WO1991001642A1

WO1991001642A1 - Biocontrol compositions

Info

Publication number: WO1991001642A1
Application number: PCT/GB1990/001237
Authority: WO
Inventors: Brian Robert Kerry; Franciscus Antonius Anna Maria De Leij
Original assignee: Agricultural Genetics Company Ltd.
Priority date: 1989-08-11
Filing date: 1990-08-08
Publication date: 1991-02-21
Also published as: GB8918400D0; AU6169990A

Abstract

The invention relates to Verticillium chlamydosporium strain AC, a sample of which has been deposited at the Culture Collection of the CAB International Mycological Institute, (IMI) on the 1 August 1989, under the accession number CMI CC 334168, or a derivative, variant or mutant thereof having nematicidal activity, and its use in the control of nematodes.

Description

BIOCOH ROL COMPOSITIONS

This invention relates to the control of agricultural pests and more particularly to biological control agents for the control of nematodes.

Root knot nematodes (Meloidoqyne spp. ) attack many important agricultural and horticultural crop plants, including tomatoes, tobacco, peanuts, vegetables, sugar cane and grapes, and present a serious problem to the farmer and grower. At present, control of nematodes is achieved by the use of chemical nematicides, but many of these are considered undesirable for environmental reasons, or are too expensive to provide economic control in many crops. The present invention arises from the search for biological control agents which are environmentally more acceptable than currently available nematicides.

Nematodes have many natural enemies, including fungi, bacteria, rickettsias, collembola, mites and other nematodes. The largest and most studied group of these are the fungi, especially the fungal parasites of nematode females and eggs which provide a degree of natural suppression of nematode populations. Among these, Nematophthora gynophila and Verticillium chlamydosporiurn are the two main fungi involved in the natural decline of the cereal cyst nematode (Heterodera avenae), and Cylindrocarpon destructans and V. chlamydosporium are the main parasites of the beet cyst nematode (Heterodera schachatii). Natural decline, however, is a slow and uncertain process and fails to provide an acceptable solution of nematode problems, particularly root-knot nematodes, which are not known to be supressed by natural decline.

Attempts have been made ro manipulate natural populations of V. chlamydosporium in soil in order to parasitise the cyst nematode Heterodera schachtii but the results of this experiment have not been successful in the field.

The fungus V. chlamydosporium is very variable and in the course of our research, many different strains have been isolated from nematodes, or from soil infested therewith, of which the great majority do not possess the properties necessary to provide effective biological control of nematodes under practical conditions of crop cultivation.

Using certain selective media and special selection criteria, detailed below, we have now discovered a distinct strain of V. chlamydosporium which is effective in controlling Meloidoqyne species. This strain was deposited on the 1 August, 1989, with the Culture Collection of the CAB International Mycological Institute, Ferry Lane, Kew, Richmond, ^' Surrey, TW9 3AF, England, unήer the following designation and Accession Number: -

V. chlamydosporium strain AC : CMI CC 334168

Apart from its biological activity against nematodes this strain is typical of the species of Verticillium chlamydosporium. Strain AC grows readily on many microbiological media and average growth rates on potato dextrose agar (PDA) are 0.75 mm/day at 18°C, or 1.45 mm/day at 25-C. It is recognised that the strain we have deposited may be modified or manipulated by spontaneous, chemical or physical mutagenesiε, protoplast fusion, transformation and other similar techniques and as such, any mutants, variants or derivatives of this strain are within the scope of this invention.

The criteria that must be fulfilled for selection of useful strains are as follows:

1. The strain must be pathogenic to nematodes.

2. The strain must have no negative effects on the growth of crop plants.

3. The strain must survive well in soil and establish and maintain itself on host plant roots.

4. The strain must readily produce chlamydospores.

The ability to produce chlamydospores is an important criterion in selection because we have found that chlamydospores contain sufficient internal energy reserves to establish in the soil without the need for a supplementary food source, whilst an additional energy base is essential to help hyphal fragments and conidia derived from shaken liquid cultures to establish in soil.

The application of an effective fungal inoculum, containing sufficient endogenous nutrient reserves to establish in the soil without the addition of an supplementary nutrient source is a major advantage because:

a) In some soils a food base encourages the - _. -

development of a competitive microflora that may limit the establishment of V. chlamydosporium.

b) In some tests the food base applied with the fungus has supported much growth in soil and prolonged saprophytic development without initiating nematode infection. Significant levels of nematode infection only occurred after the energy source had been depleted.

c) Screening is simpler without the series of control treatments (eg. uncolonised food base, autoclaved colonised food base, and food base colonised by a non-nematophagous fungus) required to separate the effect of the fungus from that of adding the food base.

d) Application of chlamydospores results in as good or better establishment of the fungus in soil than application of the actively growing fungus applied on a foodbase and provides more consistent control.

e) The lack of a bulky food base makes handling the fungus more simple and results in a marked reduction in the weight of material added to soil, thereby improving the practicality of using V. chlamydosporium for nematode control.

Verticillium chlamydosporium is a facultative parasite which can be established in soil in the absence of nematodes and may be used in the following ways:

a) At low nematode densities to prevent the build-up of damaging nematode populations, ie. as a preventative measure.

b) In combination with nematicides to give control of plant damage and long-lasting nematode control after more than one generation when nematicide efficacy is reduced.

c) In combination with resistant cultivars on which the rate of selection of resistance-breaking females may be greatly reduced by fungal parasitism. Thus the useful life of a resistant cultivar could be significantly extended.

d) In combination with a tolerant cultivar which is able to withstand damage caused by nematodes, but which, without the fungus, may support the build-up of large nematode populations.

Chlamydospores, conidia, hyphal fragments etc of useful V. chlamydosporium strains can be prepared as a seed dressing, dust, granule, pellet, wettable powder, emulsifiable concentrate and the like, formulated in combination with various agricultural acceptable carriers, sticking, binding agents and surface active agents, which are normally employed to facilitate the delivery and distribution of the active ingredient. The rate of incorporation of these for ulants may vary considerably depending on their nature, typically ranging from 0.01% to 99.9% of the final mixture. The composition may contain a single nematicidal fungal strain, or a mixture of 2 or more useful strains.

Seed dressings and dusts may comprise the fungus with finely divided solids such as talc, attapulgite clay, kieselguhr, chalk, diatomaceous earths, calcium phosphates, calcium and magnesium carbonates, and other inorganic and organic solids such as peat, which act as carriers, with adhesive sometimes being employed to bind the material to the seed.

Granules may comprise porous or nonporous particles such as attapulgite clay or sand, for example, which serve as carriers for fungal spores etc. The particles may either be impregnated with the nematophagous fungus or coated with the fungus, adhesives and carriers sometimes being employed.

The nematophagous fungal strains of the present invention may be made into liquid concentrates by emulsification in suitable liquids and into solid concentrates by admixture with talc, clays and other known solid- carriers used in the pesticide art. The concentrates are compositions containing an effective amount of the fungus and inert material, which includes surface-active dispersing, emulsifying and wetting agents commonly used in the art.

Many variations of spraying and dusting compositions known in the art may be used by substituting said nematophagous fungal strains of this invention into compositions known or apparent to the art.

The nematophagous fungal strains of this invention may be formulated with other active ingredients, including other nematicides, insecticides, acaricideε, fungicides, plant growth regulators, fertilizers, etc. In using the compositions to control nematodes, it is only necessary that an effective amount of the fungus be applied to the locus where control is desired, generally a soil locus where agricultural crops are grown. When applied to soil, it is advantageous to mix or incorporate the fungus into the soil. Liquid compositions may be injected into the soil or sprayed on the surface. Solid compositions may be applied by broadcasting or in bands.

Previous workers have isolated strains of V. chlamydosporium and shown these to be pathogenic to a single Meloidoqyne species, either M. arenaria (Godoy, e al 1983) or M. incognita (Kerry 1988). Surprisingly we have found isolate AC is able to control four different nematode species, M. arenaria, M. incognita, M. hapla and M. javanica. Such broad spectrum control has not previously been described for V. chlamydosporium nor has this fungus previously been described as a pathogen of M. hapla or M. javanica.

The present invention comprises V. chlamydosporium strains effective for the control or elimination of Meloidoqyne species of nematodes, especially strain AC and their use as bio-control agents. Procedures for the selection of V. chlamydosporium strains which have useful activity against the target nematode species, and the use of one such strain to control Meloidoqyne infections of plants are exemplified in the following examples.

Example 1. Infection of nematode eggs on aga_τ

A plug (5 mm diameter) taken from a nutrient agar culture of the fungus was placed in the centre of a Petri dish (5 cm diameter) containing distilled water agar (0.8%). When the agar was colonised, usually after 2 weeks, a 0.5 ml aliquot of an egg suspension containing about 300 eggs was pipetted into the colony. Five dishes were inoculated for each fungal isolate; dishes containing only nematode eggs were set up as controls. The dishes were incubated at 18°C for 3 weeks before they were scanned and the proportion of eggs infected determined (Table 1).

The considerable range of rates of infection of nematode eggs in this test highlights the need for careful^' selection of potential biological control agents. In general isolates giving 40% or higher infection of M. incognita eggs (18 out of 103, including strains AC, AE, AF, AG and AH) are considered worthy of further investigation.

TABLE 1 The infection of M. incognita eggs exposed to 103 different isolates of V. chlamydosporium on distilled water agar

Nematode species Mean % Standard Range

Infection Error

Root-knot nematode, 29 1.1 4-63

Meloidoqyne incognita

R-rample 2 Colonisation of the rhizosphere

Barley seeds were coated with approximately 10- conidia /seed and air dried before planting in sterile vermiculite. After 7 days 2 roots were removed from each seedling and cut into 1 cm sections. The sections were placed on agar plates and incubated to allow fungal development and the proportion of root segments colonised by V. chlamydosporium determined.

The range in root colonisation by the 103 isolates tested varied from 0-100%, with 60 colonising >70% of the roots. Of the 18 isolates shown to infect 40% or more M. incognita eggs in Example 1, 11 (including strains AC, AE, AF, AG and AH) colonised >70% of the roots, whilst the other 6 colonised <30%. Isolates colonising >70% of the roots and 40% or more eggs were considered for futher investigation.

Example 3 Ability to produce Chlamydospores

Although we do not rule out the use of any part of the fungus for the control of nematodes we have found that the best results are obtained with the use of the chlamydospores. Culture methods which favour the formation of high yields of these spores are therefore highly preferred. The chlamydospores are preferably separated from other parts of the fungal biomasε before use.

During the course of this research programme the ability of different strains to produce chlamydospores has been found to vary considerably. Therefore the ability to produce chlamydospores has been found to be a particularly important selection criterion. The range of yields of chlamydospores has been shown to be 0 - 4.7x10^ chlamydospores/g of grain in a solid culture. Six of the 10 strains selected for good pathogenicity and good root colonisation as in Examples 1 and 2 were found to produce greater than 8.6x10- chlamydoεpores/g grain (Table 2), these included strains AC, AF, AG and AH, with strain AC producing the highest yields on solid media TABLE 2 Summary of Chlamydospore yields on solid media

Strain No. Chlamydospores / g grain

AC 4.7 x 10⁷

AF 1.7 x 10⁷

AG 8.6 x 10⁵

AH 3.4 x 10⁷

a) Method to produce chlamydospoires on solid media:

1. Mix 30 g (ca. 100 ml) milled grain (wheat or barley) with an equal volume of coarse sand, and wash on a 53 urn aperture sieve.

2. Partially dry the mixture and put into a 250 ml flask, cover with a foil cap and autoclave at 15 p. s. i. (one bar) for 1 hour.

3. Remove the foil cap in a laminar flow unit and allow the mixture in the flask to dry until it is friable, usually after 30 ins - 1 hour.

4. Inoculate the flask with a plug (d. 5 mm) taken from an agar culture of the fungus, replace the foil cap, and incubate. The optimum temperature for growth and sporulation depends on the isolate being cultured: 18°C for isolates AA and AB and 25°C for isolate AC.

5. Shake the flask vigorously once a day for the first 5 days to distribute the fungus throughout the sand/grain mixture, then leave the flask undisturbed for the hyphae to continue growing and to sporulate.

6. At the time of optimum chlamydospore production (2-3 weeks after inoculation) wash the culture mix on a 53 urn sieve with a fine spray of water. Chlamydospores will pass through and can be collected on a 10 urn aperture sieve

Example 4 Control of M. arenaxia

Soils were inoculated with three different strains of V. chlamydosporium, strains AC, AD and AE on colonised sand/bran, at a rate of 1% (w/w) (sand/bran mixture contained approximately 2% chlamydospores, with some hyphae and conidia), before planting with young tomato plants. 2 weeks later the pots were inoculated with M. arenaria larvae. The establishment of the fungal isolates in the soil, on the root surface and on the surface of developing galls was monitored by plating samples of soil washings or macerated sections of washed root and gall tissue onto selective media. After 7 weeks pots were assessed for the control of M. arenaria (Table 3).

TABLE 3 The effect of three strains of V. chlamydosporium on post-cropping populations of M. arenaria

Treatment No. of eggs and % Eggs infected juveniles/g soil

Control 137 0

Strain AC 29 32

AD 124 0

AE 164 0 From the three isolates tested only strain AC had a significant (p<0.001) effect on the numbers of healthy eggs and juveniles of M. arenaria, which were more than 80% fewer in soil treated with this fungus than in soil treated with uncolonised sand/bran. Also, strain AC was the only one which could be re-isolated from nematode eggs at the end of the experiment, when approximately 30% of the eggs were infected. Only strain AC was found to increase significantly around the tomato roots, and the growth of strain AC, but not the others, was greater on galled root tissue than on uninfected roots.

Example 5 Control of M. incognita by isolate AC

In another test, 3 more isolates of V. chlamydosporium AF, AG and AH were compared to strain AC for their ability to colonise soil, roots and control M. incognita populations in pots. These isolates were selected for their ability to infect M. incognita eggs on agar, their ability to colonise roots and their ability to produce chlamydospores as described in examples 1, 2 and 3 (Table 4).

TABLE 4 Results of tests described in examples

Strain

AC AF AG AH

Soils were inoculated with 10,000 chlamydospores/g of soil (0.2 mg/g soil ie. 0.02%) and planted with 4 week old tomato plants. After allowing the plants to establish for 2 weeks the pots were inoculated with 1000 M. incognita larvae. Pots were kept at 25-30°C for 7 weeks then assessed for colonisation of soil and roots by the fungus, plant growth and control of nematode multiplication (Table 5).

TABLE 5

Treatment -??-

Control 0 0 0 0 53.4

Strain AG 47 4.3xl0⁴ Strain AH 54 1.5xl0⁴

Suprisingly, only strain AC was found to infect a significant proportion of nematode eggs and provided considerably greater control of nematode multiplication than any of the other isolates tested. Strains AC and AF were found to establish significantly better in soil than the other two isolates, although strain AC colonised the plant roots more effectively than AF, which was found to have a detrimental effect on plant growth. Strain AC was therefore found to have an unusual combination of properties which achieved a suprisingly high level of control of M. incognita and M. arenaria. The level of control acheived by strain AC was considerably greater than that acheived by any other isolate tested and could provide effective control of Meloidoqyne populations in practical situations.

Example 6 Control of 4 Meloidoqyne species

Soil was mixed with 10,000 chlamydospores/g of soil of V. chlamydosporium strain AC and planted with tomato plants. Fots were then inoculated with 1000 juvenile nematodes from one of the following 4 species, M. hapla, M. javanica, M. incognita, M. arenaria. Pots were kept at 25-30°C for 7 weeks. The level of control of the nematode population was then assessed (Table 6).

Strain AC was found to significantly reduce nematode populations of all four nematode species. V. chlamydosporium has not been previously described as a pathogen of M. hapla or M. javanica, hence this broad spectrum control of several nematode species by a single isolate of V. chlamydosporium is a novel feature of strain AC.

TABLE 6 Effect of V. chlamydosporium strain AC on

M. hapla, M. javanica. M. incognita, M. arenaria

No. lavae e mass

Example 7 Control of M. incognita

Strain AC was compared to the chemical nematicide aldicarb, for the control of M. incognita on tomatoes. Chlamydospores of strain AC were thoroughly mixed with soil at either 1,000 or 10,000 chlamydospores/g soil. These treatments were compared to the aldicarb drench (1.5 ppm) and untreated controls. Pots were kept at 25- 30°C for upto 20 weeks. The level of control of nematode populations and plant galling was then assessed (Table 7).

After one nematode generation both rates of V. chlamydosporium inoculum significantly (p<0.001) reduced the population of M. incognita, reducing the number of larvae produced by approximately 80% compared to the uninoculated control. This level of control was comparable to the aldicarb drench.

After two to three nematode generations V. chlamydosporium strain AC still reduced nematode populations by 80%, when applied at a rate of 10, 000 chlamydospores/g soil, which was significantly (p<0.05) better than the aldicarb treatment. At a rate of 1, 000 chlamydospores/g soil strain AC was not significantly different from the aldicarb treatment.

TABLE 7 Effect of V. chlamydosporium strain AC and aldicarb on damage caused by M. incognita after two nematode generations.

Treatment Galling Index (1 - 10)

Control 7.3

Aldicarb 4.3

Strain AC, 1, 000 spores 4.5

Strain AC, 10, 000 spores 4.0

The level of plant damage or galling caused by M. incognita was assessed using an arbitary 0-10 scale, on which plants scoring 0 have no galls and plants scoring 10 are severely galled and dead (Table 7). Both rates of application of V. chlamydosporium strain AC significantly (p<0.001) reduced galling, and were comparable with the aldicarb treatment after two to three nematode generations.

Example 8 Control of M. incognita by V. chlamydosporium in combination with Pasteuria penetrans.

The effect of combining V. chlamydosporium strain AC with another nematicidal agent, P. penetrans was investigated by inoculating soil with either 10³ (L) or 10⁴ (H) spores/g of strain AC (ACL, AC. H) or P. penetrans (Pp. L, Pp. H), either alone or in combination. The soils were planted with young tomato plants and left for 2 weeks before being inoculated with 250 M. incognita larvae per pot. Seven weeks later the pots were harvested and the control of M. incognita determined (Table 8).

The results show that both strain AC and P. penetrans at both densities significantly reduced M. incognita populations compared to the control, although only strain AC was as effective as aldicarb when used alone. Strain AC and P. enetrans used in combination at 10⁴ spores/g provided significantly better control of M. incognita after 2 generations than either alone.

TABLE 8 Control of M. incognita by Strain AC and P. penetrans, alone or in combination

Treatment no. larvae %control no. larvae .control /pot (xlOO) /pot(xlOO) 1st harvest 2nd harvest

Control 78.0 2, 086

82 894 57

References

Godoy, G. , Rodriguez-kabana, R. and Morgan-Jones, G. (1983) Fungal parasites of Meloidogyne arenaria eggs in an Alabama soil. A mycological survey and greenhouse studies. Nematropica, 13 : 201-213

Kerry, B. R. (1988) Two microorganisms for the biological control of plant parasitic nematodes. Proceedings of the Brighton Crop Protection Conference : Pests and Diseases 603-607.

Claims

1. Verticillium chlamydosporium strain AC, a sample of which has been deposited at the Culture Collection of the CAB International Mycological Institute, (IMI) on the 1 August 1989, under the accession number CMI CC 334168, or a derivative, variant or mutant thereof having nematicidal activity.

2. A nematicidal composition of Verticillium chlamydosporium strain AC, or a derivative, variant or mutant thereof having nematicidal activity together with a carrier or diluent therefor.

3. A nematicidal composition according to claim 2, including a gum, sticker, adhesive and/or surfactant.

4. A nematicidal composition according to claim 2 or 3, in which the Verticillium chlamydosporium is in the form of chlamydospores, conidia and /or hyphal fragments.

5. A nematicidal composition according to any of the claims 2 to 4 for the control of Meloidogyne species of nematodes.

6. A nematicidal composition according to any of the claims 2 to 4 for the control of Meloidogyne incognita, M. arenaria, M. avanica and M. hapla.

7 A method for the control of Meloidogyne species of nematode which comprises the application to plant seeds, roots, soil, compost or growing environment of Verticiliu chlamydosporium strain AC, or a derivative, variant or mutant thereof, or nematicidal composition according to any of the claims 2 to 6.