EP0766513A1

EP0766513A1 - Nematophagic agent

Info

Publication number: EP0766513A1
Application number: EP95923388A
Authority: EP
Inventors: Elisabeth Panchaud
Original assignee: IDRO 2000 SA
Current assignee: IDRO 2000 SA
Priority date: 1994-06-15
Filing date: 1995-06-14
Publication date: 1997-04-09
Also published as: JP3849882B2; MX9606395A; NO965337L; FR2721175B1; CA2191961A1; BR9508020A; NZ288651A; WO1995034209A1; AU2796095A; US5811092A; FR2721175A1; OA10338A; AU688919B2; NO965337D0; JPH10501547A; CN1156954A

Abstract

Nematophage agent intended to combat nematodes of the Meloidogyne, Heterodera and Ditylenchus Myceliophagus genus, characterised in that it is selected within a group of six strains of Arthrobotrys conoides Drechsler (42A, 42A', 42B, 42Br, 42T and 42Vl, deposited at the national collection of cultures of micro-organism (Institut Pasteur) under the numbers I-1425 to I-1430, and application process consisting in incorporating the nematophage agent into the ground wherein the cultures are grown by means of grains which have been seeded previously with the inoculum of the nematophage agent.

Description

New nematophagous agent against nematodes of the genus

Meloidogyje

Technical area

The present invention relates to a new nematophagous agent intended for combating nematodes of the genus

Meloidogyne, and a method of combating the proliferation of these nematodes by using new strains of the fungus ArtΛro-otrys conoides Drechsler.

State of the art

It has long been known that phytophagous nematodes cause considerable losses to crops, estimated at 3 billion francs each year. The most widespread and most damaging species in the world belong to the genus Meloidogyne. This genus better known to farmers under the name of "gall nematodes" in fact induces the formation of galls on the root system of attacked plants. These extremely polyphagous nematodes attack almost all crops and are therefore responsible for catastrophic yield reductions of the order of 50 to 70%. In many areas, Heterodera carotae is a dangerous pest specific to carrot crops for which its bites trigger abnormal production of lateral rootlets and which makes carrots unmarketable. Another nematode, Ditylenchus myceliophagus, can cause significant damage to agaric crops in mushroom houses.

To fight against these pests, farmers have several means which, in intensive cultivation, boil down to:

- either a preventive disinfection of the soil before cultivation using fumigants (Methyl Bromide, Chloropicrin, Dichloropropene ...). These products, with a very broad spectrum of action, sterilize the soil and thus destroy the ecological balance of the environment. In addition, in light soil, their residues can pass into the water table, and in heavy soil their effectiveness is reduced by their poor diffusion either to curative treatments on certain crops in place using systemic products, carried by the sap (carbamates or organophosphates), this for non-consumed productions such as flower crops or nurseries. These products are dangerous for both animals and humans because they pass into the plant, leaving toxic residues there. These two techniques only work on the first thirty centimeters of soil. Nematodes which can be found at a greater depth will again invade the treated area during the following culture. This implies a continual repetition of disinfections. In addition, because of their toxicity to humans, several chemical nematicides tend to be banned in many countries, as is the case in the Netherlands, Switzerland, Germany.

Statement of the invention

This is why, the object of the invention is to use a natural agent to fight against nematodes, which is not dangerous for man and his environment.

The main object of the invention is therefore a new nematophagous agent intended to combat nematodes of the genus Meloidogyne, Heterodera and Ditylenchus myceliophagus, chosen from six strains of the fungus ArtiiroJbotrys conoides Drechsler.

Another subject of the invention is a method of combating nematodes of the genus Meloidogyne, Heterodera and

Ditylenchus myceliophagus consisting of incorporating the Art Kingotrys conoides Drechsler fungus into the soil where the cultures are carried out by means of seeds sown beforehand by the inoculum of the fungus.

Brief Description of the Figures The objects, objects and characteristics of the present invention are the subject of the following description for which the accompanying drawings illustrate certain aspects of the invention.

FIG. 1 schematically represents a conidiophore and its conidia of strains c \ * Arthrobotrys conoides according to the invention.

FIG. 2 is a schematic representation of the traps formed by the strains of Arthrobotrys conoides according to

The invention for capturing nematodes. FIG. 3 represents a table giving the result obtained with an electrophoresis gel used to differentiate the different strains of ArtiiroJotrys conoides.

Detailed description of the invention

The six strains of ΛrtήroJotrys conoides Drechsler which are the subject of the invention come from different places. The main criterion for verifying that the different strains belong to the same species is the measurement of conidia (reproductive organs of these fungi).

As illustrated in FIG. 1, the conidia are grouped by 20 or 30 on the conidophore. The conidiophores can continue to develop beyond a first head of conidia and after a certain length of growth form a second group of conidia and so on until the development of 5 to 6 additional groups.

The 42A strain originates from Fada N'Gourma in Burkina Faso, it is constituted by a mycelium composed of hyaline cloisonné hyphae.

The conidiophores end in a head of about 20 tight conidia; on older crops they continue to develop and may successively produce 5 to 10 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and consist of two cells of different sizes, the total length of the conidia is on average 27 μm.

This strain was the subject of a deposit with the National Collection of Culture of Micro-organisms (CNCM) at the Institut Pasteur, where it was registered under the number 1-1425.

The 42A 'strain originates from Leguéma in Burkina Faso.

The conidiophores end in a head formed from 3 to 20 tight conidia; on older crops, they continue to develop and can successively produce 3 to 5 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are made up of two cells of different sizes, the total length of the conidia is on average 30 μm.

This strain was the subject of a deposit with the CNCM (Institut Pasteur), where it was registered under the number 1-1426.

The 42B strain comes from the Baarn mycotheque in the Netherlands.

On young cultures (less than a month old), conidiophores are rare and end in a head formed from 3 to 20 tight conidia; the formation of additional conidial groups is rare. The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are made up of two cells of different sizes, the total length of the conidia is on average 28 μm.

This strain was the subject of a deposit with the CNCM (Institut Pasteur), where it was registered under the number 1-1427.

The 42Br strain comes from Brazil. O 95/34209 PC17FR95 / 00785

The conidiophores end in a head formed from 10 to 20 tight conidia. They continue to develop and successively produce 5 to 10 additional groups of conidia. The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and consist of two cells of different sizes, the total length of the conidia is on average 23 μm.

This strain was the subject of a deposit with the CNCM (Institut Pasteur), where it was registered under the number 1-1428.

The 42T strain was collected at Tours Indre-et-Loire (France).

The conidiophores end in a head formed from 10 to 20 tight conidia. On the old cultures, they continue to develop and successively produce 3 to 5 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are made up of two cells of different sizes, the total length of the conidia is on average 28 μm.

This strain was the subject of a deposit with the CNCM (Institut Pasteur), where it was registered under the number 1-1429. The 42V1 strain was taken at Villeneuve Loubet Alpes-Maritimes (France).

The conidiophores end in a head formed from 10 to 20 tight conidia. On older crops, they continue to develop and successively produce 20 to 30 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and consist of two cells of different sizes, the total length of the conidia is on average 23 μm. This strain was the subject of a deposit with the CNCM (Institut Pasteur), where it was registered under the number 1-1430. The mycelium of nematophagous fungi has the ability to produce capture organs that trap nematodes. The mycelial traps of the six strains of ArthroJ otrys conoides according to the invention are in the form of hyphal loops which by anastomosis become more or less complicated networks as illustrated in FIG. 2.

Of the six strains studied, only the strain 42A spontaneously forms traps. The other 5 form traps induced by the presence of nematodes in the culture medium. Whether spontaneous or induced, the traps are made up of hyphal loops which anastomose become more or less complicated networks.

The study of the genome of the six strains by the RAPD technique makes it possible to differentiate unambiguously the six strains thanks to an oligonucleotide.

The use of an electrophoresis gel made it possible to reveal the results of FIG. 3 where the bands have been diagrammed, and where the control is used to determine the size of the DNA fragments. The different strains of Arthrobotrys conoides can be used industrially to fight against nematodes using the methods described below.

The use of a seed-based culture medium allows good preservation of the fungus. The latter takes refuge inside the seed and therefore becomes less sensitive to attack from the outside environment. In addition, each seed spread on the ground is the starting point for a new mycelial colony. The seeds are soaked for 24 hours in excess water before being sterilized. Then the seeds are sown with the mushroom inoculum.

The best species to use as support are those that allow maximum growth to be achieved in the shortest time. A second criterion is the number of propagules per gram of support medium reached at the time of maximum growth, the propagation corresponding to a minimum fragment of mycelium or even a single conidium.

The species that can be used are among grasses, legumes, or oilseeds. Thus, we can use hemp, sunflower and rice for which the time to reach maximum growth is 14 days. Oats, sorghum, barley, wheat, lentils, corn, canarygrass, soybeans, beans, lupins, chickpeas, and beans can still be used. Among the above species, those which make it possible to obtain a maximum number of propagules, that is to say approximately 3.10 ⁷ / g, are lupine, lentil, corn, bean, canary seed, chickpea and rice.

For a better distribution of the fungus, it is preferable to spread, with equal weight, small seeds. For this reason and others, the seeds to use preferably are those of canarygrass, lentil and corn. In the case of corn, the incorporation into the soil is satisfactory at the rate of 2 g per liter of soil. Due to the time required to obtain maximum growth, the fungus on its support is preferably incorporated into the soil about 15 days before planting.

The different strains of Arthrobotrys conoides can also be used in the form of a lyophilized product. It is recalled here that lyophilization is a drying technique by sublimation of ice from solutions or suspensions of tissue, previously solidified by freezing. This technique commonly used for the conservation of micro¬ organisms in National Mycothèques, implies great respect for the operating conditions:

. low temperature to ensure product freezing. reduced pressure to ensure sublimation of the ice absence of interstitial fluid during sublimation of the ice.

To fulfill these conditions, it suffices to induce an imbalance between the pressure and the temperature at the level of the product to be freeze-dried by raising the temperature and to prevent the equilibrium from being restored by evacuation of the steam.

Lyophilization of fungi can not be done on the mycelium too sensitive to temperature and pressure variations, we must therefore make the fungus fruit to obtain conidia which can be assimilated to resistance organs capable of being lyophilized. This need to use conidia eliminating the use of any liquid medium, the fungus is grown on an agar medium based on soy flour.

In order to eliminate the drawback presented by the presence of agar-agar (agar) and by the weight of the culture medium (soy flour), a sheet of cellophane paper is interposed between the agar medium and the inoculum of the mushroom. This paper, while separating the fungus from the agar medium, has the particularity of allowing the fungus to feed through the surface of the paper.

Once the mycelium has invaded the entire surface of the culture medium, the paper is removed and soaked in a liquid containing vitamins and microelements. In the liquid, the mycelium and the conidia detach from the cellophane paper. The whole consisting of the mycelium, the conidia and the vitamins and microelements is then lyophilized. The presence of vitamins and micro-elements will allow better recovery in the field.

The originality of this technique consists in the preliminary separation of the fungal fraction from the nutritive medium which was used for its culture. Thus, the lyophilized fungus is in the form of a powder which can be diluted in water, therefore more easily usable by the farmer. In this form, the fungus can be used in the same way as a chemical, by mechanical spreading. In addition, this formulation has the advantage of considerably reducing the quantities of product to be spread on the ground and allows better and longer conservation of the product. Due to the time required to obtain maximum growth, the fungus Arthrobotrys conoides is preferably incorporated into the soil to be treated about 15 days before planting.

The effectiveness of the different strains of Arthrobotrys conoides against Meloidogyne, Heterodera and Ditylenchus myceliophagus was first tested in in vitro tests carried out in a Petri dish. To carry out these in vitro tests, the different strains of Artήroiotrys conoides were cultured on agar medium (Corn meal agar: 1, Agar-agar: 1) in Petri dishes. When the fungus has invaded all of the agar, 200 nematodes are aseptically placed in each box of mushrooms including control boxes containing only agar medium. After a period of 1 hour, 2 hours, 8 hours, and 24 hours, the agar contained in the dishes is inverted on a 100 μm mesh sieve, the bottom of which is flush with the water of an underlying Petri dish. This arrangement allows nematodes not trapped by the fungus to actively cross the sieve fabric to reach the water. The non-trapped nematodes are then counted under the magnifying glass.

In the case of Meloidogyne and Ditylenchus myceliophagus, the six strains separate into 2 groups: strains A, A 'and B trap between 60% and 100% of nematodes in 24 hours, the other three strains being below this threshold .

In the case of Heterodera carotae, the six strains trap between 50% and 75% of the nematodes in 24 hours.

It has also been observed that the development of the fungus was rapid (on average 1.5 cm per 24 hours). It can therefore be used satisfactorily in practice as a nematophagous agent in cultures vegetable and floral as well as in nursery plants.

In the context of tests relating to the ecology of new strains of nematode predator fungus, it has been observed that they develop well in soils with a salinity of not more than 3 g / l.

It has also been noted, moreover, that the predatory fungus invades the medium much more intensely when it is introduced at a high dose, but this does not prevent it from colonizing the substrates appreciably even at a dose of 10% of the volume of soil. .

The predatory activity of the different strains of Arthrobotrys conoides 42 has also been confirmed by a certain number of tests of practical applications, some results of which are given below for information.

I. Glasshouse test against Meloidogyne

The strains studied fall into 2 categories with different trapping capacity: A, A 'and B trap on average 80% of nematodes in 24 hours; Br, T and VI only trap 30% of Meloidogyne in 24 hours.

To carry out this test, we took the 2 most representative strains in each category, A and VI. This test was carried out in 50 ml tubes containing 30 ml of soil. The strains of mushrooms cultivated on a medium based on cooked and sterilized cracked corn were incorporated into the soil at a dose of 100 g / m ² .

The tubes were treated as follows: Control soil not containing predatory fungus

A1 Soil containing strain A, the nematodes are inoculated on the same day as the fungus is incorporated into the soil.

VU Soil containing the Vl strain, the nematodes are inoculated on the same day as the fungus is incorporated into the soil.

A15 Soil containing strain A, the nematodes are inoculated 15 days after incorporation of the fungus into the soil.

V1 15 Soil containing the strain V1, the nematodes are inoculated 15 days after incorporation of the fungus into the soil.

A1 + V1 15 The strain V1 is incorporated into the soil (in half dose: 50 g / m ² ) 15 days before inoculation of the nematodes, and the strain A is added in half dose on the same day as the nematodes.

On D-Day, 30 Meloidogyne larvae are deposited in each tube. After 24 hours, the tubes of each series are taken out and their content is analyzed in order to know the number of trapped nematodes. This manipulation is repeated every day until all the nematodes are fully trapped.

The results obtained presented in the following table show that, whatever the strain of ArtiiroJotrys conoides, if the fungus is incorporated 15 days before cultivation (therefore before the activation of nematodes by its host plant), trapping varies significantly during the first few days, but this difference quickly fades.

% average of% average of% average of nematodes trapped with nematodes trapped with nematodes trapped in

D + 1 D + 2 D + 3

A1 46.14 66.67 90.28

SEEN 32.09 59.44 87.22

A15 63.12 80.28 94.44

V1 15 61, 8 86.11 93.06

A1 + V1 15 44.92 88.89 95.06 II. Greenhouse test in mini-pots with Meloidogyne hapla

The purpose of this test is to highlight the action of the fungus Arthroiotrys conoides on the nematode Meloidogyne hapla as well as to specify the quantity of product useful per m ² . The 6 strains of Arthrobotrys conoides were tested with this nematode. The test was carried out in a mini-pot containing 300 g of soil, the plant used was a St Pierre tomato, a variety very sensitive to nematodes.

Each pot was contaminated with 300 Meloidogyne hapla or 100 nematodes per 100 g of soil, which corresponds to the upper threshold of an average infestation in the fields.

The results were read using a coloration of the roots with eosin, this vital coloration making it possible to count the masses of first generation eggs from the infesting larvae which have entered the root system.

Arthrobotrys conoides is a predatory fungus which, in the absence of nematodes, can be maintained in the soil by digesting organic matter (saprophagous nutrition). This is why to the sterile soil of the station is added a sterile organic amendment (without micro¬ organisms) which allows a better development of the fungus during the first 15 days of the test.

Calculation of treatment doses: the number of propagules equivalent to 10 ⁸ propagules / m ² of soil is taken as a basis. However, for the 6 strains of ArfchroJotrys conoides, this figure is reached on average with 15 g of preparation based on crushed and autoclaved corn seeds. To leave some room for maneuver, the average dose tested common to the 6 strains was 40 g / m ² , the second test dose was twice that of 80 g / m ² . 5 repetitions were made for each strain and for each dose, without forgetting a control without fungus. Results: for the average test dose common to the 6 strains and corresponding to 40 g / m ² the results are as follows:

A A 'B Br T VI Witness

Mass number 65 150 145 150 125 123 163 eggs / plant

% efficiency of 60% 8% 11% 8% 23% 24% mushroom

The best results have been obtained with strain A.

At the double test dose of 80 g / m2 the results were as follows:

2A 2A '2B 2Br 2T 2V1 Control

Mass number 32 116 133 146 94 77 163 eggs / plant

% efficiency of 80% 28% 18% 10% 42% 53% mushroom

As for the 40 g / m ² dose, the best results were obtained with strain A, but strain VI at this dose is also effective. To obtain rapid and reliable results, it is better to use strain A at a dose of 80 g / m ² when it is produced on a medium based on cooked cracked corn.

III. Pot test on cultivation of carrots attacked by Heterodera carotae

This test was carried out with strains A, B, T and VI. To make this test, the different strains were cultivated on a substrate based on cracked cooked corn. The quantities of mushroom brought in were rigorously adjusted according to the strains so that there is always the same number of propagules per liter of soil (10 ⁸ propagules / 100 1). For strains A and B the inoculation dose was 60 g / 100 l of soil. For strain T the inoculation dose was 190 g / 100 1. Finally for strain VI, a dose of 80 g / 100 1 of sol was used. A culture of carrots not treated with fungus served as a control.

This test made it possible to count the cysts of Heterodera carotae resulting from the generation of the infesting larvae.

A B T Vl Witness

No. of 39 49 96 40 106 cysts

% efficiency of 63% 54% 9% 62% mushroom

We note that with Heterodera carotae as with Meloidogyne hapla, the strains which give the best results are strains A and VI.

IV. Greenhouse test on tomato cultivation

This test was carried out in a greenhouse, in an agricultural environment with Arthro & otrys conoides 42A. It is intended to evaluate the dose of predatory fungus to be spread on the ground.

For this test, the fungus is grown on a medium based on cracked corn. The content of this medium is 10 ⁷ propagules / g.

The plot available to us is 120 m ² . It was divided into three zones of 40 m ² each, which are separated from each other by planks in order to isolate them well. The first plot constitutes the control area, it is followed by an area treated with 50 g of preparation of 42A per m ² , then a plot containing 100 g of preparation per m ² .

Before setting up this test, a first analysis was made on soil sampled between the culture in place (salad).

It was carried out by placing young sensitive tomato plants (St Pierre variety) in this soil to reveal the presence of nematodes. One month after planting, the roots are colored with Lactophenol Blue-Cotton (from GUIRAN, 1966). This coloration highlights the juveniles of Meloidogyne having penetrated into the roots of the tomato.

We found 620 Me2oidogyne larvae / 10 g of roots, which corresponds to an average infestation. This average infestation rate is ideal for testing the nematophagous fungus. Indeed, like all means of biological control, it acts slowly and cannot manage to regulate a very strong swarming of nematodes on a single crop campaign. ArtήroJbotr s conoides 42A was applied 15 days before planting the tomato plants. This 15-day period allows the predatory fungus to start colonizing the soil and therefore to be operational when the crop is set up. Indeed, the tomato plants emit radical exudates into the soil, which activate the hatching of Meloidogyne eggs and allow the young larvae to reach the roots.

The tomato plants are distributed in four rows, in the two middle rows (in order to avoid border effects), 10 feet of sensitive tomatoes (St Pierre variety) have been distributed, these plants will be uprooted for analysis .

The last samples were taken at the end of the culture. Sensitive plants were uprooted and the roots analyzed.

For this, the radical system once washed is ground in a 1% solution of calcium hypochlorite at 12 * chlorometric. This ground material is passed through a series of sieves in order to remove the fragments of plant tissue. The Meloidogyne eggs from the root are collected on a 5 μm sieve and counted with a binocular magnifier. They will constitute the infesting potential for the next crop.

Control plot Plot at 50g / m ² Plot at 100g / m ²

Number of eggs per 10 g of 44 581 25 838 17 725 root

It is good to clarify certain points:

- a Meloidogyne female can give, depending on the climatic conditions, from 300 to 3000 eggs, an infestation is considered to be average when the number of larvae per 10 g of root is between 100 and 1000.

There is a significant decrease in the infestant potential (more than half) between the control plot and the plot treated with 100 m ² of ArtiîroJ otrys conoides 42A.

For plants available in lumps, pots or containers, the nematophagous fungus is directly incorporated into the root ball, bucket or container.

The sowing by the predatory fungus is carried out from the start in the pressed clods which are used for the manufacture of market garden plants. By operating in this way, it appeared that in the 20 to 30 days when the newly transplanted plants in the clods remain in the greenhouse in hot and humid conditions, the predatory fungus has time to grow and invade all of the clod which then behaves like an inoculum when it is placed in culture. This variant of the process according to the invention, applicable to the deliverable plants O 95/34209

17

in clods, buckets, containers or the like has been found to have the following decisive advantages:

The soil usually used for making clods is a mixture rich in humus, at neutral pH, it is therefore particularly favorable for the development of Arthroiotrys conoides 42. We have also found that the plant's root system is literally enveloped in the mycelial felting which does not however absolutely disturb its growth. The root system is therefore protected from attack by nematodes, even if the root ball is planted in particularly contaminated soil. It thus appears that the young plant is well protected from the start of the vegetation, but it is generally when a plant is young that it most fears attacks by nematodes.

The various experiments carried out have shown that the root ball constitutes an important inoculum, from which the fungus very easily reaches the surrounding soil. The preparation of such lumps, buckets, container or equivalent does not raise the slightest technical problem with the use or manufacture of plants. It suffices in fact to bring the granule into the mixture for example using a hopper. The planting of the plants thus prepared is also carried out in a very usual manner.

In summary, the method of the invention can be applied to all vegetable, floral, or nursery plants, although some of these plants do not suffer too much from Meloidogyne nematodes. It can also be used on carrot crops which are sensitive to Heterodera carotae, or against Ditylenchus myceliophagus in the case of the layer fungus Agaricus bisporuε. In the latter case, the nematophagous fungus can be added to the compost after pasteurization.

Claims

O 95/34209 PCÏ7FR95 / 0078518REVENDICATIONS

1. Nematophagous agent intended to combat nematodes of the genus Meloidogyne characterized in that it is chosen from the group of six strains of Arthrobotrys conoides Drechsler consisting of strains 42A, 42A ', 42B, 42Br, 42T and 42V1.

2. Nematophagous agent according to claim 1, characterized in that said group of six strains comprises the strains deposited in the National Collection of Cultures of Microorganisms (Institut Pasteur) respectively under the numbers: 1-1425, 1-1426, 1 -1427, 1-1428, 1-1429 and 1-1430.

3. Method for combating nematodes of the genus Meloidogyne, Heterodera and Ditylenchus myceliophagus in cultures, consisting in incorporating into the soil where the cultures are carried out, the nematophagous agent according to claim 1 or 2.

4. The method of claim 3, wherein the nematophagous agent is incorporated into the soil by means of seeds previously seeded with the inoculum of said nematophagous agent.

5. The method of claim 4, wherein said seeds are selected from the group consisting of mountain climbers, lentil and corn.

6. The method of claim 5, wherein the nematophagous agent is previously seeded on a medium based on cracked corn containing at least 10 ⁷ propagules of said agent per gram.

7. The method of claim 5 or 6, wherein the nematophagous agent is previously seeded on a medium based on cracked corn, cooked and sterilized, said seeded medium then being incorporated into the soil at a dose of 100 g / ² .

8. The method of claim 5 or 6, wherein the nematophagous agent is previously seeded on a medium based on cracked corn, cooked and sterilized, said the seeded medium is then incorporated into the soil at a dose of 2 g per liter of soil.

9. The method of claim 3, wherein the nematophagous agent is previously lyophilized before being incorporated into the soil in powder form.

10. The method of claim 4, wherein the nematophagous agent is previously put to fruit on a culture medium for the development of conidia before being lyophilized.

11. The method of claim 10, wherein a sheet of cellophane paper is interposed between the nematophagous agent put to fruit and the culture medium so that the elements of said nematophagous agent containing the conidia are collected by soaking said sheet of cellophane in a liquid.

12. The method of claim 3, wherein the nematophagous agent is incorporated directly into the clod, the bucket or container which is used to deliver seedlings from nurseries or vegetable crops.

13. Method according to any one of claims 3 to 12, wherein said nematophagous agent is incorporated into the soil about 15 days before planting.