Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/145—Fungal isolates
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi

Definitions

• the present invention relates to a new nematophagous agent intended for combating nematodes of the genus
• 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.
• chemical nematicides tend to be banned in many countries, as is the case in the Netherlands, Switzerland, Germany.
• 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.
• 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
• FIG. 3 represents a table giving the result obtained with an electrophoresis gel used to differentiate the different strains of ArtiiroJotrys conoides.
• the six strains of ⁇ rt ⁇ roJotrys 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).
• 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 ⁇ m.
• 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 ⁇ m.
• the 42B strain comes from the Baarn mycotheque in the Netherlands.
• 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 ⁇ m.
• 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 ⁇ m.
• 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 ⁇ m.
• 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 ⁇ m.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the seeds to use preferably are those of canarygrass, lentil and 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.
• 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 Myco reviews, implies great respect for the operating conditions:
• 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.
• 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.
• the paper is removed and soaked in a liquid containing vitamins and microelements.
• 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 lyophilized fungus is in the form of a powder which can be diluted in water, therefore more easily usable by the farmer.
• the fungus can be used in the same way as a chemical, by mechanical spreading.
• 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 agar contained in the dishes is inverted on a 100 ⁇ m 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.
• strains A, A 'and B trap between 60% and 100% of nematodes in 24 hours, the other three strains being below this threshold .
• the six strains trap between 50% and 75% of the nematodes in 24 hours.
• 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.
• the tubes were treated as follows: Control soil not containing predatory fungus
• VU Soil containing the Vl strain the nematodes are inoculated on the same day as the fungus is incorporated into the soil.
• 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.
• 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.
• 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.
• Mass number 65 150 145 150 125 123 163 eggs / plant
• strain A 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.
• strains A, B, T and VI 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 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.
• 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 .
• 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 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 ⁇ m sieve and counted with a binocular magnifier. They will constitute the infesting potential for the next crop.
• 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.
• 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.
• 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.
• 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 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 bisporu ⁇ . In the latter case, the nematophagous fungus can be added to the compost after pasteurization.

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• 1994
  • 1994-06-15 FR FR9407553A patent/FR2721175B1/fr not_active Expired - Fee Related
• 1995
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