when condia adhere to a nematode cuticle. The adhesive nature of the spore differs to some extent between genera. The ability of conidia of some species to attract nematodes (Jansson, 1982a, b) has been considered of ecological significance for these fungi. However, one species in the genus Harposporium produces conidia which initiate infection by lodging in the buccal cavity or the gut of the nematode host. However, plant parasitic nematodes are unable to ingest these conidia.

Importance of endoparasitic fungi in biocontrol Zoosporic endoparasitic fungi depend on free water for their activity, which limits the effectiveness of these fungi in biocontrol. Attempts to produce endoparasitic fungi with adhesive knobs in large quantities for introduction into soil may not be successful because of their limited growth in culture. Moreover, their poor competitive saprophytic ability, and the susceptibility of their spores to mycostasis, will also make it difficult to establish them in a new environment. Similarly, endoparasites with ingested conidia are not suitable as biocontrol agents. Assessment of potential of these fungi in biocontrol has not received much success, as in the case of Nematoctonus (Giuma & Cooke, 1974), but use of Drechmeria coniospora gave positive results (Townshend et al., 1989). Another fungus, Hirsutella rhossiliensis, has shown the ability to suppress nematode density (Tedford et al., 1993; Timper & Brodie, 1994), although variability among isolates of this fungus was observed (Tedford et al., 1994). Two zoosporic fungi, Catenaria auxiliaris and Nematophthora gynophila, have a role in regulating population dynamics of Heterodera schachtii and H. avenae in some soils (Crumpet al., 1983; Kerry et al., 1980). Some other endoparasites which have some role in biocontrol are Acrostalagmus, Harposporium, Myzocytium and Haptoglossa, etc. (Morgan-Jones & Rodriguez-Kabana, 1987).

Predacious fungi There are more than 50 species of predacious fungi which capture and kill nematodes in soil, decaying organic matter and other places. The nematodetrapping habit is found in several genera of Hyphomycetes and some species may be found in Zoopagales. With age, these fungi exhibit reduction in nematode-trapping efficiency. For effective control, it is necessary that their limited period of activity coincides with the period of nematode invasion of crop roots. Peak fungal activity is reached only 12-15 days after introduction. A number of attempts have been made to assess their potential as

Control of nematodes by fungi: a review biocontrol agents of nematodes. It is believed that the activity of these organisms might be stimulated by the addition of organic matter to the soil. Predacious fungi form different types of traps, such as adhesive branches, adhesive-network traps, adhesive knobs, non-constricting rings and constricting rings, to capture nematodes.

Importance of predacious fungi in biocontrol Predacious fungi have been shown to be poor competitive saprophytes and are susceptible to antagonism from other soil fungi (Cooke, 1964; Mankau, 1962). These fungi are also unable to overcome soil fungistasis. Predacious fungi have an inability to compete over a wide range of soil conditions and are sensitive to changes in the environment. Moreover, sufficient growth of these fungi is difficult to obtain to affect nematodes in the field conditions. The chances of successful colonization and exploitation of predacious fungi by introduction in a particular soil microhabitat are small (Cooke, 1968). Failure to achieve any measurable improvement in crop yield following inoculation of these fungi may have been due to the fact that maximum fungal growth did not coincide with juvenile emergence. Trials conducted with predacious fungi to control nematodes have mostly given negative and inconclusive results. Experiments with Dactylaria thaumasia along with organic matter and Arthrobotrys spp. to control soil populations of Globodera rostochiensis had no effect on yield and nematode population (Duddington et al., 1956). However, use of Arthrobotrys spp. to control nematodes gave some positive results (Slepetiene et al., 1993; Vouyoukalou, 1993; Dias & Ferraz, 1994), while the application of Dactylella and Monacrosporium to nematode-infested soil also induced some interesting results (Mankau & Wu, 1985). Despite the poor performance of some predacious fungi as biocontrol agents there are some examples of their success. Cayroi et al. (1978) have developed a commercially prepared isolate of Arthrobotrys sp. to protect mushrooms against Ditylenchus mvceliophagous, and this gave about 40% control when used with compost. Another commercial preparation of an Arthrobotrys isolate gave good protection against root-knot nematodes on tomato (Cayrol & Frankowski, 1979). These results give better hope for their use in biocontrol of nematodes.

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gal colonization like the egg masses of root-knot nematodes. Fungal growth of these fungi is known to be enhanced in the rhizosphere. The nematode cysts and eggs released into soil are highly vulnerable to deterioration and colonization. Once in contact with cysts or egg masses, these fungi grow rapidly and eventually parasitize all eggs that are in early embryonic developmental stages. Apparently, when juveniles are formed the parasitic activities of these fungi are generally reduced. It is generally believed that these fungi have a better role in biocontrol of nematodes than the two earlier groups discussed. Although large numbers of opportunistic fungi are known, Paecilomyces lilacinus and Verticillium chlamydosporium have been studied by many workers. Because of the importance of these fungi, the recent work on these fungi in biocontrol has been summarized in tabular form (from 1991 onwards, Tables 1 and 2).

Paecilomyces This fungus is primarily a saprophyte, being able to compete for and use a wide range of common substrates in soil (Domsch et al., 1980). In the laboratory test this fungus infects eggs of M. incognita and destroys the embryos within 5 days (Jatala, 1986). The infection process begins with the growth of fungal hyphae in the gelatinous matrix and eventually the eggs of the nematodes are engulfed by the mycelial network. The colonization of eggs appears to occur by simple penetration of the egg cuticle by individual hyphae aided by mechanical and/or enzymatic activities (Jatala, 1986). The serine protease produced by P. lilacinus might play a role in penetration of the fungus through the eggshell of the nematodes (Bonants et al., 1995). This fungus penetrates eggs of Meloidogyne spp. at a much faster rate than it does those of Globodera and Nacobbus. Laboratory experiments indicate that P. lilacinus grows well at temperatures between 15 and 30°C, with the optimum being between 25 and 30°C. Its adaptability to a wide range of soil pH makes it a rather competitive organism in the agricultural soils. Moreover, P. lilacinus is compatible with many fungicides and nematicides (Villanueva & Davide, 1983). Paecilomyces lilacinus gets established in the soil, grows and disseminates quite rapidly and, within a short period of time, becomes the dominant species where it is applied. Recent studies clearly indicate that P. lilacinus is a successful biocontrol agent for nematodes (Table 1).

Opportunistic fungi Opportunistic fungi can colonize nematode reproductive structures and have the ability to deleteriously affect them. Nematodes belonging to the Heteroderid group and at the sedentary stages of their life-cycle are vulnerable to attack by these fungi either within the host roots or when exposed on the root surface or within the soil. Obese females/cysts become increasingly susceptible to fun-

Verticillium Verticillium chlamydosporium has been shown to be capable of preventing egg hatching of M. arenaria and to colonize eggs by hyphal penetration (Morgan-Jones et al., 1983). Both eggshell and juvenile cuticle were found to be physically disrupted, and fungal hyphae readily proliferated endogenously within eggs and juveniles. The main type of destruc-

Z. A. Siddiqui, L Mahmood

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Table 1. Effect of Paecilomyces spp. on nematode reproduction and plant growth

Fungus

Nematode

Effect of fungus on nematode and plant growth

References

P. lilacinus

M. javanica

P. lilacinus P. lilacinus

M. incognita R. reniformis M. javanica

P lilacinus

M. javanica

Stephan et al. (1991) Vicente et al. (1991) Walia et al, (1991) Zaki & Maqbool (1991)

P. lilacinus

M. incognitaacnta

P. marquandii

Meloidogyne spp.

P. lilacinus

M. incognitarace 3

P. lilacinus

M. incognita race 3

P.. lilacinus

M. incognita

P. lilacinus

M. incognita

P. lilacinus

M. incognita R. reniformis

P. lilacinus

Meloidogyne spp.

P. lilacinus

R. reniformis

P. lilacinus

M. incognitarace 3

P. lilacinus

M. javanica

P. lilacinus

M. incognita

P. lilacinus

M. javanica

P. .lilacinus

M. javanica

P. lilacinus

R. reniformis

P. lilacinus

M. incognita

P. lilacinus

Heterodera cajani

P. lilacinus

M. javanica

Fungus reduced number of nematodes on cucumber. Reduced number of nematodes on water melon. Fungal treatment resulted in better top growth of okra. Fungus at 2 g/pot being most effective when used 1 week before nematode inoculation. Medium and high doses as seed dressings significantly reduced root galling. This fungus is one of the natural soil organisms that contribute to nematode control. Fungus treatment caused greater reduction in nematode multiplication than seed treatment by ascorbic acid on chickpea. P. lilacinus caused greater reduction in nematode multiplication than Bacillus licheniforrnis on chickpea. There was a decrease in number of galls, egg/egg mass, final soil population in plants treated with fungus. Reduced nematode population on brinjal. Significantly more and heavier fruits were obtained when fungus was added 1 week before planting of pepper Shoot weight of brinjal was increased by 17% with fungus treatments. P. lilacinus was better than green manuring in reducing nematode multiplication on pigeonpea. Treatment with this fungus was better than Bacillus subtilis in reducing nematode population on chickpea. Significantly control gall formation on tomato and okra. Controlled nematode population and increased plant growth. Suppressed 65-83% nematodes on tomato and aubergine. Four grams fungus per kg soil was found to be the optimum dose for the reduction of nematodes. This fungus had detrimental effects on nematodes both in greenhouse and field conditions. P. lilacinus caused greater plant growth than Bacillus subtilis. P. lilacinus was less effective than Verticillium chlamydosporium in reducing nematode multiplication on pigeonpea. P. lilacinus along with V.chlamydosporium and Trichoderma harzianum caused greater reduction in nematode multiplication in chickpea.

Khan et al. (1992) Marban-Mendoza et al. (1992) Siddiqui &Mahmood (1992a) Siddiqui & Mahmood (1992b) Pandey & Trivedi (1992) Trivedi (1992) Vicente & Acosta (1992) Zaki & Maqbool (1992) Mahmood & Siddiqui (1993) Siddiqui & Mahmood (1993) Parveen et al. (1993) Ekanayake&Jayasundara (1994) Ibrahim(1994) Zaki (1994)

Walters & Barker (1994) Gautam et al. (1995) Siddiqui & Mahmood (1995b) Siddiqui et al. (1996)

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Control of nematodes by fungi: a review

tive activity is thought to be enzymatic disruption and physiological disturbances brought about by biosynthesis of diffusable toxic metabolites (Morgan-Jones & Rodriguez-Kabana, 1985). Verticillium chlamydosporium is reported to secrete several proteases (Segers et al., 1994). It hydrolyses proteins from the outer layer of the eggshell of M. incognita and exposes its chitin layer. Several isolates of V. chlamydosporium and V. lecanii secrete proteases which are pathogenic to nematodes, while plantpathogenic species of Verticillium do not secrete proteases (Segers et al., 1994). Recently Verticillium has been used in nematode control by a number of workers and gave successful results (Table 2). ½rticillium lamellicola and V. leptobactrum were also effective parasites of the 14 taxonomically diverse fungi tested by Godoy et al. (1982), while V. lecanii was found to be a promising parasite of mature eggs of Globodera pallida (Uziel & Sikora, 1992).

Verticillium chlamydosporium is a parasite of cyst and root-knot nematodes (Freire & Bridge, 1985), but can serve as a saprophyte in the absence of a nematode host. This fungus is one of the main causes of the natural decline of cereal cyst nematode populations under cereal monoculture (Kerry et al., 1982). The fungus can reduce nematode populations more than 90% when introduced into the field soil (De Leij et al., 1993b). It is promising that a combination of this fungus with a nematicide did not restrict fungal establishment in the soil or affect nematode control by the fungus. Miscellaneous opportunistic fungi Crump (1987) suggested Cylindrocapon destructans was an important parasite of nematodes, with some interesting results (Vinduska, 1984; RodriguezKabana & Morgan-Jones, 1986). It was the most common parasite of Heterodera schachtii in England

Table 2. Effect of Verticillium spp. on nematode reproduction and plant growth

Fungus

Nematode

Effect of fungus on nematodes and plant growth

References

V. chlamydosporium

Meloidgyne s p p .

De Leij & Kerry (1990)

V.. chlamydosporium

H. schachtii

V. chlamydospofium

M. arenat4a

V. chlamydosporium

H. schachtii G. pallida

V. chlamydosporium

M. incognita

V.. chlamydosporium

M. incognita M. javanica M. arenaria H. schachtii

Fungus reduced multiplication of all the four species on tomato plant when 2000 propagules/g of the fungus were added to the soil in the pot test. Fungus caused about 75% control of nematodes. V. chlamydosporium isolate was effective against M. arenaria to reduce nematode multiplication. Caused 75 and 76% reduction in eggs of first generation of H. schachtii and G. pallida, respectively. Establishment of fungus in soil was dependent on the initial inoculum of fungus used. Control with V.chlamydosporium was approximately the same for the three nematode species. Fungal parasitism had influence on nematode reproduction. Nematode control was greater in peaty sand than in other soil types. Control of nematodes was greater in soil layers that were well aerated than those likely to be poorly aerated. Young white cysts were colonized to 23% with this fungus. This fungus had little impact on root-knot nematode population. Treatment with V.chlamydosporium was more effective than P lilacinus in reducing nematode multiplication. Use of V.chlamydosporium with P lilacinus and Trichoderma harzianum was best in reducing nematode multiplication on chickpea. V. chlamydosporium with Trichoderma harzianum and Glomus mosseae caused highest reduction in nematode multiplication on pigeonpea.

V. chlamydosporium V. chlamydosporium

M. incognita M. hapla

V. chlamydosporium

M. incognita

Verticillium spp.

Heterodera trifolii

V. chlamydosporium

Meloidogyne spp.

V. chlamydosporium

11. cajani

V. chlamydosporium

M. javanica

V. chlamydosporium

H. cajani

Crump (1991) De Leij & Kerry (1991) Crump & Irving (1992) De Leij et al. (1992a) De Leij et al. (1992b) Muller (1992) De Leij et al. (1993b) De Leij et al. (1993a) Hay & Skipp (1993) Mertens & Stirling (1993) Siddiqui & Mahmood (1995b) Siddiqui et al. (1996) Siddiqui & Mahmood (1996)

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Z. A. Siddiqui, I. Mahmood

and has had a dominant role in the decline of its population. Gliocladium spp. has performed well in controlling nematode population and has the ability to invade females (Hay & Skipp, 1993). Different species and biotypes of this species vary in their efficacy in control of nematodes. Species of Fusarium, Exophiala and Acremonium were also found consistently associated with nematodes, and in some instances reduced nematode multiplication. Conversely, the level of parasitism of these fungi is generally low and some species of these fungi are also plant pathogenic. MASS PRODUCTION OF FUNGI

Endoparasitic fungi generally have poor growth outside the nematode host (Barron, 1977). Members of the Deuteromycetes, however, can be grown on artificial media, but attain only minimal growth (Durschner, 1983). Liquid culturing of fungi for mass production of spores and mycelium has often been considered best for biological control (Papavizas et al., 1984). Cornmeal and potato dextrose media have been advised for mass culturing of endoparasitic fungi such as Drechmeria coniospora, Verticillium balanoides and Harposporium anguiUulae (Lohmann & Sikora, 1989). Production of large amounts of these fungi has an advantage as a biocontrol agent because they do not rely on an energy source, as do egg-parasitic and nematode-trapping fungi. However, the pathogenicity of conidia produced in artificial medium was found lower than that of the conidia from parasitized nematodes (Lohmann & Sikora, 1989). Crump and Irving (1992) used wheat, bran, barley grain and wheat straw along with coarse sand for the production of V. chlamydosporium. De Leij et al. (1993a) produced inocula of V. chlamydosporium on a moist mixture of sand and milled barley grains (1:1 v/v). The inoculum of V. chlamydosporium produced in the laboratory is sufficient for experimental purposes, but for commercial use fermentation technology has to be advanced to produce largescale inocula (Kenney & Couch, 1981). Jatala (1981) suggested culture of P. lilacinus on cereal grains for field application, but grains are expensive. Sharma and Trivedi (1987) tested some oil cakes and waste materials for mass culturing of P. lilacinus. However, Zaki and Bhatti (1988) used Gram seeds and Neem leaves for mass culturing of this fungus. Recently Siddiqui and Mahmood (1994) suggested culture of P. lilacinus on leaf extracts and leaf residues for nematode control. FORMULATION OF FUNGI

Granular formulation is generally considered to be most suitable for microorganisms that are to be applied to soil. There has been considerable interest

in encapsulating biocontrol agents in gellants such as sodium alginate (Fravel et al., 1985). Kerry (1988) showed that hyphae of V. chlamydosporium grew approximately 1 cm from alginate-bran granules and suggested that a granular formulation may be suitable for this species. Diatomaceous earth granules impregnated with 10% molasses, lignitestillage granules and alginate-clay pellets have all proved suitable carriers for biocontrol agents (Backman & Rodriguez-Kabana, 1975; Jones et al., 1984; Fravel et al., 1985). Paecilomyces lilacinus formulated on alginate pellets or diatomaceous earth granules showed promising results in nematode control (Cabanillas et al., 1989). A whey-powder-based blend of predatory fungal microcultures stabilized in seaweed extract known commercially as Nemout has been used for the control of plant-parasitic nematodes (AI-Hazmi et al., 1993; Ibrahim, 1994). Sikora and Schuster (1989) formulated the egg-parasitic fungi, Acremonium sordidulum and Fusarium sp., into Na-alginate in a mixture containing 1 ! broth with approximately 1.5 g fungus, 20 g alginate and 20 g milled bran, to control G. pallida. Some commercial preparations of these fungi have been marketed, but the products have never been used widely. Arthrobotrys robusta is commercially formulated as Royal 300 ~ (Cayrol et al., 1978), while Royal 350 ® is a similar product containing A. superba (Cayroi & Frankowski, 1979; Cayrol, 1983). These products had quality control problems and gave inconsistent results. Matskievich et al. (1990) suggested bio-preparation of Arthrobotrys spp. on composted straw-manure, turf-manure, sawdust manure and other materials. Two strains of the predatory fungus Arthrobotrys are available as a pesticidal agent 'Nematofagin-BL' in Russia (Matskievich, 1993). Both strains are effective in reducing nematode populations on various crops. Stirling and Mani (1995) produced granular formulations of Dactylella candida and Arthrobotrys dactyloides in alginate by encapsulating different quantities of fungal biomass. The best formulations were those in which granules were formed after the fungus had been encapsulated in alginate. These formulations produced a network of traps in soil which were maintained for at least 10 days and extended 5-10 mm from the granule. Development of practical control systems Although it is confirmed that fungi have great potential in the biological control of nematodes, the use of fungi by farmers in the field is lacking. A major problem is the absence of commercial interest in the biological control of nematodes. It is not possible for the farmer to develop a technology for the mass culture of fungi for the biocontrol of nematodes in the field. Farmers can use fungi only when the cultures are made available for them at a commercial scale, either by private sectors or by Government organisations. Presently, a lot of sub-

Control of nematodes by fungi: a review strates have been tested for the mass production of fungi, out of which straws of wheat, bran and barley are available at a very low cost. These can be used for culture of V. chlamydosporium (Crump & Irving, 1992). However, some oil cakes, waste material, Gram seeds and leaves of several plants are good substrates for P. lilacinus (Sharma & Trivedi, 1987; Zaki & Bhatti, 1988; Siddiqui & Mahmood, 1994). These substrates are generally cheap and easily available. Leaves of several plants are available even without any cost. The need at the present time is to use these substrates for the production of fungi which have potential as biocontrol agents against nematodes. Fungi having potential as biocontrol agents should be produced at a factory level and should be distributed to farmers. Culture of fungi on leaf residues appears to be more economical than on the other substrates, for field application. The leaves of several plants are readily available and only the cost of labour involved for the collection of leaves would be required. Some of the plants tested (Siddiqui & Mahmood, 1994) are widely distributed, but if tested plants are not available in some areas, leaf residues of some other plants may be tested for the mass culturing of fungi. The spores of most fungi can survive for some years and farmers can easily use such cultures for nematode management. Even more, the efficacy of P lilacinus cultured on leaf residues is the same as on the potato dextrose media (Siddiqui & Mahmood, 1994). Thus production of fungi at a factory level could give a boost to the fungai biocontrol of nematodes. CONCLUSIONS AND FUTURE PROSPECTS

Although a large number of fungi have been reported to reduce nematode density, only a few of them have shown their efficacy as efficient parasites of nematodes. Even the efficient parasites do not have all the desired characteristics of a good biocontrol agent, but their application has given promising results. Out of the fungi tested as biocontrol agents, P. lilacinus, V. chlamydosporium, D. coniospora, H. rhossiliensis and Arthrobotrys spp. gave significant control in field and pot tests in various agroclimatic conditions, with few failures. The tests with P lilacinus and V. chlamydosporium have met with greater success than those with others. Some techniques have been developed to estimate the number of fungi in soil, but few have been used to predict whether a soil would effectively suppress nematode multiplication (Crump, 1987). The methods currently available are too time-consuming to be widely used. There is an urgent need to develop a new methodology for rapid analysis of soil. Recently, a method has been developed for determining the pathogenicity of fungus isolates against nematodes in vitro (Carneiro & Gomes, 1993). This method may be used for selection of

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effective fungus isolates which might be used for nematode biocontrol. Studies on the environmental factors which effect the activity of biocontrol agents are also needed. These data may then be used to predict with the help of a computer the effect on nematode multiplication. In most cases, several tonnes of fungal inoculum are required for field application. Few studies have indicated that seed treatment for biocontrol is as effective as soil application, although the seed treatment with these fungi might be beneficial to reduce the quantity of fungal inoculum required. There is a need to conduct some field experiments to confirm earlier results so that seed treatments with fungal biocontrol agents may be recommended to farmers. Treatment with a single biocontrol agent may not provide sufficient control in some cases, but more success can be achieved by the combined application of biocontrol agents (Siddiqui & Mahmood, 1993; 1995b), because these biocontrol agents attack their host at different stages in its life cycle (Kerry, 1987). Chitin application is known to increase microbial activity and sometimes has resulted in a significant control (Rodriguez-Kabana et al., 1984). Chitin increases the activity of nematophagous fungi which parasitize eggs and females of root-knot and cyst nematodes. Use of chitin with nematophagous fungi has been recommended for nematode control. Biological control can also be used with other methods of nematode control, such as soil solarization (Walker & Wachtel, 1988), plant resistance (De Leij & Kerry, 1991) and low rates of application of nematicides (B'Chir et al., 1983) (especially organophosphate and carbamates) to achieve significant nematode control. In some cases use of organic amendments with fungi has also been recommended to achieve better nematode control. Although most of the fungal biocontrol agents are free from health hazards, P. lilacinus has caused concern with mammalian toxicity. This fungal species causes eye infections and facial lesions in humans and infections in domestic animals (Chandler et al., 1980), particularly when eyes are physiologically stressed or injured prior to infection or when tissues have been weakened by previous surgery (Pettit et al., 1980; Minogue et al., 1984). So there is an urgent need for testing of biological agents before their use, to ensure that they are free from health hazards (Sayre, 1986).

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