WO2023084126A1

WO2023084126A1 - Method for sampling, isolating, selecting, propagating and inoculating indigenous microbial populations of soils

Info

Publication number: WO2023084126A1
Application number: PCT/EP2022/082038
Authority: WO
Inventors: Olivier Besnard
Original assignee: Bes, Claude
Priority date: 2021-11-15
Filing date: 2022-11-15
Publication date: 2023-05-19
Also published as: FR3129060A3; FR3129060B3

Abstract

Disclosed is a method for sampling, isolating, selecting, propagating and inoculating indigenous microbial populations of soils. The method, which increases the quantity and/or quality of plants by improving the indigenous biological capacity of soils in a plot of land by obtaining fungal isolates, comprises: - a step (1) of taking soil samples from a plot of land; - a step (2) of isolating fungal spores of the genus Trichoderma spp; - a step of separately culturing isolated spores on selective media; - a step (3) of selecting the strains of these cultures that perform best for at least one agronomic property of interest; - a step (4, 5) of optimizing the quantitative and qualitative production of spores of each selected strain through solid-state fermentation on plant products or residues; - a step (6) of formulating the obtained inocula; and - a step of inoculating the formulated inocula by spreading and/or burying in the soil in the plot of land in order to restore the indigenous populations.

Description

PROCESS FOR COLLECTION, ISOLATION, SELECTION, MULTIPLICATION AND INOCULATION OF INDIGENOUS SOIL MICROBIAL POPULATIONS

TECHNICAL AREA

The present invention relates to the field of obtaining fungal isolates more particularly selected to be beneficial to the revitalization of soils and plants.

PRIOR TECHNIQUE

To date, no complete process, similar to that of the present invention, has been tested and developed. It consists of taking samples from the soil of the same plot, the isolation of "beneficial" fungal populations from the soil, the selection in the laboratory of the most efficient clones for their agronomic properties, multiplication by fermentation in a solid medium, and restitution by inoculation to the plots to be treated with the aim of increasing crop yields by improving the native biological capacities of the soil.

SUMMARY OF THE INVENTION

Addressed to farmers, plant managers or anyone responsible for maintaining plants, the proposed process aims to increase the quantity and quality of plants by improving the native biological capacities of the soil.

To this end, the present invention relates to a process for increasing the quantity and/or quality of plants by improving the native biological capacities of the soils of a plot by obtaining fungal isolates, which comprises:

- a step of taking soil samples from the plot;

- a step for isolating fungus spores of the genus Trichoderma spp;

- a step of separate culture of spores isolated on selective media;

- a step of selecting the strains of these most efficient crops for at least one agronomic property of interest;

- a step for optimizing the quantitative and qualitative production of spores of each selected strain, by fermentation in a solid medium, on residues or plant products;

- a step of formulating the inocula obtained; And

- a stage of inoculation of inocula formulated by spreading and/or burying in the soil of the plot in order to restore the multiplied native populations.

In embodiments, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the stimulation of root growth. In embodiments, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the release of organic phosphorus.

In embodiments, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the solubilization of the mineral phosphorus.

In embodiments, during the step of selecting the strains of these best performing crops, at least one agronomic property of interest is the ability to degrade organic matter.

In embodiments, during the step of selecting the strains of these best performing crops, at least one agronomic property of interest is mycoparasitism.

In embodiments, during the step of selecting the strains of these best performing crops, at least one agronomic property of interest is resistance to soil fungicides.

In embodiments, during the step of selecting the strains of these best performing crops, at least one agronomic property of interest is the induction of the natural defenses of the plants.

In embodiments, during the step of formulating the inocula obtained, nutritional supplements are added.

In embodiments, during the step of isolating spores of fungi of the genus Trichoderma spp, each spore is uniquely isolated, such that the step of culturing isolated spores separately produces populations of clones.

In embodiments, during the selection step, a step is performed

(4) analysis of the antipathogenic activity of Trichoderma.

In embodiments, the antipathogenic activity of the Trichoderma is analyzed on Sclerotinia homeocarpa, Fusarium graminearum and/or Microdochium nivale.

In embodiments, during the selection step, a step is performed

(5) analysis of the ability to colonize soil organic matter by Trichoderma.

In embodiments, during the optimization step, a step (6) of fermentation in a solid medium of the Trichoderma is carried out.

In embodiments, during the step of formulating the inocula obtained, a flow of dry air is applied and a dry fermented product is obtained, containing the biomass of the fungus essentially in the form of conidiospores, which can be used directly in solid form or after washing and filtration in liquid form, as a biological agent.

According to another aspect, a method which is the subject of the invention consists in obtaining isolates fungi, more particularly those known to be beneficial for the revitalization of soils and plants, essentially chosen from the family of molds and more particularly from the genus Trichoderma. It is characterized in that it includes the implementation of all of the following steps: a) a sampling phase, in the soils and all types of plant growing media, of several corings; b) phases of isolation and purification of molds of the genus Trichoderma spp, on selective media in the laboratory, with the aim of generating a “mycotheque” of different clones; c) the selection of the most efficient strains for several agronomically interesting properties, in particular for the stimulation of root growth, the release of organic phosphorus and the solubilization of mineral phosphorus, the ability to degrade organic matter, mycoparasitism, the resistance to soil fungicides as well as the induction of natural plant defences; d) optimization of the quantitative and qualitative production of spores of each clone selected by a fermentation process in a solid medium, on residues or plant products, in particular by sterile quality control procedures; e) the formulation of the inocula obtained with nutritional supplements for conservation and multiplication in the environments to be colonized, f) a method of inoculation of the said formulas by, injections, spreading or/and burial in the soil, in order to restore the populations natives multiplied; g) post-implantation control of the good development of the reintroduced populations, in particular by metagenomic methods, guaranteeing exact traceability of the setting in a maximum state of colonization of the soil.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 represents, in the form of a flowchart, the steps of a particular embodiment of the method which is the subject of the invention.

DESCRIPTION OF EMBODIMENTS

The process according to the invention aims to obtain strains of Trichoderma sp showing interesting agronomic properties, allowing:

- rapid growth so that they can colonize all types of soil or growing media in which they will be buried;

- an ability to metabolize phytohormones involved in the multiplication of root cells (particularly auxins), inducing a larger possible adsorption surface of soil nutrients; - solubilization properties of the phosphorus element incorporated in the residual fertilizer minerals or in the mother rock (phosphatase);

- an aptitude for saprophytic colonization that can be measured by various processes, and which translates the possibility of a good degradation of organic matter of all types of properties, allowing the release of phosphorus or nitrogenous elements, incorporated in dead organic matter (phytases, cellulases, chitinase): these forms of phosphorus and nitrogen then being made mobile and absorbable by the plant;

- a mycoparasitic power, the estimation of which consists in selecting strains with respect to different organelles (sclerotia, acerviculi, pycnidia, etc.) of different pathogens;

- an ability to resist various pesticides (fungicides, herbicides and soil insecticides), so as not to be destroyed by the co-use of synthetic molecules, as well as the possible metabolization of these by degradation of said metabolites ;

- the production of siderophores, molecules capable of chelating cations, in particular feric cations, and other trace elements;

- the ability to stimulate the defenses of the plants that one wishes to treat, in particular by measuring in them the increased activities of peroxidases, phenylamoniases, etc.

- stimulation of the enzymatic activities of degradation of recalcitrant molecules concentrated in the soil, in particular residues of herbicides, insecticides and fungicides, heavy metals, hydrocarbons (HAP), plastics (polyethylene PE, polyethylene terephthalate PET, etc. ) or other brominated, chlorinated molecules, etc.

In each of the specific cases, with the aim of obtaining the strains presenting the best possible growth and production of the said molecules involved, various tests are carried out to evaluate the performance of each strain, with the aim of selecting the most suitable for development. best in poor or rich, wet or dry, low or high temperatures. The strains thus selected will then be pre-cultivated, in agar media in Petri dishes to obtain a basic inoculum, then massively multiplied by fermentation in a solid medium on plant residues. It is a method of culture under sterile conditions of the strains of Trichoderma allowing the quantitative and qualitative production of the spores by fermentation on a solid medium (acronym "FMS"). The formulation of the inocula obtained with nutritional supplements and a method of inoculation of the said formulas by spreading and/or burying in the ground in order to restore the indigenous populations multiplied. Finally, the post-implantation control of the good development of the reintroduced populations is implemented, in particular by metagenomic methods, guaranteeing an exact traceability of the good implantation and colonization of the soil. Step No. 1: Agronomic sampling and isolation of samples:

The samples are taken from moist soil (grassed soil, market gardening, vineyard, arboreal or field crops, etc.) on the basis of samples of dimensions one to three cm in diameter, preferably two cm, and one to ten cm depth, preferably five cm, per plot to be diagnosed, the minimum number of which is 16 samples spread over the entire surface of the plot to be analyzed. The samples are grouped together in the same bag.

The three compartments (soil, plant and water) are separated. The isolation technique used is that of direct incorporation of the soil into a selective isolation medium and in the case of soil organic matter, the isolation technique used is that of dilution suspensions. The cultures are then incubated for several days. The colonies are then examined and isolated.

Step No. 2: Isolation of Trichoderma:

For the isolation of Trichoderma populations, the isolations are purified, i.e. samples containing only one spore are obtained, the only guarantee of the same genetic heritage of each. A separate culture is carried out for each dissociated spore on a rich agar medium such as PDA or rolled oats in a Petri dish. Thereafter, the cultural aspect of these strains is to be identified in order to determine the summary classification of the isolated strains.

As soon as the sample is received, part of the soil is put in an oven to determine its humidity level. The plant parts and the soil are put in sterile water with Twinn, to detach the spores. After observation under the microscope and counting with the Malassez cell, the number of total spores/ml of solution is determined. The dilution necessary to obtain approximately 100 spores is thus calculated.

A small quantity of earth is sprinkled and immediately dispersed in a selective “general purpose” isolation medium which is poured and maintained under supercooling in Petri dishes (37°C to 40°C) by stirring until solidification. In the case of soil organic matter, the technique of suspension dilutions used is as follows: After sieving and grinding in the presence of sterile water, various successive dilutions of the suspension obtained are incorporated into the isolation medium. To do this, 10 ml of each of the dilutions are poured into an Erlenmeyer flask containing 90 ml of selective isolation medium kept supercooled in a water bath (between 37°C and 40°C). After homogenization, the 100 ml are distributed in petri dishes placed under optimal conditions for isolation of Trichoderma. After an incubation time of three to seven days, the dilution showing a sufficient number of colonies but without confluence is identified. Once the dilution has been chosen, the colonies can be isolated. The specific nutrient media are: - TME medium from Papavizas (1982): a mixture of glucose (one g), agar (20 g) and distilled water (800 ml) is autoclaved. 200 ml of V-8 are added, the pH is preferably between 3.8 and 4.0. To the still lukewarm medium, neomycin sulphate (100 mg), bacitracin (100 mg), penicillin G (100 mg), chloroneb (100 mg), nystatin (20 mg), chlortetracycline HCl (25 mg), sodium propionate (500 mg).

- TSM medium from Elad et al. (1981): This medium consists of MgSO4.7 H ₂ O (0.2 g); K2HPO4 (0.9g); KCI (150mg); NH4NO3 (1.0g); glucose (3.0g); Chloramphenicol (250mg); Fenaminosulf (300mg); quintozene (200 mg); rose bengal (150 mg); agar (20 g); distilled water (1000 ml).

- Medium from Davet (1979): Ca(NO ₃ ) ₂ (1.0 g); CaCl _2.2H ₂ O (1.0 g); _KNO3 (250mg); MgSC, 7H ₂ O (250 mg); KH ₂ PC>4 (125 mg); sucrose (2.0g); citric acid (50mg); agar (25 g); distilled water (1000 ml). After autoclaving, the pH is adjusted to 4.5 with 1 N HCl and streptomycin sulphate (30 mg), vinchlozolin (2.5 mg) and allyl alcohol (0.5 ml).

In the end, regardless of the medium used (depending on the desired objective), four Petri dishes are used, allowing culture:

- Plant parts

- Of the earth

- Suspension intended to count the spores

- Diluted suspension to obtain 100 spores.

The isolation of the Trichoderma populations sampled is carried out after growth of the Trichoderma population colonies in a Petri dish at 24° C., in the light for three to seven days, purification is carried out by monosporal isolation of each of the colonies.

To do this, for each colony, the green spores are taken, then suspended in sterile water in order to dissociate them. A separate culture is carried out for each dissociated spore (under a microscope) on a rich agar medium such as PDA or oat flakes in a Petri dish. Thereafter, the cultural aspect of these strains is to be identified in order to determine the summary classification of the isolated strains. The characteristics are color evolution of the colonies, their morphological aspect (aggregates, reverse, shape of the mycelium, etc.). Microscopic observation at low magnification reveals the type of trees and their shapes. At higher magnification, the organization of the conidiogenous structures is studied (ramifications, conidiophore, phialides, conidiospores). The optimal growth temperature of Trichoderma clones is determined on conventional media such as malt-agar, PDA, beet pulp, oatmeal, with a pH between four and seven. Step N° 3: Selection of fertilizing clones:

Organic phosphorus release:

The technique for determining the ability of Trichoderma to solubilize different forms of Phosphorus consists in using a completely synthetic medium and using the form of Phosphorus to be studied as the sole source of this element. NBRIP medium (30 g/l European Bacteriological Agar, 10 g/l Dextrose, 5.0 g/L MgCL ₂ , 0.25 g/l MgSO ₄ , 0.2 g/l KCI, 0.1 g/l ( NH ₄ ) ₂ SO ₄ , 5.6 g/LK ₂ HPO ₄ ) is thus modified, replacing K ₂ HPO ₄ by the source of P to be studied: P ₂ O ₅ (superphosphate used in most 2 .29 g/l), Cas(PO ₄ ) ₂ (insoluble mineral phosphorus incorporated at a rate of five g/l), or even CeH O^Pe (organic phosphorus, phytates, added at 3.55 g/l).

The spores of the strains that have grown on the PDA are collected in sterile water and inoculated on the selective Phosphorus solubilization medium then placed in an oven in order to observe and compare the growth rates. If a strain is unable to solubilize the P form in question, the mycelium does not expand and form new spores.

Step No. 4: Analysis of the antipathogenic activity of Trichoderma:

4-1: Direct selection in Petri dish:

In Petri dishes, it is easy to observe the pure fungus and its mode of colonization. In these filled with a solution of the PDA type, a strain of Trichoderma and one of the pathogen are implanted. Subcultures are made with cookie cutters 0.8 cm in diameter. On the bottom of the Petri dish, a diameter is drawn, and the pieces of agar containing spores of the chosen fungus are placed one cm from the edge of the dish. Progression of growth is observed, but only the result after one month gives the verdict.

Trichoderma are then divided into three classes:

- surpassing the pathogen when Trichoderma takes over the pathogen and sporulates on its mycelium,

- stopping the growth of said pathogen, when no superiority between the antagonists is observed,

- surpassed by the pathogen when the pathogen mycelium is observed to pass over that of Trichoderma.

4-2: Estimation of parasite activity:

We use the collection of monosporous Trichoderma isolates obtained previously. The inoculum necessary for the tests is prepared in Roux flasks (200 g of sand, six grams of oatmeal, 30 ml of distilled water; two autoclaves at 24 hours interval). The flasks are placed in an oven at 28° C. for two weeks; during this period, they are shaken manually on the ninth day to improve the colonization of the medium: they are kept for another two weeks at laboratory temperature and in daylight. Their contents are then air-dried under aseptic conditions for three days. After homogenization, a sample of the powders thus obtained is taken to determine their propagule content (DAVET, 1979). The rest is stored at 5.0°C in airtight boxes and constitutes the inoculum.

Targeted pathogenic fungi:

The strains of pathogens (e.g. Sclerotinia homeocarpa, Fusarium graminearum, Microdochium nivale, etc.) will have been isolated on grass. The pathogens are inoculated on PDA medium in Petri dishes and cultured at 24°C. The preservation organelles (scerotes, pycnidia , acervicules, etc.) are collected by scraping the surface when the culture medium has dried out, after five or six weeks of incubation.They are stored in a dry place at laboratory temperature.

The soil: the soil comes from the samples. It is a sandy-loamy soil with a pH (Ca C)21 = 6.7, containing 1.94% organic carbon. The quantity required for the tests is sieved, then stored in the laboratory in a closed polyethylene bag.

Confrontations between sclerotia and antagonists:

In the tests comprising the entire collection, an inoculum consisting of 10 ml of dried inoculated sand is mixed with 200 ml of soil.

In other tests, carried out with reduced samples of 12 clones, the inoculum is adjusted so as to have exactly a proportion of 150 propagules per gram of soil. This concentration represents, for most clones, a volume similar to the previous one; for some, however, the volume needed is significantly different (from 2 to 60 ml). 150 mg of pathogen are added to this mixture. The whole is homogenized, moistened to 70% of the retention capacity and placed in a polyethylene bag sealed with adhesive tape. The bags are kept at 22°C or 28°C for three weeks.

The contents of the bags are sifted under a stream of water. 100 pathogen organelles are collected per bag (250 µm mesh screen). When they are pierced, the organelles are considered destroyed and counted straight away, because preliminary tests have shown us that they were, in this case, completely parasitized and incapable of germinating. The other organelles are cultured on acidified PDA medium (100 mg/l of citric acid), after superficial disinfection with 5% sodium hypochlorite for three minutes. The cultures are observed after five days of incubation in an oven at 28° C. and two days in daylight, at laboratory temperature. Only those organelles which exclusively supply a colony of Trichoderma sp. Organelles that are colonized but which manage to germinate are not taken into account. Each test is repeated at least three times.

Ratings:

A "pathogen destruction rate" equal to

(n1 + n2) / N x 100, formula in which: n1 is the number of invaded pathogens, n2 the number of pure colonies of Trichoderma sp. And

N the total number of pathogens counted.

The analyzes of variance are made after transformation of the percentages into arc sines and the means are compared by the method of NEWMAN & KEULS. The correlations between rankings are calculated by the SPEARMAN method.

4-3: Hydrolysis of carboxymethylcellulose

Cultures in liquid medium are filtered through muslin, then through filter paper, and the filtrate is immediately used. The enzymatic activity is assessed by the colorimetric method of NELSONOS IMGOY (THOMAS & EDNORY, M 1958). The reaction medium consists of 1 ml of filtrate and 1 ml of 1.2% carboxymethylcellulose solution in a 0.05 M citrate buffer at pH=4. The assay of the glucose released is carried out after six hours of incubation at 38°C. Each analysis is repeated at least four times.

Calculations

Correlation analyzes are made by SPEARMAN's rank correlation method

Step No. 5: Analysis of the ability to colonize soil organic matter by Trichoderma:

Saprophytic colonization of straw

The inoculum is mixed with the sieved earth at a rate of 0.1% (vol./vol). In practice, this proportion is obtained by weighing, taking into account the apparent densities of the soil and of the inoculum. The mixture is homogenized dry for eight minutes in a mechanical homogenizer (Turbula). To 200 ml of soil thus inoculated, two grams of wheat straw chopped into fragments of about two cm are added, then water so as to reach 60% to 70% of the retention capacity. After stirring with a spoon, the whole is placed in a polyethylene bag, the opening of which is sealed with adhesive tape. The bags are incubated in an oven at 28° C., in the dark. Four days later, the straw is collected, washed under running water, superficially disinfected with sodium hypochlorite for two minutes, rinsed and cut into 5 mm long fragments. For each bag, 100 of these fragments are cultured on a selective medium for Trichoderma (DAVET 1979). After one week of incubation at 28° C., the number of colonized straw fragments is counted. For each clone, at least four repetitions are made.

Estimation of the development of Trichoderma in competition by the agar pellet method.

This method consists of mixing increasing proportions of inoculum with soil. A 12 g sample of each mixture is spread at the bottom of a Petri dish. 20 ml of supercooled agar water is poured over it and the dish is given a rapid circular motion so as to homogenize the whole. After cooling and solidification, pellets 8 mm in diameter are cut out with a punch and placed on PDA medium, at the rate of four, arranged in a cross, per Petri dish. The dishes are incubated in the dark at 22°C or 28°C for five days and then left for two days in the light at laboratory temperature before being scored. Five boxes of four pellets are inoculated for each treatment. The tests are repeated two to five times, depending on the series.

Concentration ranges: In a series of tests covering the entire collection of Trichoderma available, identical quantities of inoculum are mixed with soil regardless of the clones, in proportions of 6%, 24% and 90% in weight, regardless of their actual richness in propagules. In a second series, the propagule content of each inoculum is first evaluated, according to the technique of DAVET (1979), then quantities adjusted to the soil are mixed so as to have the same propagule content for each clone.

Scoring: Each pellet is scored from "0" (no development) to "4" (the entire surface around the pellet is colonized by Trichoderma). The scores of the 20 pellets corresponding to each treatment are then added up, the maximum score therefore being 80. It is then possible to calculate the theoretical proportion of inoculum (or the quantity of propagules/g of soil) necessary to obtain the score 40, half of the maximum mark. These values, called C 40, can be determined graphically, by constructing the curves representing the scores obtained as a function of the concentrations. It is also possible to linearize the curves, after having expressed the concentrations in logarithms and obtaining the desired values by calculation. In both cases, we end up with a classification of the clones, the most competitive being those with the lowest C 40 value. It is also possible to classify the clones in a third way, by attributing to each of them a score equal to the total of the different scores obtained for each of the concentrations tested.

Estimation of saprophytic development by the method of filter paper traps.

The method has been described in detail previously (CAMPOROTA, "Evolution of the saprophytic behavior of strains of Trichoderma spp. in soils - Study technique" Revue Agronomie, 3 (6), 607-609, 1983). It consists in burying in the ground, moistened to 70% of its retention capacity, traps made up of rounds of filter paper 10 mm in diameter, impregnated with RICHARDS medium, i.e. for one litre: KH2PO4: 1 g; MgS04.7HsO: 0.5 g; FeSO4.7HsO: 5 mg; ZnSO4: 5 mg; MnCh: 2mg; galactose: 20g; glycocoll: 2.25 g; added fungicides (rose bengal: 100 mg; streptomycin sulphate: 100 mg; benomyl: 5 mg; copper sulphate: 5 mg; prothiocarb: 50 mg; pentachloronitrobenzene: 50 mg).

For each of the concentrations, four Petri dishes 90 mm in diameter are filled with a constant quantity of mixture in which, after humidification, 10 traps are placed vertically per dish, spaced 20 mm apart in all directions. After incubation for five days at 25°C, these traps are rinsed, drained and placed on the selective medium of DAVET (1979). The colonization of the traps by Trichoderma is noted after 48 hours of incubation at 25°C.

Quantification: The soil analyzed is diluted by mixing with greenhouse soil disinfected three times at 100°C, so as to have concentrations of 100%, 10%, 1%, 0.1% and 0.01%. The regression line linking the percentage of colonization of the traps and the corresponding concentrations is calculated. We then solve the equation of the straight line for a colonization of 50%: this makes it possible to determine the weight of soil necessary for 50% of the traps to be colonized under the test conditions. This weight is called “Colonization Unit 50” (UC 50) which is expressed, in order to be able to compare different soil samples with each other, in number of CU 50/g of soil.

Experimental Protocol: The saprophytic behavior, in the soil to be tested, of ten of the clones in the collection is compared. Two analysis dates are retained: at the time of introduction of the inoculum into the soil (D 0) and after 30 days of incubation of the inoculated soil (D 30). The richness in propagules of the inoculum produced on sand is determined here after spreading 10 mg in ten Petri dishes containing malt agar medium, in four repetitions. The soil is inoculated at a rate of 15 “fertile grains”/g and constitutes the initial stock which will then be diluted with sterile soil. After humidification, each batch is divided into two parts: one is analyzed immediately, the other is stored at 28°C in a closed crystallizer. by a perforated plastic film to be analyzed after 30 days.

Step No. 6: Fermentation process in a solid medium for Trichoderma:

This method is characterized in that it comprises firstly cultivating said selected strains of Trichoderma sp, by a mass culture method carried out on PDA (Potato Dextrose Agar) agar medium at 39 g/l of distilled water, mixed in 500 ml Pyrex bottles in the autoclave. Autoclaving at 121°C for 30 minutes. Once the medium has cooled, add citric acid (concentration of 0.2 g/l) and appropriate antibiotics (Triton and allyl alcohol). Inoculation into the still liquid mass of a small quantity of sterile water containing the Trichoderma inocula then mix and pour into a Petri dish. Once solidified, said boxes are left to incubate in an oven, under controlled temperature, aeration and humidity conditions. The latter are incubated at 25°C for three to four days, and during this period, visual tests of growth rate are carried out.

The implementation of the process requires the use of a closed static bioreactor inside which is inserted a solid absorbent support consisting of various lignocellulosic plant residues chosen according to the agronomic circumstances (beet pulp, wheat straw, corn seeds, etc.), finely ground (size less than 0.5 mm) then the mixture is homogenized and autoclaved at 120°C for one hour impregnated with a nutrient solution (100 g of dry matter contains: (NF jjSC , 9.75 g; urea, 2.38 g; KH2PO5, 5.0 g; Distilled water, 100 ml; pH of the solution obtained, adjusted to 4.2.) and of the inoculum (from fermented Petri dishes ) of the filamentous mushroom(s) chosen according to the desired final product.

The humidity of the product thus obtained is reduced to 70%. 20 g of this product are introduced into each of the incubators which are placed in a 29° C. water bath: compressed air saturated with water ensures aeration of the culture medium with a flow rate of 7 l/h. Fermentation is triggered and controlled by a device for real-time measurement of environmental conditions (T°C, humidity, air flow and CO2) and continuous analysis of the growth of the micro-organism, its respiration, the number and spore germination. This device is controlled, in particular by controlling the duration of application of the water stress, by injecting more or less dry air into the bio-reactor at a precise moment of the culture of the microorganism in order to induce the phase of conidiogenesis . The flow of dry air applied at the end of the process makes it possible to obtain a dry fermented product, containing the biomass of the fungus essentially in the form of conidiospores which can be stored for several months without loss of viability under perfectly controlled aseptic conditions. Shutdown and recovery occurs when the moisture content of the substrate is below 9%. These spores can be used directly in solid form or after washing and filtration under liquid form, as a biological agent.

The control of the moisture content is carried out by weighing samples before and after passing through the microwave. The number of spores is counted by Malassez cell counts of successively diluted solutions.

The concentrations in number of spores per gram of dry support are deduced therefrom. The counting of the number of spores and the control of the humidity rate are carried out every two days. The results obtained are archived in a workbook, on control sheets and growth curves are performed.

Germination control is a measure of the germination rate of each ferment. It is carried out by spreading out known quantities of the number of spores per Petri dish containing PDA, and observation with a magnifying glass a few hours later, of the number of germination starts. The ratio of germinated spores/total spores introduced is deduced therefrom.

The formulation is most often requested in liquid form. The support is therefore washed with hot water + Twinn to detach the spores, then filtered through several sieves up to 100 μm. Humic acids are added at a rate of 2.5 g/L.

The formulation of Trichoderma with agronomic properties of interest to the customer from the strains he has in his field can then be re-inoculated at a rate of 20-25 L/Ha at a concentration of 5x10 ⁸ spores/mL.

Step No. 7: Process for characterizing and monitoring Trichoderma populations

In parallel, the selected Trichoderma strain is characterized molecularly. To do this, the strain is cultured in Potato Dextrose liquid medium (200 g of potatoes in 1 L brought to the boil, then filtered through carded cotton, then supplemented with 20 g of glucose. Adjustment to 1 L then autoclaving for 20 min at 120 °C) in 100mL Erlenmeyer flasks: 20mL of Potato Dextrose are introduced into the Erlenmeyer flask, as well as a 5 mm diameter implant from the strain that has grown on PDA. After 48 hours of incubation at 20°C and shaking at 120 rpm (revolutions per minute), the mycelium is harvested by filtration on sterile Miracloth, dried and frozen at -80°C with liquid nitrogen for storage until use. . A ground material in fine powder is used to extract genomic DNA by the method of Atoui et al (2012). The DNA is then amplified by PCR with primers ITS1 and EF1 following the protocol described by Al-Sadi et al (2015). The sequence of the PCR product is then placed in the pGEM-T Easy plasmid according to the supplier's recommendations, and bound by the heat shock method in thermocompetent bacterial cells of the DH5a strain of Escherichia coli selected on Petri dishes of medium LBA: Luria Bertani Agar. The plasmid DNA is then sent for sequencing to companies specializing in this field.

Classical and molecular counting method:

All sampling is carried out in the same way as previously (examples 1 and 2), at different dates after inoculation, making it possible to measure the dynamics of Trichoderma populations over time.

These are again placed in culture, isolated, purified and amplified by PCR, put under plasmid and sequenced. In this way, by comparing the DNA sequences of the strain which has fermented for multiplication and the majority Trichoderma DNA in the soil, it is possible to demonstrate that the high-density Trichoderma strain in the soil after inoculation does indeed come from Trichodynamization.

Of course, those skilled in the art will be able to carry out the invention as described and represented by applying and adapting known means without it being necessary to describe them. It may also provide other variants without departing from the scope of the invention as determined by the content of the claims.

Claims

1 . Process for increasing the quantity and/or quality of plants by improving the native biological capacities of the soils of a plot by obtaining fungal isolates, characterized in that it comprises:

- a step (1) of taking soil samples from the plot;

- a step (2) of isolation of fungus spores of the genus Trichoderma spp;

- a step of separate culture of spores isolated on selective media;

- a step (3) of selecting the strains of these most efficient crops for at least one agronomic property of interest;

- a step (4, 5) for optimizing the quantitative and qualitative production of spores of each selected strain, by fermentation in a solid medium, on residues or plant products;

- a step (6) for formulating the inocula obtained; And

2. Method according to claim 1, in which, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the stimulation of root growth.

3. Method according to one of claims 1 or 2, in which, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the release of organic phosphorus.

4. Method according to one of claims 1 to 3, in which, during the step of selecting the strains of these best-performing cultures, at least one agronomic property of interest is the solubilization of the mineral phosphorus.

5. Method according to one of claims 1 to 4, in which, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the ability to degrade the organic material.

6. Method according to one of claims 1 to 5, wherein, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is mycoparasitism.

7. Method according to one of claims 1 to 6, in which, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is resistance to soil fungicides.

8. Method according to one of claims 1 to 7, in which, during the step of selecting the strains of these best-performing crops, at least one agronomic property of interest is the induction of the natural defenses of the plants. .

9. Method according to one of claims 1 to 8, in which, during the step of formulating the inocula obtained, nutritional supplements are added.

10. Method according to one of claims 1 to 9, wherein, during the step of isolating fungus spores of the genus Trichoderma spp, each spore is isolated in a unique way, so that the step of culture separated from isolated spores produces populations of clones.

11. Method according to one of claims 1 to 10, in which, during the selection step, a step (4) of analyzing the antipathogenic activity of the Trichoderma is carried out.

12. Method according to claim 11, in which the antipathogenic activity of the Trichoderma is analyzed on Sclerotinia homeocarpa, Fusarium graminearum and/or Microdochium nivale.

13. Method according to one of claims 1 to 12, in which, during the selection step, a step (5) of analyzing the aptitude for colonization of the organic matter of the soil by the Trichoderma.

14. Method according to one of claims 1 to 13, in which, during the optimization step, a step (6) of fermentation in a solid medium of the Trichoderma is carried out.

15. Method according to one of claims 1 to 14, wherein, during the step of formulating the inocula obtained, a flow of applied dry air is applied and a dry fermented product is obtained, containing the biomass of the fungus essentially in the form of conidiospores, which can be used directly in solid form or after washing and filtration in liquid form, as a biological agent.