WO1994028725A1 - Nematophagous fungi composition - Google Patents

Abstract

Biocontrol compositions that are efficacious against plant-parasitic nematodes are described. The compositions consist preferably of at least two living nematophagous fungi selected such that at least two of the following classes of fungi are present: endoparasitic fungi, nematode trapping fungi, egg parasitic fungi. Preferably the nematophagous fungi are grown in a liquid cultivation medium.

Description

NEMATOPHAGOUS FUNGI COMPOSITION

1. TECHNICAL FIELD OF INVENTION

This invention relates to biocontrol compositions, processes for their production and methods of treating target pests with such biocontrol formulations. More particularly the compositions are for target pests that are plant parasitic nematodes, especially root-knot nematodes.

2. BACKGROUND OF THE INVENTION Plant-parasitic nematodes, in particular root-knot nematodes (Meloidogyne spp.), are a serious problem for most agricultural crops. Root-knot nematode is widely distributed throughout tropical, sub-tropical and temperate regions where it causes serious losses to many important agricultural, horticultural and ornamental crops. Susceptible crops among tree and vine fruits include almond, grape, kiwi fruit, nectarine, passionfruit, pawpaw, peach and plum. Susceptible vegetable crops include bean, (Mung, French, Navy), beetroot, capsicum, carrot, celery, cucurbits (cucumber, melon, pumpkin), eggplant, lettuce, okra, onion, potato, sweet potato and tomato. Susceptible ornamentals include carnation, chrysanthemum, dahlia, gerbera, gladioli, protea, and rose. Susceptible field crops include aloe vera, clover, cowpea, kenaf, lucerne, lupin, pigeon pea, peanut, soybean, sugarcane, tea and tobacco. Other horticultural crops that are susceptible include banana, ginger, pineapple and strawberry.

It has been estimated that the overall average annual crop yield reduction due to damage by plant-parasitic nematodes is more than 10%. In 1984 this resulted in over $100 billion of crop loss in effected geographic areas.

Plants infected with root-knot nematode show symptoms such as severe galling of roots, stunting and poor growth, and cracking and rotting of tubers and rhizomes. The symptoms caused by Meloidogyne are produced in response to the presence of juvenile nematodes, which hatch from eggs and migrate through the soil to invade the plant. Once inside the plant tissue, these juveniles become sedentary, establish a permanent feeding site and then swell to become sphere-shaped females. The females have a high reproductive capacity, laying as many as 2000 eggs in an egg mass on the root surface. Since the females are relatively protected within roots, juveniles and eggs are the only stages available for attack by soil-borne antagonists.

For the last thirty years, root-knot nematode has been controlled in the majority of intensive cropping systems by the regular application of chemical nematicides. However, these chemical control strategies are now at risk because many widely used nematicides are being abandoned for health, environmental or efficacy reasons. For example, with chemical soil fumigants the adverse effects and present status are as follows:

DBCP

Ground water contamination and mutagenic Deregistered in Australia in 1979.

EDB

Ground water contamination and carcinogenic - Deregistered in most countries in 1983 and to be phased from use in Australia. 1,3D

Volatilisation problems

Use suspended in California Methyl Bromide

Degradation of ozone layer.

Likely to be restricted in the next few years. Methyl Sodium

Odour problems - Restricted in built-up areas With respect to organophosphates and carbamates (eg fenamiphos, aldicarb, carbofuran, oxa yl, ethoprophos) these all have high mammalian toxicity, are toxic to birds and can contaminate ground water. It is believed these will eventually lose their effectiveness because of resistance and enhanced microbial degradation. Their use is restricted overseas in areas with shallow water tables.

Host plant resistance is a potential solution to the problem and is available in some crops. However its widespread use has been hampered by a lack of resistant genes, their specificity and the inability to identify reliably the large number of Meloidogyne species and races which occur in the field.

Biological control is a preferable method of control because it utilises organisms that are a natural component of the soil environment.

Much of the relevant prior art in relation to biological control of nematodes has been summarised in a recently published book (A) Biological Control of Plant Parasitic Nematodes (Stirling, G.R.) CAB International (1991).

This book and its cited literature make it clear that there are three groups of nematophagous fungi with biological control potential.

(i) Endoparasitic Fungi

These fungi infect their hosts by producing encysting zoospores, adhesive spores or ingested conidia. They include Catenaria anguillulae, Drechmeria coniospora, Verticillium balanoides, Hirsutella rhossiliensis and Ne atoctonus spp.

(ii) Nematode-trapping Fungi These fungi consist of a sparse mycelium which has been modified to form organs capable of capturing nematodes. They include members of the genera Arthrobotrys, Dactylella, Monacrosporium, Geniculifera and Duddingtonia.

( ϋ) Egg-Parasitic Fungi

This miscellaneous group of fungi have the capacity to invade the female and / or egg stage of nematodes. They include Verticillium chlamydosporium, Paecilomyces lilacinus, Verticillium lecanii, Dactylella oviparasitica, Fusarium spp. and Cylindrocarpon spp.

Relevant prior art in relation to each of these groups of nematophagous fungi is cited throughout the Stirling book

(A) particularly in Chapter 4, pages 50 - 75. Prior art that is relevant includes the following:

Endoparasitic Fungi

(B) Sayre, R.M. and Keeley L.S. Nematologica 15, 492 - 502 (1969) .

(C) Townshend et al. Journal of Nematology 21, 179 - 1983 (1989) . (D) Eayre C.G. et al. Plant Disease 71, 832 - 834 (1987).

(E) Jaffee B.A. and Muldoon A.E. Journal of Nematology 21, 505 - 510 (1989) .

(F) Mclnnis T M and Jaffee B.A. Journal of Nematology 21, 229 - 234 (1989) . (G) Lackey B.A. et al. Phytopathology 82, 1326 - 1330 (1992) . (H) Eayre c. G. et al. Plant Disease 71, 832 - 834 (1987 )

Prior art reference (E) demonstrates that natural populations of Hirsutella rhossiliensis can suppress nematode populations in some situations. A range of endoparasitic species including Catenaria anguillulae (reference B), Drechmeria coniospora (reference C) and Hirsutella rhossiliensis (references D, F, G, H) can also be grown in culture, or on nematodes, and introduced into soil to achieve some nematode control.

Nematode-trapping Fungi

There is a large amount of relevant prior art in relation to the use of the nematode-trapping fungi for biocontrol and this has been summarised in reference (A) , pages 127 - 135. Numerous examples exist of the culture of nematode- trapping species on a solid organic substrate (eg. cereal grains or bran) and their introduction into soil as a fungus / substrate preparation. Similarly, these fungi have often been grown in broth and applied to soil as a liquid or as dried spores. In all these cases, efficacy has never been entirely satisfactory and these production systems have therefore never been commercially successful.

The most relevant prior art in relation to the use of the nematode-trapping fungi for biocontrol is as follows:

(H) Cayrol J.C. et al. Pepinieristes, Horticulteurs,

Maraichers Revue Horticole 184, 23 - 30 (1978). (I) Cayrol J.C. and Frankowski. Pepinieristes, Horticulteurs, Maraichers Revue Horticole 193, 15-23 (1979) . (J) Rhoades H.L. Nematropica 15, 1 - 7 (1985). (K) Cayrol J.C. Revue de Nematologie 6, 265 - 273 (1983).

References (H), (I) and (J) demonstrate that formulations of the nematode-trapping fungi have been produced. However, they have either been ineffective (reference J) or they have failed because of quality control problems and inconsistent performance (references H, I and K) .

Egg-Parasitic Fungi

Relevant prior art in relation to the use of the egg- parasitic fungi for biocontrol has been summarised in reference (A) at pages 137 - 145. The most relevant prior art involves the following:

(L) Rodriguez-Kabana et al. Nematropica 19, 155 - 170 (1984) . (M) Hewlett et al. Journal of Nematology 20, 578 - 584 (1988) . (N) Cabanillas E. and Barker K.R. Journal of Nematology

21, 115 - 120 (1989) . (0) Schuster R.P. and Sikora R.A. Fundamental and Applied Nematology 15, 257 - 263 (1992).

(P) De Leij F. and Kerry B.R. Revue de Nematologie 14, 157

- 164 (1991) . (Q) Stirling et al. Phytopathology 69, 806-809 (1979). (R) Patent Specification PCT / GB90 / 01237. (X) Patent Specification PCT / AU90 / 00325.

These references demonstrate that Verticillium chlamydosporium (references L, P and R) , Paecilomyces lilacinus (references M, N and X), Dactylella oviparasitica (reference Q) and Acremonium sordidulum (reference 0) can be mass produced on a solid organic substrate such as bran or added to alginate granules and introduced into soil to achieve some nematode control.

References (R) and (X), refer to biocontrol formulations based on the egg-parasitic fungi Verticillium chlamydosporium and Paecilomyces lilacinus respectively.

Reference (R) refers to the production of chlamydospores on solid media and the incorporation of the fungus into liquid concentrates by emulsification in suitable liquids and onto solid concentrates by admixture with clay, talc and other known solid carriers. It is noted that reference (R) refers only to certain specific isolates of Verticillium chlamydosporium. Reference (X) refers to the nematicidal activity of specific strains of P. lilacinus (strains 251, 252, 253 and 254) when present at 10 - 10 spores/g. These strains have specific allozyme profiles and cardinal temperatures. Methods of production and formulation are not described, but methods in common use are referred to; eg P7, Lines 9-10; P7 Lines 28-29.

Close examination of the relevant prior art shows that most of the attempts to utilise nematophagous fungi for biocontrol purposes have utilised a single fungus.

Occasionally, more than one fungus has been used, but in these cases the fungi have had the same mechanism of action (eg. parasitism of eggs). Reference (S) is the only one prior art that involves the use of a number of organisms with different mechanisms of action.

(S) Japanese Patent 61210006 in the name of Katakura Chikkarin.

Reference (S) involves the use of a nematode-inhibiting material comprising one or more of a number of organic substances, together with several nematophagous fungi, including Arthrobotrys oligospora, Monacrosporium ellipsosporum, Dactylella oviparasitica, Verticillium chlamydosporium and Phoma macrostoma. Nematicidal activity was observed when these fungi were used together with large amounts of organic matter (equivalent to about 2 tonnes/ ha) . The fungi were not tested without organic matter. In reference (X) it is suggested that certain P. lilacinus strains are parasites of many plant-parasitic nematodes and that activity occurs against larvae, adults and cysts. However as the experiments were carried out without adequate controls it is not possible to compare mortality in the presence and absence of the fungus. Many nematodes would die or become stressed after being placed in petri dishes for 2-10 days as described in the specification. Evidence of nematicidal activity therefore relies on a number of glasshouse and field experiments (Examples 4-12). Although statistical evidence of yield increases is provided (eg Tables 7, 11 and 13) no statistical evidence of nematode control is provided. Reference (R) provides more convincing evidence that the V. chlamydosporium isolates referred to are capable of causing significant mortality to root-knot nematode. The results of several glasshouse experiments are presented in which statistically significant reductions in nematode populations were obtained.

From the above review of the prior art, whilst there have been numerous attempts over many years to utilise nematophagous fungi for biological control purposes there is no successful commercial biocontrol composition in use. Most attempts at biological control have involved root-knot nematode as the target pest. In almost all cases where nematophagous fungi have been used, they have been grown on a solid substrate and introduced into soil as a non- formulated fungus / substrate mixture. Occasionally, they have been incorporated into an alginate granule or formulated by other means.

Despite the work that has been done with the nematophagous fungi over the last fifty years, the most recent review of the prior art (reference A) makes it clear that there is not yet any example of the widespread successful use of these fungi in commercial agriculture. There are a number of reasons for this.

The fungi have been mass produced on solid substrates such as bran, rice hulls or cereal grains. This production method has many disadvantages, it is expensive as it cannot be readily scaled up to a commercial operation and it is inefficient, as optimum environmental conditions for growth of fungi cannot be provided. The end product is subject to contamination and therefore cannot be stored readily and is difficult to apply in the field with conventional farm machinery because it has a low bulk density and therefore requires excessively high application rates of more than 1 tonne / ha and quality control is very difficult. It is the object of this invention to provide biocontrol compositions and methods of producing biocontrol compositions that have improved efficacy against nematode pests.

3. SUMMARY OF THE INVENTION

This invention provides in one form a plant-parasitic nematode biocontrol composition comprising at least two living nematophagous fungi selected so that at least two of the following classes of fungi are represented, endoparasitic fungi, nematode-trapping fungi and egg- parasitic fungi.

Preferably the composition comprises at least one egg- parasitic fungi.

Preferably the egg-parasitic fungi are selected from the group consisting of Verticillium chlamydosporium, V. lecanni, Paecilomyces lilacinus, Dactylella oviparasitica, Fusarium spp. and Cylindrocarpon spp., more preferably V.chlamydosporium and P.lilacinus and most preferably the egg-parasitic fungi is V.chlamydosporium, especially strain LS53.

Preferably the nematode-trapping fungi are selected from the group consisting of genera Arthrobotrys, Dactylella, Monacrosporium, Geniculifera and Duddingtonia, more preferably Dactylella Candida and Arthrobotrys dactyloides and most preferably the isolates are 023 or A4 respectively.

Preferably the endoparasitic fungi are selected from the group consisting of Catenaria anguillulae, Drechmeria coniospora, Verticillium balanoides, Hirsutella rhossiliensis and Nematoctanus spp. and more preferably Hirsutella rhossiliensis. Preferably the fungi in the composition are multiplied in a liquid fermentation medium before being formulated into a biocontrol composition.

In an alternative form the invention provides a method of preparing a plant-parasitic nematode biocontrol composition comprising living nematophagous fungi, the method comprising:

a) growing the nematophagous fungi in a liquid cultivation medium to produce a biomass.

b) processing the biomass into a biocontrol composition.

Preferably the process involves the further step of preparing granules from the biomass. Preferably the form of the biomass fungus is morphologically distinct and more preferably it is in the form of hyphal fragments.

In a further form this invention provides a method of treating a target pest by applying an effective amount of a biocontrol composition a described above or prepared by a process as described above.

4. DETAILED DESCRIPTION OF THE INVENTION

The method of preparing the biocontrol composition according to this invention is applicable to both naturally occurring and genetically manipulated nematophagous fungi.

Suitable nematophagous fungi for the present invention were preferably selected according to the following protocol. (a) Egg-Parasitic Fungi

Forty-six Queensland soils were surveyed for fungi capable of parasitising eggs of root-knot nematodes (Stirling, 1991). The only species recovered were Verticillium chlamydosporium and Paecilomyces lilacinus. To identify isolates with the best biocontrol potential, 26 isolates of P. lilacinus and 13 isolates of V. chlamydosporium were screened for parasitic activity against eggs of Meloidogyne javanica using three different tests. Within each test, the number of eggs parasitised by different isolates of the same fungus varied considerably, suggesting that isolates differed in virulence. Two isolates of V. chlamydosporium (LS53 and LS106) and one isolate P. lilacinus LS39 were highly parasitic in all three tests. In general however, V. chlamydosporium tended to be more virulent than P. lilacinus and is our preferred egg-parasitic nematophagous fungi. Detailed methods and results are presented by Stirling in reference A. Whilst we have found the isolates mentioned above to be particularly useful, the invention is not limited to such isolates and the invention can be applied to a wide range of fungi.

b) Nematode-Trapping Fungi

In order to select appropriate nematode-trapping fungi for manufacture Australian isolates were screened for activity against nematodes on agar and in soil (Galper et al - simple screening methods for assessing the predacious activity of nematode - trapping fungi [Nematologica, 1994]). Three simple methods were used to assess trapping activity in soil. Results were compared to activity on agar. It was found that Dactylella Candida isolate 023 and Arthrobotrys dactyloides isolate A4 , which formed detachable rings and knobs or constricting rings, consistently reduced the number of M. javanica juveniles recovered from soil and these are our preferred nematode- trapping nematophagous fungi. However network forming species tended to be inconsistent in performance. Detailed methods and results are presented by Galper in the above reference.

c) Endoparasitic Fungi Suitable isolates were selected using standard techniques as described in reference (A), Chapter 8.

The fermentation process which we prefer to use is a batch fermentation process although any other fermentation process such as a fed batch or continuous process or fermenter design could be utilised. For best results it should meet the criteria set out below.

An actively growing culture of a nematophagous fungus such as Verticillium chlamydosporium, Dactylella Candida, or Hirsutella rhossiliensis on an agar plate of media such as Potato Dextrose Agar or Corn Meal Agar is used to inoculate a liquid medium which meets the nutritional requirements of the particular fungus being grown. A medium may contain a carbon source such as glucose or sucrose or molasses, and a nitrogen, mineral and vitamin source such as corn steep liquor, soya bean meal, fish meal or cotton seed meal. The carbon source should preferably constitute up to 30% of the medium and the nitrogen/mineral/vitamin source up to 30% of the medium. More preferably, the medium should contain between 10 and 20% carbon source and 5 to 10% nitrogen/mineral/vitamin source. It is important to the working of this aspect of this invention that the growth medium is liquid. By this we mean that while minor amounts of solid material may be present the carbon source of nutrient is predominantly from liquid or medium soluble sources of carboyhydrate such as glucose or sucrose. This contrast to conventional solid substrate cultivation where the solid substrate is the carbohydrate source. Whilst in our preferred embodiments the liquid medium is substantially free of solid non dissolved organic substrates we can tolerate in some processes up to 50% solid organic material though preferably this is usually no more than 10%. Also permitted within the scope of our invention is a minor amount of solid substrate refermentation after granules have been prepared. The pH of the medium should preferably be between pH 3 and pH 9, but more preferably between 5 and 7.

A suitable liquid medium should promote the growth of biomass to a level of at least 0.1-1.0% dry weight of the medium.

The medium should also promote growth of the right morphological form of the fungus, preferably hyphal fragments approximately 10 to 1000 urn long which are easily incorporated into a formulation and/or survival structures such as spores which promote better shelf life.

The preferred medium is one that is completely soluble in water but may which also contain particles of up to 2mm in suspension.

For large scale commercial operation the media ingredients should be consistently available in at least one tonne quantities at a cost which enables products to be produced at a price which is cost effective.

The temperature for cultivation should preferably be between 10°C and 40°C, more preferably between 20°C and 30°C and even more preferably between 25°C and 28°C depending on the nematophagous fungus. For example for V.chlamydosporium the preferred temperature is 25°C, for D.Candida and A.dactyloides 26°C and H.rhossiliensis 27°C.

The stirring rate in the fermenter required for adequate growth of the fungus will vary as a function of the size and design of the fermentation vessel, the fermentation medium and the nematophagous fungus being grown. The fermentor design and stirring rate that is chosen should be sufficient to ensure a shear force which will prevent the fungus from clumping and should promote the growth of short hyphal fragments and or conidia or other morphologically distinct structures. This ensures greater homogeneity and thus ensures better incorporation into the formulated biocontrol product. The stirring should also ensure that proper mixing is achieved so that adequate oxygen is transferred to the fungus for growth.

Aeration rate will also vary with the size, design of the fermentation vessel, fermentation medium and the nematophagous fungus but should be adequate to supply oxygen to the growing nematophagous fungus.

The time required to completion of fermentation will also vary with such parameters as the size of the fermentation vessel, fermentor design, medium, nematophagous fungus, aeration rate and stirring rate, and should be sufficiently long to ensure maximal biomass production without any deleterious effect to the nematophagous fungus. The process variables should be optimised in such a way as to ensure that the fermentation is carried out in a minimum time frame so as to overcome contamination problems and reduce costs without affecting the quality of the nematophagous fungal biomass produced. Unless the fermentation biomass is going to be used as inoculum it should otherwise be grown to late log phase.

In order to achieve commercial quantities of biomass fermentors with volumes of 5 to 500,000 litres, but more preferably between 5 and 200,000 litres would be utilised. It is envisaged that preferably the entire contents including fermentation medium of a mid to late log phase cultures would be used in a scale up process which utilises preferably 1 to 20% culture as inoculum, more preferably 1 to 10% culture for a larger fermentation vessel. The process is not restricted to the use of the entire fermentation biomass however. Any of the processes described below could be carried out on the fermentor biomass before utilising it as inoculum. Late log phase may typically be achieved between 16 hours and 10 days depending on the particular nematophagous fungus. For example, it takes 30 hours for V.chlamydosporium and 50 - 70 hours for D.Candida and A.dactyloides. It is often preferred that the final fermentation be left for an additional time period to maximise the production of biomass. We have also found that the efficacy of the final product is sometimes enhanced when growth of the fungi takes place over a five or six day period compared to shorter or longer periods.

The nematophagous fungal biomass produced during the fermentation process is preferably used directly since this reduces process waste, ensures that the formulation produced contains the right quantity of moisture (as described in the formulation processes below) and due to the lack of processing is therefore a low cost option. It also alleviates most of the process waste disposal problems. If necessary, the fermentation biomass could be processed in a number of ways examples of which include the following.

The biomass may be concentrated by processes such as filtration or centrifugation or drying to achieve the desired number of propagules after addition to the final product, or, to reduce the moisture content of the final product, or, to eliminate the fermentation medium which may contain secondary by-products which may reduce the storage life of the fungus, or, to remove nutrients which may provide a nutrient source in the formulation which when introduced into soil encourages competing micro-organisms especially plant pathogens to out compete the nematophagous fungus and render it less effective as a biocontrol agent. After concentration the fungus can be incorporated into a product directly or resuspended in a liquid to a volume less than, equal to or greater than the original volume. This liquid may be water, saline, a sugar solution or any other liquid which provides a nutrient source or encourages better viability of the fungus and/or extended storage life.

The biomass may alternatively be processed via homogenisation, milling, filtration, high density liquid centrifugation or combinations of some or all of these or other techniques so that specific morphological fractions of the fungal biomass such as hyphal fragments, spores, chiamydospores, adhesive spores are separated from each other and utilised in a formulation individually or combined in specific ratios to optimise the efficacy and /or storage ability of the final formulated product. The processing steps utilised may also be used to increase the number of propagules per gram of final formulation by breaking apart individual hyphae and therefore providing more point sources for growth or may be used to improve homogeneity by ensuring that the majority of the fungal biomass is of similar particle size which after formulation ensures that each batch of product consistently contains the right quantity of nematophagous fungus.

The biomass may be treated by drying in air, or using equipment such as a vacuum oven, oven, fluid bed dryer with optional protectants such as skim milk powder, inositol, sucrose or molasses at a concentration of up to 20% which may enhance or maintain the survival of the organism.

The nematophagous fungal biomass produced during the fermentation process which may or may not be processed as described above can be utilised to make biocontrol formulations. The formulation process involves the manufacture of a product which incorporates the nematophagous fungal biomass in conjunction with substances such as carriers or fillers (examples of which are given below) in such a way as to protect the fungus during storage. The end product which results from the formulation process should also be in a form which can be applied in the field. More preferably, the end product should have the characteristics set our below.

Each batch of product manufactured should consistently contain a standard number of individual propagules per gram for best results. The exact quantity required to ensure adequate shelf life and efficacy in the field is dependant on the nematophagous fungus. For example, the product composition we prefer to produce for V.chlamydosporium

Q contains about 10 colony forming units (CFU)/g while the D.Candida and A. oligospora compositions contain about 10 CFU/g.

The product should preferably not contain a level of nutrients high enough to provide a nutrient source for competitive micro organisms already present in the soil to which the product will be applied as their growth may interfere with the growth of the nematophagous fungus and thus affect the efficacy of the product. The exact quantity of nutrients depends on their type and the formulation type and the nematophagous fungus in the formulation. Typically compositions contain less than 50%, preferably less than 10% and more preferably less than 1% solid organic nutrient matter. The compositions are applied to soil at a rate of 200 Kg/hectare as opposed to the prior art where organic matter is applied at a rate of greater than 2 tonnes per hectare (Ref A) .

The product should preferably be able to retain viability and efficacy during storage at temperatures of at least 25°C for more than 6 months.

The product should preferably be of such a physical form that it can be applied through conventional farm machinery. It should preferably be a granular product which consists of granules in the size range of 0.05 to 4.0 mm but more preferably between 0.25 and 2.5 mm and even more preferably between 0.25 and 1.5 mm. The granules should preferably have a crush strength of more than 150 g, ie when placed on a scale they should be able to withstand crushing until a weight of more than 150 g is applied to them. This ensures that they do not break when being packaged, transported and utilised in the field. The moisture content of the final product will vary with the nematophagous fungus used and the formulation type but should be in a range of 0-50% and even more preferably between 1 and 5 %. Examples of different formulation processes and resulting end-products are given below.

1. A liquid or solid form of the fungal biomass or specific morphological fractions thereof derived in any of the ways described above can be added to other ingredients and granulated with or without the addition of extra granulating fluid such as water, saline (which may preserve the osmotic balance of the fungal cells), acidic solution (which may reduce contamination problems), in suitable granulating equipment such as an Eirich mixer, tableting press, pan granulator or high speed agglomerator (such as a Schugi Flexomix) or a low pressure extruder (such as a Fuji Paudal Basket granulator or radial extruders). The resulting granule in the appropriate size range as described above can then be left as is or dried either in air or using equipment such as a vacuum drying oven, fluid bed dryer, or oven to a moisture content as described above, which preserves shelf life without interfering with the viability and efficacy of the nematophagous fungal product. The crush strength should preferably be as described above.

In the kaolin and gum arabic formulation, kaolin acts as a carrier or bulking agent for the active ingredient which is the fungus. Its specific crystalline structure, which in turn affects its water holding capacity, ensures that the fungus retains good viability between 4°C and 25°C with time, provided that the formulation itself is at an appropriate moisture content of between 1% and 5%.

Gum arabic is required to bind the formulation together, but also ensures that the granule achieves the right hardness so that it can be commercially produced and transported without destruction or damage. A moisture content of between 1% and 5% ensures that the fungus retains viability because contaminants cannot grow while the growth rate of the fungus itself is slowed considerably. Granules in the range of 0.25 - 1.5 mm have been found to maintain good viability during storage and have been proven to be a suitable size for commercial application and efficacy.

2. A liquid or solid form of the fungal biomass or specific morphological fractions thereof derived in any of the ways described above can be absorbed onto preformed granules such as vermiculite, attapulgite, Drisorb, Trufeed, corn cobs and rice hulls preferably of a size range of 0.05 to 4.0 mm but more preferably between 0.25 and 2.5 mm and even more preferably between 0.25 and 1.5 mm. The absorption process can be assisted by mixing in equipment such as an agglomerator or Eirich mixer . In addition, binders such as methyl cellulose, with or without other formulation ingredients such as clays or silica powders can be used to assist the fungus in adhering to the pre-formed granule or to coat it to assist with shelf life and efficacy.

The moisture content of the final product will vary with the nematophagous fungus used and the formulation type but should be in a range of 0-50% and more preferably between 1-5%. This moisture content may be achieved through the addition of moisture to the granule initially or by drying the granule in air or using such equipment as a vacuum drying oven, fluid bed dryer or oven. The granules should preferably have a crush strength of more than 150 g at the end of the process.

3. A liquid or solid form of the fungal biomass or specific morphological fractions thereof derived in any of the ways described may be incorporated in such a way as to form a liquid formulation based on liquids such as water, saline, vegetable or mineral oil or any other organic or inorganic liquid. The liquid ingredients can be combined in such a way as to form a suspension concentrate or emulsifiable concentrate. The liquid preserves the shelf life of the nematophagous fungus by limiting the oxygen which is available for metabolism.

4. A liquid or solid form of the fungal biomass or specific morphological fractions thereof derived in any of the ways described can be incorporated with substances such as polyacrylamide, carrageen, gelatin or alginate in such a way as to form a gel. This gel can then be dried as described previously to an appropriate moisture content or could be utilised in any of the processes described above to form a different type of product. For example, the gel could be added to a carrier such as clay or diatomaceous earth or silica and processed to form a granule.

5. A liquid or solid form of the fungal biomass or specific morphological fractions thereof derived in any of the ways described could also be incorporated with ingredients to form a foam. For example, the fungus in conjunction with a wetting agent such as Teric BL - 8 (ICI Surfactant) and a liquid such as water could be vigorously agitated in a piece of equipment such as a lightning mixer to form a foam.

6. Any of the formulations described above may be fermented in a solid or liquid fermentation system under appropriate moisture and temperature conditions and used as is or re-formulated using any of the processes described above. For example, the nematophagous fungal biomass produced using a liquid fermentation process could be immobilised in an alginate bead which could then be fermented in liquid for a period of time to increase the percentage of fungus within the bead. The alginate beads could then be harvested from the fermentor and further processed in any of the ways described above or as described by Stirling and Mani in "Activity of Nematode Trapping Fungi Following Their Encapsulation in Alginate" Nematologica 1994. The formulated product may be packaged in air or under vacuum or under nitrogen or any other gas, which facilitates a good shelf life in any container or bag deemed suitable. Examples include plastic or metal drums or containers in which the product is packaged in water soluble or water insoluble plastic bags. The formulated product could also be stored in contact with a liquid such as water. We prefer to package the formulations in plastic bags vacuum sealed to 98% vacuum. It could also be stored in contact with solid materials such as silica gel which because of its moisture absorbing properties would preserve the formulation at the right moisture content. It should preferably be able to be stored at temperatures of up to 25°C for a period of at least 6 months but may be refrigerated if required.

Formulated biocontrol compositions are applied to seeds, transplants or other planting material, to plants or to soil. Applications are made prior to, at, or after planting, and the products are applied in a manner which is designed to ensure that the biological component comes into contact with the target nematode. Products can be applied by conventional or other farm machinery (eg. granule applicators, rotary incorporation's, spray equipment) or via irrigation water at application rates suitable for use with conventional or other farm machinery.

The invention will be further described by reference to preferred embodiments in the following examples.

Example 1: This example describes the preparation of a biocontrol granule composition based on the egg-parasitic fungus Verticillium chlamydosporium

A 1 cm section of an actively growing culture of the fungus on a potato dextrose agar plate which has been grown for 7 days, was used to inoculate 1000 mis of glucose/peptone/yeast (15g/L 2g/L 5g/L) broth of pH 6.0 in a 2000 ml flask. The flask was then shaken at 200 rpm and 25°C for 5 days after which time it was in mid to late log phase and contained about 10 propagules per ml, the major component of which were blastospores (Tables 1 and 2).

Table 1 shows the average dry weight of 3 replicate 5 ml samples taken during the growth of Verticillum chlamydosporium in GPY medium in 2000ml flasks. Not only is this a commercially acceptable yield but the formulation produced from biomass harvested at this time has been shown to be superior in activity when tested for growth from granules in soil. This liquid culture (1000 mis) is then used to inoculate 16 litres of glucose/peptone/yeast (15g/L, 2g/L, 5g/L) broth at pH 6.0 in a Setric Genie Industrial Fermenter. The fermenter medium containing 10 mis of Dow Corning 1520 anti foam had previously been sterilised at 121°C for 30 minutes.

Table 2 shows the average number of blastospores per ml of three replicate samples taken during the growth of V. chlamydosporium in GPY Medium in 2000 ml flasks.

After inoculation, aeration was set at 0.6 vvm per hour but was increased to 1 vvm after 21 hours. The stirring rate was set at 200 rpm initially but was progressively increased to 600 rpm as the viscosity of the medium increased due to growth of the nematophagous f ngus. Total fermentation time to late log phase was about 30 hours in the fermentor rather than 5 days as seen in flasks (Tables 1 and 2), unless the biomass was going to be used as inoculum for a larger fermentation vessel in which case it would have been harvested after about 20 hours. The resulting biomass was then utilised at a 5% level to inoculate larger volumes of fermentation medium. The scale- up process was repeated until the desired quantity of fungal biomass was achieved.

The biomass preferred above was used directly from the fermentor as this process had facilitated the growth of short hyphal fragments and conidia which are easily incorporated in to a formulation in a homogeneous manner.

The preferred formulation of Verticillium chlamydosporium is a granule in the size range of 0.25 - 1.5 mm with a crush strength of 150 g and a moisture content between 1 and 5%. The granule contains a filler (kaolin HR1 clay) and a binder (gum arabic) in the ratio of 95:5. The granules were prepared by the following method.

950 g of kaolin and 50 g of gum arabic were placed in an Eirich mixer and mixed for 30 seconds on speed 1. To this was added 250 mis of fermentor biomass which had been taken directly from the fermentor. The mixer was energised for about 60 seconds until granulation was achieved. In an alternative method the mixture was mixed for 30 seconds and then extruded through a die face containing 0.5 or 1.0 mm pore spaces to form granules of this diameter. The 1mm die face was preferred as these granules have been shown to maintain good viability in storage for up to 18 months at 25°C. Also, there is very little waste in this extrusion process. If granules are produced without extrusion then approximately 20% of the formulation is lost because of incorrect sizing.

Granules produced by both methods were then dried at up to 40°C in a fluid bed drier until a moisture content of 1 - 5% was achieved. The granules were then sieved so as to retain only those between 0.25 and 1.5 mm.

After production, the product was analysed to ensure that it met the criteria listed below. This ensured that it has good storage stability and will perform well when used in the field.

(a) Number of propagules per g of formulation (10 - 10 CFU/g) .

(b) % viability of granules as determined by plating on to Tap Water Agar (should be 100%). Growth in mm from the granule is also recorded as a measure of potency and should be 3 - 4mm in 4 days. (d) A pot test where formulations are mixed in to soil and placed in pots which are then inoculated with M. javanica eggs. After two weeks a tomato seedling is planted in each pot and after 4 weeks the plants are harvested and the % of egg masses parasitised is recorded and should be at least 50%.

The product was vacuum packed and stored at 4° C as this has been shown to facilitate a good shelf life and ensure that the product maintains efficacy during storage.

Efficacy of Final Product

Numerous glasshouse and field experiments were carried out and these showed that when Verticillium chlamydosporium was introduced into field soil as a formulated granule, it parasitised the eggs of root-knot nematode. When tomato is used as a host plant, as many as 75% of the first generation eggs are parasitised with application rates of 1% w/v. Similar levels of mortality were also observed in eggs produced by later generations of the nematode.

The experimental method used was as follows:

A field experiment was carried out in a grey sandy loam soil (56% coarse sand, 29% fine sand, 8% silt, 7% clay) at Applethorpe, Queensland. Four beds each 0.8 m wide were formed and an irrigation line with emitters spaced 75 cm apart was placed in the middle of each bed. Treatments (Table 3) were applied by removing 12.5 litres soil from a hole 40 cm diameter and 10 cm deep centred on each emitter and mixing the appropriate treatment with the soil. Treatments were replicated 12 times and set out in a randomized block design.

Bacteria were produced by inoculating all four isolates into sterile sand containing 1% potato starch and incubating for 18 hours. Horse manure-compost was prepared by sieving dry horse manure and garden compost through a 5 mm sieve and then mixing them together in equal volumes.

Nematode inoculum was prepared by finely chopping the roots of 22 tomato plants containing newly formed egg masses of M.javanica. The number of nematodes on the roots was quantified by recovering eggs from sub-sample by treatment with 1% NaOCl for 5 minutes. The appropriate quantity of roots were then mixed with clean sand so that the inoculum contained approximately 200 eggs/ml. At the same time as treatments were applied, 125 ml of nematode inoculum (approximately 25 000 eggs) was added and incorporated into each plot.

Beds were covered with plastic mulch after treatments and nematode inoculum had been applied. Five days later, a tomato seedling (cultivar Floradade) was planted next to each emitter. Seedlings had been grown in Speedling ^v- cells and four days prior to transplanting, those to be planted into soil treated with V.chlamydosporium were pre- inoculated with the appropriate fungi. V.chlamydosporium was applied by sprinkling 0.2 g granules on the surface of each cell.

About 5 weeks after planting, four of the 12 replicates were harvested and root galling was assessed as follows:

0 = no galls 1 = 1-20 galls

2 = 20-100 galls

3 = <33% of the roots galled

4 = 33-75% of the roots galled

5 = >75% of the roots galled, some multiple galls

A sample of ten egg masses were removed from each root system and examined for the presence of parasitized eggs. A 1 g sample of roots was then macerated with a mortar and pestle and the population density of V.chlamydosporium on roots was determined by dilution onto a selective medium.

Soil samples were collected at the conclusion of the trial, 10 weeks after planting with an Oakfield tube as described previously and nematodes were extracted from a 200 ml sub- sample with a Baermann tray. Sub-samples (lg) were taken from treatments 3, 4, 8 and 15 and processed for V.chlamydosporium as described previously. Roots were then removed and rated for galling as follows:

0 = no galls

1 = 1-25% of the roots galled

2 = 26-50% of the roots galled. Minimal multiple galling 3 = 50-75% of the roots galled, with some multiple galling

4 = 76-99% of the roots galled, with severe multiple galling

5 = 100% of the roots galled. Multiple galls deteriorating A sample of ten egg masses from treatments 3, 4, 8 and 15 was examined for parasitism and population densities of V.chlamydosporium on roots were determined as described previously. Roots from these treatments were then placed on a mister for 4 days to extract Meloidogyne juveniles. Eggs remaining on roots were recovered by immersing roots in 1% NaOCl for 3 minutes and then retrieving the eggs on a 38 mm sieve.

Data from the four replicate plants that were harvested 5 weeks after planting (Table 3) showed that plants in some treatments (eg treatments 2 and 8) had a significantly lower root gall index than one of the controls. Differences in galling were less clear-cut at 15 weeks because of variation between the two controls.

Data from plots treated with V.chlamydosporium (Table 3) showed that after 8 weeks, the fungus was present in plots to which it had been added. Population densities had declined in soil at 15 weeks, whereas populations on roots had increased. In V.chlamydosporium treatments at both sampling times between 1/3 and 2/3 of the egg masses contained parasitized eggs.

Example 2. Production process for a biocontrol composition based on the nematode-trapping fungus Dactylella Candida

(i) Fermentation

A 1 cm² section of an actively growing culture of the nematode-trapping fungus on corn meal agar, which had been grown for 7 days, was transferred to 1000 ml of glucose (15g/L)/corn steep powder (lOg/L) medium pH 7.0, in a 2000 ml flask. The flask was shaken on a rotary shaker at 26°C and 200 rpm for 6 days. This flask was then used to inoculate 16 litres of the same medium in a Setric 20B

Industrial Fermentor. Before addition of the inoculum, the medium containing 15 mis of Dow Coming 1520 anti foam was sterilised for about 25 minutes at 121°C.

After inoculation of the fermenter, the stirring rate was set at approximately 450 rpm and the aeration rate at 0.78 wm per hour. The temperature was set at 26°C. The total fermentation time was approximately 50 hours (unless the biomass is to be used to inoculate a larger fermentation vessel in which case it should be left for only 40 hours). The resulting biomass was then utilised at a 5% level to inoculate larger volumes of fermentation medium. The scale- up process was repeated until the desired quantity of fungal biomass was achieved.

The biomass was taken from the fermentor and centrifuged at 10,000 rpm for 10 minutes at 10°C to concentrate it ten¬ fold as this fungus does not produce spores in liquid and this step ensures that the composition contains at least 10⁶ CFU/g.

A granule in the size range of 0.25 - 1.5 mm with a crush strength of 150 g and a moisture content between 1-5% was prepared. The granule contained two fillers, (Kaolin HR1 clay) and finely milled vermiculite and a binder (gum arabic) and was prepared as follows. 36 g of gum arabic was added to 300 is of concentrated biomass in an Eirich mixer and mixed for 30 seconds on speed 1. To this was added 175 g of finely milled vermiculite which was mixed in for a further 30 seconds. 539 g of kaolin was then added and the mixture mixed on speed 2 for 30 seconds. The mixture was then extruded through an extruder with a 1mm diameter pore space to form granules.

The granules were then placed in sterile bags which were aerated for a period of three days. The granules were then removed and air dried to between 1 and 5% moisture content over a period of about 6 hours.

After production, the product was analysed to ensure that it meets certain standards. This ensures that the product will maintain viability during storage and perform well in the field.

(a) Number of propagules per g of formulation should be at least 10 .

(b) % viability of granules as determined by plating on to Tap Water Agar should be 100%. Growth in mm from the granule is also recorded as a measure of potency and should be 3 - 4mm in 4 days.

(c) Growth in a soil slide test where granules are placed on a slide and covered with a lOOu mesh and soil. Growth from the granules is examined after 5 and 10 days and the number of traps produced is recorded. The more traps the more effective the composition.

(d) Cup test where formulations are mixed in to soil and placed in cups which are then inoculated with M. javanica eggs. After 2, 4 and 6 weeks the number of nematodes in the soil are counted and for our preferred compositions at least 50% reduction should be achieved relative to the controls. (e) A pot test where formulations are mixed in to soil and placed in pots which are then inoculated with M. javanica eggs. After two weeks a tomato seedling is planted in each pot and after 4 weeks the plants are harvested and the number of galls on the roots counted. Preferably at least a 75% reduction in galling compared to the control is achieved.

(ii) Packaging and Storage

The product is vacuum packed and stored at temperatures of between 4°C and 25°C.

Efficacy of Final Product

When granular formulations of Dactylella Candida are introduced into field soil, hyphae grow from the granules and these hyphae produce traps. These structures trap nematodes and evidence that this occurs has been obtained. Laboratory tests showed that when formulated granules containing Dactylella Candida are added to field soil at 1% w/w, the number of root-knot nematode juveniles is reduced by more than 80%.

Example 3: This example illustrates production process for a biocontrol composition based on the endoparasitic fungus Hirsutella rhossiliensis

The fungus was maintained on corn meal agar at 27°C. A portion of the agar plate containing actively growing fungus was transferred to a 0.05% solution of the surfactant Tween 80 in water and then shaken to dislodge and suspend spores from the fungal biomass.

The spore suspension was then filtered to purify the spore component of the biomass and retained.

3.0 x 10⁴ spores were added to 100 ml of media containing 0.5g of fish meal and 2.0g of glucose in a 250 ml flask. The flask was inoculated at 27°C on an orbital shaker at 100 rpm for 11 days.

The resulting biomass was then utilised at a 5% level to inoculate larger volumes of fermentation medium. The scale- up process was repeated until the desired quantity of fungal biomass was achieved.

The biomass was washed with sterile water to remove excess nutrients and then resuspended in 4.5 mm KCI solution. It was then macerated until a homogenous product is obtained.

The macerated biomass was added to vemmiculite in the weight ratio of 1:2 and then dried to a moisture content between 5 and 20%. Although the process described above has some similarity to that described in reference G and H, this process is additional. The fermentation is done in a medium which improves biomass yields by more than 100%. Instead of using solid fermentation on vermiculite impregnated with nutrients, washed biomass is added to vermiculite. This overcomes the problem of competition from fast growing saprophytic fungi which is referred to in reference H.

After production, the product was assessed for colony forming units/g by preparing a dilution series in water and plating it out on to agar medium. The product should contain at least 10 colony forming units/g.

The product was stored under vacuum at temperatures of up to 30°C.

Efficacy of Final Product

When vermiculite formulations of Hirsutella rhossiliensis were introduced into soil, the fungus sporulates and these spores infest root-knot nematode juveniles. In laboratory tests with field soil, formulated granules (applied at 1% v/v) reduced nematode numbers by more than 75%. Example 4: This example is a comparative example which illustrates the effect of different fermentation technologies.

The following compositions were prepared on Arthrobotrys dactyloides (A4) and were solid substrate re-fermented.

4.1 Culture homogenised on day 6, re-fermented, washed prior to formulation

4.2 Culture homogenised on day 6, re-fermented, not washed

4.3 Culture homogenised on day 7 before use and washed 4.4 Culture homogenised on day 7 before use and not washed

The same four treatments were carried out again except that the formulation was not solid substrate re-fermented and these were designated 4.5, 4.6, 4.7 and 4.8 respectively.

The samples were homogenised using a hand-held homogeniser on low speed for 60 seconds before being re-fermented for one day. The samples which were homogenised immediately prior to formulation were treated the same was except that they were not re-fermented.

Formulation

Same as for Example 1. Only half of the formulations were solid substrate fermented after extrusion as described in the treatment section above.

Quality Control and Formulation Assessment

All equipment and formulation ingredients were sterilised before use. All formulation ingredients were sprinkled on to CMA to test for contamination. Samples of the formulation directly after mixing, directly after extruding and after solid substrate fermentation were also tested for contamination. A CFU count and % viability and potency determinations were carried out on the dry granules.

Samples of the formulations were stored in air and under vacuum at 4°C and 25°C for long term viability testing. The remaining formulations were vacuum sealed and stored at 4°C for efficacy testing, the results of which are set out in Table 4. Table 4

These results show that

(a) re-fermentation showed no obvious difference in granule viability or CFU count or spore rating in comparison to cultures that were homogenised immediately prior to formulation.

(b) washing the culture before incorporation in to the formulation showed no obvious effect on all parameters tested (including degree of contamination of the granules) regardless of whether the formulation was solid substrated or not.

(c) solid substrate fermented granules were more contaminated than those that were not solid substrate fermented.

(d) compositions 4.1 - 4.4 gave significantly better gall control than their counterparts 4.5 - 4.8 that involved solid substrate fermentation. This shows that some solid substrate fermentation after formulation can be beneficial. Example 5: This example demonstrates the improved efficacy of compositions according to the present invention when formulated as granules compared to a liquid drench.

Thirty pots were filled with 50:50 Bundaberg soil: Nematology sand. V.chlamydosporium granules as prepared in Example 1 were added to two groups of six pots at a rate of 1% and 4% w/v and all pots were then planted with tomato seedlings (cv. Tiny Tim) . Twelve days later, pots were inoculated with 4000 M.javanica juveniles. As this inoculation was not successful, the pots were re-inoculated with 4000 M.javanica (70% juveniles: 30% eggs) after a further 3 weeks. The third and fourth groups of pots received the same quantity of V.chlamydosporium granules as groups one and two, respectively, except that the fungus was applied by adding the appropriate quantity of granules to 100ml water, shaking on a wrist action shaker for 2 minutes and then decanting the liquid onto the pots. The third group received a suspension made from 3.33g granules applied 2, 3 and 4 weeks after the second nematode inoculation, while the fourth group received 13.33g granules at the same time. The fifth group was not inoculated with V.chlamydosporium.

Eleven days after the last drench was applied, a core of soil was taken from each pot to estimate the V.chlamydosporium populations. Serial dilutions were prepared from a lg sub-sample and 0.1ml aliquots were plated onto a selective medium. Very little fungus was isolated so the V.chlamydosporium soil density was checked again 3 weeks later. Plants were harvested 15 weeks after planting and samples of ten egg masses were examined for parasitism. A second sample of ten egg masses was set on mini hatch plates and incubated for 7 days. The number M.javanica juveniles hatched from these egg masses was recorded. Roots were placed on a mister for 8 days to extract M.javanica juveniles.

The results are set out in Table 5. When V.chlamydosporium granules were mixed with the soil at planting the estimated population of the fungus at 14 weeks was significantly higher than when applied as a drench.

There was more egg mass parasitism when V.chlamydosporium was applied as a granule at planting, rather than as a drench and fewer juveniles were recovered from egg masses in these granule treatments.

Table 5 Treatment

1% V.c at plantin 4% V.c at plantin 1% V.c at drench 4% V.c at drench Control

Example 6: This example shows that addition of various sources of organic material to soil does not effect the efficacy of a granular biocontrol formulation of V.chlamydosporium.

A 50:50 Bundaberg soil: Nematology sand mix was amended with three sources of organic matter - horse manure/compost at 1% w/v and barley straw and lawn clippings at 20% v/v. Unamended soil was used as a control. V.chlamydosporium granules as prepared in Example 1 were mixed with the soil at 0 and 2% w/v. This resulted in a 4 x 2 factorial, each replicated five times. Pots were planted with tomatoes (cv. Tiny Tim) and inoculated with M.javanica juveniles 3 weeks later. As this inoculation was unsuccessful, pots were re-inoculated with nematodes. Ten weeks after granules were added a core of soil was taken from each pot. The V.chlamydosporium population was determined by preparing serial dilutions from a lg sub-sample and plating 0.1ml aliquots onto a selective medium of Kerry et al

(1990). Since V.chlamydosporium was not recovered, the fungal population was checked again 4 weeks later. Plants were harvested 15 weeks after planting and samples of ten egg masses were examined for parasitism. The V.chlamydosporium population density on the roots was determined by chopping roots into 1 cm pieces and placed in a lg sub-sample in a Stomacher for 1 minute. A serial dilution was prepared and plated as above onto the selected medium.

Roots were stored at 4 C for 10 weeks and eggs were then retrieved by immersing roots in 1% NaOCl for 3 minutes. The results are set out in Table 6.

In treatments with V.chlamydosporium there were higher populations of the fungus recovered from roots when horse manure/compost and lawn clippings were added to the soil. However, the presence of organic matter did not affect the number of egg masses parasitised or the soil population of V.chlamydosporium.

The low recovery of V.chlamydosporium from the soil was not consistent with previous experiments. However, soil samples were taken later than in other experiments.

* equivalent means with transformed means (arcsine of square root in parentheses.

Example 7: This example illustrates the improved efficacy as a biocontrol composition when different fungi are combined. The compositions were prepared and tested according to the methods in Example 1 giving the results set out in Table 7. The composition with VC was applied at 1% w/v, M2 + A4 was applied as fermentation biomass at 0.006% and 0.008% w/v respectively.

The effect of the two nematode-trapping isolates M2 and A4 was particularly apparent when the gall ratings of individual plants were examined: 6/8 plants in composition M2 + A4 had a gall rating of 1 whereas 6/8 control plants had ratings of 3 or 4. Galling of plants harvested 8 weeks after planting to collect data on V.chlamydosporium also suggested that the nematode-trapping fungi had been effective, as the root-gall index for treatments 10 was significantly lower than the controls. Treatments containing the nematode-trapping fungi had lower gall indices than those that did not, and the gall index for composition M2 + A4 was significantly lower than one of the controls.

It is believed that the biocontrol compositions of the present invention are more reliable and efficacious than prior art compositions as when nematophagous fungi are introduced into the soil having been multiplied in an organic substrate, the substrate provides a food source for other soil micro-organisms and the introduced fungus often fails to establish in this competitive environment.

Claims

1. A plant-parasitic nematode biocontrol composition comprising at least two living nematophagous fungi selected so that at least two of the following classes of fungi are represented, endoparasitic fungi, nematode-trapping fungi and egg-parasitic fungi.

2. A biocontrol composition as deferred in Claim 1 comprising at least one egg-parasitic fungi.

3. A biocontrol composition as defined in Claim 1 or 2 comprising a nematode-trapping fungi.

4. A biocontrol composition as defined in Claim 2 wherein the egg-parasitic fungi is selected from the group consisting of Veticillium chlamydosporium, V.lecanni, Precilomyces lilacinus, Dactyella oviparasitica, Fusarium spp. and Cylindrocarpon spp.

5. A biocontrol composition as defined in Claim 4 wherein the egg parasitic fungi is V. chlamydosporium or P. lilacinus.

6. A biocontrol composition as defined in Claim 5 where in the egg-parasitic fungi is V. chlamydosporium, strain LS553.

7. A biocontrol composition as defined in Claim 3 further comprising a nematode-trapping fungi selected from genera Arthrobotrys, Dactylella, Monacro-osporium, Geniculifera and Duddingtonia.

8. A biocontrol composition according to Claim 3 wherein the nematode-trapping fungi are selected from the group consisting of Dactylella Candida and Arthrobotrys dactyloides.

9. A biocontrol composition as defined in Claim 1 wherein the endoparasitic fungi are selected from the group consisting of Caternaria anguillulae, Drechmeria coniospora, Verticillium balanoides, Hirsutella rhossiliensis and Nematoctanus spp, are preferably Hirsutella rhossiliensis.

10. A biocontrol composition according to any one of Claims 1 - 9 wherein the fungi are multiplied in a liquid fermentation medium.

11. A biocontrol composition as defined in any one of Claims 1 - 10 in the form of granules.

12. A method of preparing a plant parasitic nematode biocontrol composition comprising living nematophagous fungi, the method comprising the steps of

(a) growing the nematophagous fungi in a liquid cultivation medium to produce a biomass

(b) processing the biomass into a biocontrol composition.

13. A method of preparing a biocontrol composition as defined in Claim 12 the processing step (b) including a granulation stage.

14. A method of preparing a biocontrol composition as defined in Claim 12 or 13 wherein the biomass is grown to a level of at least 0.1 - 1.0% dry weight of the medium.

15. A method of preparing a biocontrol composition as defined in any one of Claims 12 - 14 wherein process step (b) includes combing at least two nematophagous fungi grown separately.

16. A method of preparing a biocontrol composition according to Claim 15 wherein at least one fungi is an egg- parasitic fungi and at least one fungi is a nematode- trapping fungi.

17. A method of preparing a biocontrol composition according to any one of Claims 12 - 16 wherein the cultivation temperature is between 20°C and 30°C and more preferably between 25°C and 28°C.

18. A method of preparing a biocontrol composition according to any one of Claims 12 - 17 wherein the fungi are grown in the medium to late log phase.

19. A biocontrol composition prepared by a process as defined in any one of Claims 12 - 18.

20. A method of treating a target pest by applying an effective amount of a biocontrol composition as defined in any one of Claims 1 - 11 or prepared by a process as defined in any one of Claims 12 - 18.