EP0662934A1

EP0662934A1 - Microgranulated products usable in combination with bacterial inoculums, method for obtaining them and application to agriculture

Info

Publication number: EP0662934A1
Application number: EP93920897A
Authority: EP
Inventors: Gérard CATROUX; Georges Lycée Agricole FOUILLEUX
Original assignee: Institut National de la Recherche Agronomique INRA; LIPHA SAS; LIPHA Liyonnaise Industrielle Pharmaceutique
Current assignee: Institut National de la Recherche Agronomique INRA; Merck Sante SAS
Priority date: 1992-09-18
Filing date: 1993-09-17
Publication date: 1995-07-19
Also published as: FR2695929B1; ZA936878B; AU4823093A; CA2144739A1; WO1994006733A1; FR2695929A1

Abstract

Method for improving the yield of agricultural crops, wherein an inoculum is used which contains at least one microbial culture and microgranules, said method being characterized in that microgranules comprise at least a substance capable of promoting the growth of inoculum germs.

Description

MICROGRANULES FOR USE IN COMBINATION WITH BACTERIAL INOCULUMS, THEIR OBTAINMENT AND THEIR APPLICATION IN AGRICULTURE

The invention relates to the field of agricultural crops in which seeds, plants and all other known growing media are used. In another aspect the invention is part of the general technique of soil treatment in agriculture. It is applicable with many advantages to legume crops.

In the prior art, it has already been proposed to use microbial inocula in agriculture. The purpose of this technique is to provide, when the seeds or plants are placed in the soil, bacteria that are useful or essential for the growth of the plant. The inocula used for this purpose can be liquid microbial cultures, or on a solid support. They can also be dehydrated microbial cultures, in the form of a gel or of lyophilisate.

The inocula currently in use and which are to be approved are pure cultures, free of contaminant in order to facilitate quality control, improve survival and prevent any introduction other than the authorized microbe.

Several different techniques are known for using such inocula. For example, in the case of legumes, a first technique consists in bringing the inoculum at the time of sowing by bringing it into contact with the seed. A variant consists in using pre-inoculated seeds. Another technique provides for the inoculation of the seed bed: the inoculum is then brought into the seed line, either in liquid form or in the form of microgranules, these being practically spread using devices known for this purpose to distribute phytosanitary products. The latter technique is often preferred by farmers, since the quantities of product to be handled are lower than in the case of seed inoculation. For example, it suffices to use approximately 10 kg per hectare of microgranules instead of 80 to 150 kg of seeds in the case of seed inoculation. The results already recorded in practice also show that the efficiency of the seed bed inoculation technique is also good.

By way of illustration, a technique is currently known which consists in mixing, at the time of use, dry, a certain quantity of bacterial inoculum on a solid support, such as peat, with mineral microgranules of industrial origin, such as as clays, carbonates or others, the size of which is suitable for spreading. In practice, the particle size is approximately 0.2 to 0.8 mm. Such a technique has the advantage of using inexpensive microgranules, in combination with small amounts of inocula, which can be easily stored separately. In such a technique, it is not really concerned with the fixing of the inoculum on the microgranules, the latter only serving in fact to dilute the inoculum. The nature of such microgranules can be very varied and apart from the clay and mineral materials already mentioned, mention may be made of peat, sand, polyethylene beads and other similar materials capable of being spread by agricultural machinery.

Another known technique consists in inoculating in advance and during the manufacture of granules of various nature, so as to produce microgranules ready for use. Granules containing the microorganism (s) to be inoculated are thus known, based on clay, peat, alginates, vermiculite, perlite, gels of various polysaccharides, silica, calcium sulfate. Have also been described microgranules comprising associations of constituents, for example calcite coated with peat, gels of polysaccharides and silica, mixtures peat-clay and calcium sulfate, clays and alginates, among others. This technique for producing inoculated microgranules in advance is attractive, but it is very difficult to manufacture these products under sterile conditions and to ensure storage at low temperature, given the volumes involved, the quantity of product to be used being l 'order of 10 kg per hectare for example. Furthermore, if nutrients are also introduced with the support of the inoculated microgranules, there is an increased risk of development of contaminating germs in the medium thus enriched.

The following patents and patent applications illustrate this state of the art.

The patent US-5055293 (ARONSON) relates to the biological control against the larvae of an insect infecting corn.

Granules of inert clay, or of another mineral or organic material acceptable for agricultural use, covered with a layer of Bacillus laterosporus bacteria are described there.

They can be sprayed with a solution of cells or spores which may contain nutrients and excipients, then dried.

This document does not mention the deposition of microorganisms, just before application in the field. on granules already containing nutrients.

European patent application EP-A-0295968 (PIONEER FRANCE BUT) relates to the protection of microorganisms contained in fertilizer compositions. The microorganisms are microencapsulated in a polysaccharide matrix, then the inoculum thus prepared is mixed with the fertilizer in solid form or in liquid form immediately before spreading. Note that it is only a question of mineral fertilizers for plant growth and not the addition of nutritive substrates to obtain growth in the soil of a microorganism. In addition, the microorganisms are pre-microencapsulated before mixing with the fertilizer.

European application EP-A-0203708 (AGRACETUS) relates to a process for adsorption of bacteria in the stationary phase by mixing the suspensions of bacteria with a granular support, chemically inert and porous, then drying. No nutrients are added to these granules.

Finally, European application EP-A-0479 402 (LIJIMA, RYUSUKE) relates to a method of producing a mycelial fertilizer in which thermoactinomycetes are incubated in the presence of a porous support, said support having been previously kneaded with pyrolignic acid.

The thermoactinomycetes are left in fermentation for at least two days while maintaining the temperature between 55 and 80 "C.

The microorganisms are therefore not added just before the field application of the granules.

The present invention belongs to the general technique of microbial inoculation using microgranules to be inoculated extemporaneously. She has mainly for the purpose of using microgranules which, after burial in the soil, make it possible to improve survival and obtain a multiplication of microorganisms in the inoculum.

In a first aspect, the invention relates to microgranules comprising a solid support and at least one substance capable of promoting the growth of germs in a microbial inoculum with which the micro-granules are brought into contact at the time of use.

The microgranules according to the invention are not inoculated in advance. They are used, at the time of use, in conjunction with the microbial inoculum.

In another aspect, the invention also relates to a method for improving the yield of agricultural crops, in which an inoculum containing at least one microbial culture and microgranules is used, said method being characterized in that the microgranules comprise at least a substance capable of promoting the growth of germs in the inoculum.

The method of the invention therefore consists in using microgranules containing a substance, in particular a nutritive substrate, capable of promoting the growth of the germs of the inoculum, said microgranules being prepared in advance and can be stored in the dry state. , and inoculate them at the time of use with a microbial inoculum, in order to obtain in the soil better survival and growth of this inoculum.

For the purposes of the present invention, any type of microgranules obtained by grinding or granulation, whether microgranules of mineral or organic nature, or mixtures of such granules, can be used. Among the granules of mineral origin, mention may be made of industrial clays and clay mixtures, which have given good practical results. As for microgranules of organic origin, mention may be made of various granulated or ground substrates, based on peat, compost, plant residues and the like. The technical literature contains numerous examples of usable microgranules as well as indications on the desirable particle size. It is advisable to choose grain sizes which are suitable for spreading with conventional agricultural machinery, and it is known that an appropriate dimension for the size of the microgranules is of the order of 0.2 to 0.8 mm, for example. indicative and not limiting.

According to the essential characteristic of the invention, the microgranules in question are enriched in advance, by contacting the microgranules themselves and the enrichment substrate, which consists of at least one substance capable of promoting the growth of germs. the microbial inoculum.

In practice, the enrichment of the microgranules in advance can be carried out by imbibition, immersion or spraying of the microgranules and subsequent drying, by granulation of a suspension of solid material and the enrichment substrate or by mixing the microgranules with the finely ground enrichment substrates. According to this embodiment, the microgranules prepared in advance can be stored dry, so as not to allow the development of microorganisms during storage.

As regards enrichment substances or substrates, any product capable of promoting the survival and the multiplication of germ to be inoculated, either directly by allowing or stimulating its growth, or by limiting the growth of competitive soil germs. It is possible in particular to use carbon-based substrates such as glucose, glycerol and any other source of carbon suitable for the growth of most microorganisms or else, on the contrary, use carbon-based substrates specific for the germ to be favored. It is also possible to use other products, in particular nitrogenous or phosphorous and more generally any nutrient capable of allowing the growth of the germ to be inoculated. However, according to the inventive concept described herein, it is also possible to use biocides compatible with the germ to be inoculated but capable of inhibiting or slowing the growth of competitors, such as products active in the soil against bacteria, fungi and protozoa. These types of products are known to those skilled in the art and can in particular be chosen from antibiotics, fungicides and more generally any phytosanitary product.

It has been indicated above that in the process of the invention, an inoculum containing at least one microbial culture could be used. In fact, the inoculation can call upon one or more germs, which can be brought successively or in mixture. The microgranules are inoculated by the user, at the time of use or shortly before it is used. All types of inoculum can be used, whatever their support and their packaging, for example inoculums on solid support, such as peat, liquid inoculums, gel inoculums or lyophilized inoculums.

It's inoculation techniques are known to skilled in the art and need not be described in more detail.

The invention can be applied with one or more microorganisms capable of being used in the inoculation of seeds, plants, growing media and soils. These include all microorganisms that can be used to directly promote plant growth, such as those belonging to the Rhizobium, Bradyrhizobium, Azospirillum species, as well as ecto- and endomycorrhizogenic fungi. Conclusive results have been obtained with the microorganism Bradyrhizobium japonicum. Also within the scope of the invention are microorganisms which can be used in biological control against plant diseases and pests, such as bacteria antagonizing bacteria and phytopathogenic fungi (PGPR: Plant Growth Promoting Rhizobacteria, Pseudomonas, Agrobacterium, Bacillus and other similar genera), antagonistic fungi of phytopathogenic fungi (Fusarium, Trichoderma, and other similar species) and entomopathogenic bacteria and fungi (Bacillus thuringiensis, Beauveria, and other similar species). Mention may also be made of the microorganisms which can be used in depollution and bioremediation of soils.

The invention provides advantages over the traditional use of inoculum. We know that the effectiveness of microbial inoculations in agriculture directly depends on the number of live and effective germs brought. It is also known that the immediate losses on inoculation are generally very large and are mainly due to the mortality of the germs brought on by desiccation. We are therefore trying to find methods which make it possible to increase the possible dose of microorganisms to be inoculated. But it is also necessary to limit the losses of microorganisms during use in the fields, while taking into account the physical limits, the possible quantities to be inoculated being limited by the weight of seeds as well as by the equipment available, as well as economic (production costs, storage). For this reason, it has already been proposed in the prior art to carry out the growth of the microorganism inoculated in the soil, but only after inoculation. Some attempts have been made to add nutritive substrates directly to the soil, for example a carbonaceous substrate. But it is then necessary to add to the soil, for example in the case of the treatment of a seed bed, between 20 and 200 kg of carbonaceous substrate per hectare, which corresponds to completely unrealistic amounts in field crops.

The invention makes it possible to obtain growth in the soil of the germs brought in by increasing their number, which is reflected either by an improved effect of the inoculation, or by a safety effect in the event that difficult conditions are present during the inoculation treatment, which may be the case of a partially deficient inoculum or of an abnormal inoculation mortality. Thanks to the invention, the dose of microorganisms made available to the seed or the plant can be increased or maintained under excellent safety conditions.

It has already been said previously that the use of microgranules, and more particularly of pre-inoculated microgranules, that is to say inoculated in advance during manufacture, is known in the prior art. Compared to such a technique of pre-inoculated microgranules, the The present invention provides many advantages, in particular the following:

the invention makes it possible to use commercial microgranules, generally inexpensive,

- the enrichment in nutritive substrate can be achieved without sterility requirement and is therefore less expensive,

- zero or very reduced risks of development of contaminating germs in microgranules during storage (dry storage), while such risks appear on pre-inoculated microgranules,

- Storage possible at ordinary temperature over a long period, the properties of the microgranule not being linked to the number of germs it contains and to their survival, whereas we know the unfavorable effect of temperature for the survival of an inoculum and pre-inoculated microgranules,

- possible cold storage of the microbial inoculum in small packaging separate from the microgranules, which can be packaged in bulky packaging,

- various possibilities of playing on the nature and the concentration of the substrates, the adaptation of the nature and the specificity of the substrate to the germ to be inoculated,

possibility of adding biocides disadvantaging competitors, without risk of toxicity by prolonged contact between the inoculum and the microgranule.

The invention will now be illustrated without being in any way limited by examples of implementation in the laboratory and in the field.

FIG. 1 represents the results obtained with granules enriched with Bradyrhizobium japonicum. The development of microorganisms (in ordered) is followed as a function of time (in days).

Figures 2 and 3 illustrate the growth of populations of the Pseudomonas C7R12 strain in liquid form (L), in normal clay form (A) and in enriched clay form (Ae), respectively in natural soil (Figure 2) and in soil disinfected (Figure 3).

Figures 4 and 5 show the evolution of the number of healthy flax plants (on the ordinate) as a function of the number of days (on the abscissa) respectively in natural soil (Figure 4) and in disinfected soil (Figure 5) treated with different formulations microorganisms.

EXAMPLE 1 Microqranules inoculated with Bradyrhizobium japonicum.

We will first expose all the means and modes of carrying out the experiment and then the results of it.

STRAINS

All greenhouse and field tests are carried out with the commercial strain G49 (IARI SB16,

New Delhi) from Bradyrhizodium japonicum. The crop chosen is that of soybeans.

We know that, for this plant, nodulation

(number of nodes, weight of nodes) increases when the dose of Bradyrhizobium japonicum provided increases.

Furthermore, the yield is linked to nodulation.

To follow the evolution of populations introduced into the soil, a strain of

B. japonicum resistant to antibiotics. In the laboratory, a spontaneous mutant of G49 resistant to rifampicin (100 mg / ml) and kanamycin (200 mg / ml) was isolated on a petri dish containing antibiotics, the stability of which was verified and which was named G49RfKm.

This new strain was deposited at the CNCM collection of the Institut Pasteur under Nº I-1264 on September 11, 1992.

MICROGRANULES

These are commercial products that meet the following criteria:

- density high enough to allow good descent into the microgranulator,

particle size: fairly fine and homogeneous, between 0.2 and 0.8 mm,

- pH compatible with the survival of bacteria,

- absence of toxicity vis-à-vis inoculated bacteria,

- mixing with an inoculum easy to make, retaining enough peat, and giving a homogeneous product.

We used granules with sufficient porosity and not destructuring in water, in order to retain all their properties during the impregnation phases (with the enrichment solution) and drying. This procedure does not affect the ease of distribution of the microgranules by microgranulator.

Two types of microgranules meeting these requirements have been used, one called K, based on cristoballite, and the other M, formed of calcined montmorillonite.

Table 1 gives the essential characteristics of these microgranules.

ENRICHMENT SUBSTRATES

They are essentially formed from a carbon source, a nitrogen source, and mineral elements.

The products selected for enrichment are dissolved in water. The volume should not be excessive, so as to be absorbed entirely by the porosity of the microgranules. But it must be high enough to, on the one hand lead to complete dissolution of the products, on the other hand allow homogenization of the mixture with the microgranules before its complete absorption.

This solution is brought to the dry microgranules, the whole being immediately stirred in order to obtain a uniform mixture. She is absorbed by porosity.

Subsequent drying makes it possible to leave the nutritive substrate dry in this porosity, allowing long storage, even under non-sterile conditions.

The concentrations used for the experiments vary from 16 to 96g of organic matter (expressed as organic carbon), per kg of dry microgranules.

ASSOCIATED FUNGICIDES

After a test on their effectiveness against soil fungi and their safety against inoculated strains, the retained products are added to the enrichment solution, before their incorporation into the microgranules.

SOILS AND CROP SUPPORTS USED

Field trials

Most of the work was carried out on clay soil from the INRA - Dijon domain, and a secondary part concerned sandy soil from the region. These soils were initially devoid of B. japonicum.

The essential characteristics are given in table 2.

Laboratory tests.

They were almost entirely made with the same clay soil as that used for the field trials. This soil is devoid of B. japonicum. PLANT

All the tests were carried out with the soybean variety Maple Arrow, of undetermined type and belonging to the precocity group 00.

INOCULUMS

When using the normal G49 strain, the inoculum, peat or liquid, is a commercial product ("Biodoz", from the company LIPHA).

The inocolum of the resistant G49 strain (G49RfKm) is produced in the laboratory.

A flask containing a liquid culture medium, based on malt extract, is sterile inoculated from an agar tube containing the strain. It is then placed on a stirring table (200 rpm) at 28ºC for 6 to 7 days. The culture obtained then constitutes the liquid inoculum used, which can be stored for several months at 4ºC.

For the production of peat inoculum, one starts from a sterile peat of the same origin as that of the commercial inoculum. 12 ml of liquid inoculum are added to a bottle containing 30 g of this product, at 12% humidity. After homogenization and maturation for 2 to 3 weeks at room temperature, a product is obtained which has substantially the same properties as commercial peat: humidity of 55-60%, richness of the order of 10 ⁹ seeds per gram of peat.

MICROGRANULE INOCULATION TECHNIQUES

A proportion corresponding to that of inoculation in the field was chosen, ie 4% of pure inoculum relative to the microgranules.

Taking into account the quantities necessary for the introduction into the soil, the ease of handling and the expected precision, we worked on batches of 25 g of microgranules (measured dry and without enrichment), in 250 ml flasks.

For use with the peat inoculum, the microgranules are first moistened to the desired level. Then, each batch is inoculated with 1 to 1.3 g of inoculum, the whole being homogenized, then used in the following minutes.

When used with a liquid inoculum, the microgranules, dry at the start, receive an inoculum previously diluted so as to simultaneously provide 1 ml of a pure inoculum for 25 g of microgranules, but also the amount of water necessary to be at the desired humidity (7.5 ml for K and 3.25 ml for M in most experiments). After stirring for homogenization, the microgranules are used quickly.

Soil introduction and incubation techniques

After sieving at 4 mm or 2 mm, the soils are introduced into glass bottles, either at the rate of

50 g of dry soil in a 500 ml bottle, i.e. 20 g in 250 ml. The humidity is adjusted 24 hours in advance, generally at the level of 80% of the water retention capacity.

In these bottles, 300 mg and 120 mg of microgranules are introduced respectively, which represents approximately 4500 and 1800 microgranules respectively for K, 2100 and 800 for M.

The flasks are then shaken manually so as to distribute the microgranules throughout the volume of soil.

The inoculated vials are incubated by closing them with a plastic film, intended to prevent water loss while allowing respiratory exchanges. They are then placed in the dark, generally at 20 ° C, but also at 15 ° C or 28º C for some tests.

The counts are made on the whole of a bottle after several days of incubation.

Petri dish counts

The main measures consist in counting populations of B. japonicum capable of forming colonies on petri dishes containing an agar culture medium. These counts are made at several stages of inoculation:

- inocula themselves, before use, in quantities generally of 1 ml or 1 g,

- microgranules after inoculation, in working either on an entire batch or on aliquots,

- soils having received inoculated microgranules, working on the entire contents of a bottle.

The products to be counted for their richness in B. japonicum are introduced into 100 ml of an extraction solution and placed on a stirring table (200 rpm) for 30 minutes.

Successive dilutions are then made to 1/10, and the bacterial suspensions obtained are spread on the enumeration media by one of the following two methods.

In the absence of soil, a device (Spiral System) deposits a known volume of the suspension on the agar medium of a Petri dish. This is then incubated at 28º C, then the colonies present are counted at 5 or 6 days, hence a count in cfu (units forming a colony). A calculation from the dilutions used makes it possible to determine the richness of the product to be analyzed.

In the presence of soil, the previous device is unusable, and the counts are made by the method called "double layer". A small volume, 0.1 or 0.2 ml, of a suspension to be counted is introduced into a hemolysis tube containing 3 ml of 0.7% agarized solution by supercooling (43º C). After homogenization, this mixture is spread on a Petri dish containing the agar culture medium, as well as the antibiotics and fungicides to which the G49RfKm strain is resistant.

After incubation for 7 days at 28 ° C, the colonies present on the dishes are counted, the dilutions being made so as to count between 30 and 300 colonies per dish.

All counts are made with 3 repetitions for each treatment, and the results are given with the calculated standard deviations.

Field trials

The tests set up on the soils described above are devices with 4 complete blocks.

Sowing is carried out at the end of April-beginning of May, the objective being to provide 600,000 seeds per hectare (Maple Arrow variety) inoculated at the agronomic dose, or 10 kg of microgranules per hectare, in lines spaced 30 cm apart. These are enriched and inoculated under the same conditions as for laboratory work, in batches of 1 kg, inoculation taking place in the field a few minutes before sowing.

Irrigation is carried out during the season, depending on the needs of the crop.

RESULTS

The results of the experiments are reported below.

FIG. 1 summarizes the results obtained during laboratory tests, comparing enriched (E) or unenriched (M) microgranules, inoculated with a liquid inocolum and incubated in non-sterilized soil, kept moist (H) or subjected to desiccation (S). Number of B. japonicum germs recovered after inoculation in liquid on microgranules and introduction into the soil, as a function of time and according to the drying of the soil during incubation. The legends of the figure are: N = non-enriched microgranules, E = enriched microgranules GLY3C, H = soil kept moist (82% of the water retention capacity) for 21 days, S = soil subjected to natural drying out after 7 incubation days.

From the results illustrated in Figure 1, we see that: after 7 days, the enriched microgranules make it possible to obtain a multiplication of B. japonicum by a factor greater than 10 compared to the non-enriched microgranules,

- after 21 days, the enriched microgranules maintain the number of B. japonicum at a level 10 times higher than that obtained with non-enriched microgranules when the soil is kept moist and at a level more than 100 times higher when the soil dries out.

Tables 3 to 7 summarize the results obtained during field trials. For each table, the data in the same column followed by the same letter are not statistically different at the 5% threshold according to the Newman and Keuls test.

Tables 3 and 4 show the first results obtained during field trials in 1988 and 1989 respectively. They show a significant effect of enrichment on nodulation.

Tables 5, 6 and 7 relate to tests carried out in 1990 and 1991 with various treatment conditions, as indicated at the bottom of each table.

In Table 5, the treatments corresponding to the invention (enriched microgranules inoculated with a peat inoculum) are treatments 3, 6, 8, 10.

We can see an increase in the number of nodules per plant at stage V3, general for all treatments, compared to the non-enriched microgranular control (treatment 2). This increase is significant only for treatments 6 and 10. Similarly, an increase in the nitrogen level of the seeds is obtained for treatments 3, 6, 8, 10, significant only for treatments 8 and 10. In Table 6, treatments 2, 6, 9 and 10 correspond to the invention (enriched microgranules inoculated with peat inoculum (2 and 6), or with liquid inoculum (9 and 10)).

With a peat inoculum (2 and 6 compared to 1), there is an increase in the number of nodules, not significant at stage V3, but significant at stage R4 for treatment 6 (addition of a fungicide in the enrichment).

With a liquid inoculum (10 compared to 9), a significant increase in the nodulation at V3 and R4 is obtained, as well as a significant increase in the nitrogen level of the seeds.

In Table 7, treatments 2, 3, 4, 5, (inoculated with a peat inoculum) and 10 (inoculated with a liquid inoculum) correspond to the invention.

With the peat inoculum (2, 3, 4, 5 compared to 1), non-significant increases in nodulation at stages V3 and R4 are obtained, but significant increases in the nitrogen level of the seeds. Note that this last effect is obtained with different carbon substrates.

With the liquid inoculum (10 compared to 9), a significant significant increase in nodulation (V3 and R4) and in the nitrogen level of the seeds is obtained.

EXAMPLE 2; Microqranules inoculated with Fusarium or Pseudomonas.

Among the cryptogamic diseases to which vegetable and horticultural plants are affected, vascular fusarium wilt, tracheomycosis due to pathogenic forms of the fungus Fusarium oxysporum, is one of the most problematic because there is no chemical control method to date. specific. On the other hand, biological control calling on competition between a non-pathogenic form and the specific pathogenic form of the same species of Fusarium oxysporum gives satisfactory results under the conditions of culture under greenhouse, either in artificial substrate, or in disinfected soil. The principle consists in introducing the non-pathogenic inoculum into the substrate at the time of sowing or transplanting. In addition, the antagonistic effect of non-pathogenic strains of F. oxysporum can be increased if one co-inoculates certain fluorescent bacteria belonging to the genus Pseudomonas.

The purpose of the experiments in this example is to test an original formulation method which consists in giving a competitive advantage to the inoculum by incorporating a source of nutrients in the formulation support. Population dynamics of bacteria of the genus Pseudomonas Sp. Were measured in both natural and disinfected soil. The activity of the inoculated microorganisms has also been tested in greenhouse biotests using the host plant-pathogen flax F.oxysporum f.sp. United.

MATERIAL AND METHODS

1- Stumps and plants.

Fusarium oxysporum strain Fo47B10, spontaneous mutant resistant to benomyl of the strain Fo47 used in biological control (collection number CNCM I-1363).

Fusarium oxysporum f.sp. plain, Hay strain 3, (collection number CNCM I-1362) pathogen of flax.

Fluorescent pseudomonas strain C7R12 spontaneous mutant resistant to rifampycin from the C7 layer used in biological control (collection number CNCM I-1361). Flax Linum usitatissimum, Opaline variety, susceptible to fusarium wilt.

2- The ground.

Silty clay soil sampled in the Dijon region, air dried and sieved to 2mm. (composition: sand 15%, silt 47%, clay 35%, C 1.2%, CaCO ₃ 0%, pH 6.9).

The soil is distributed in aluminum containers at the rate of 140 g of dry soil / container. The disinfected soil was autoclaved 3 times 1 hour at 120ºC 24 hours apart. The soil humidity is adjusted to pF3 (20% dry soil) during inoculation.

3. Inocula

3.1- Preparation of fungal inocula for biotests.

The mushrooms are cultivated 5 days on liquid malt medium stirred at 25ºC (Difco malt 10g, demineralized water 1000 ml). The culture is then filtered to remove the mycelium. The spores contained in the filtrate are washed 3 times with sterile water by centrifugation 20 min, 4000 g.

The inoculum of the non-pathogenic Fusarium strain Fo47B10 is prepared in the form of a liquid inoculum (suspension of spores in sterile water).

The inoculum of the pathogenic Fusarium Foln3 strain is prepared in the form of a talc formulation: the final pellet is taken up in a minimum volume of water (approximately 20% of the initial volume of the culture) and is incorporated into virgin talc (1 pellet volume -2 volumes of talc). The mixture is left to dry for 48 hours in a ventilated oven at 20ºC. The talc is then sprayed and sieved to 200 μm. It is stored at 4ºC. Its title is measured before any use. The inoculated talc is optionally diluted in virgin talc when low inoculum densities are required.

3.2. Preparation of bacterial inocula In this case, the clay is enriched with glycerol as a carbon source at the rate of 120 g of glycerol / kg of clay. The bacteria produced on King B solid medium (Peptone Difco 20g, glycerin 12.6g, K ₂ HPO ₄ 1.3g, MgSO ₄ 7H ₂ O 1.5g, Agar 15g, distilled water 1000 ml) are recovered in sterile water and washed 3 times by centrifugation (20 min, 4000g). They are incorporated into the clay at the time of their introduction into the soil.

The controls are produced by direct inoculation of the suspension of bacteria washed in a volume making it possible to adjust the humidity to the value of pF3.

4- The analyzes

Each container constitutes an experimental unit. 3 experimental units constituting 3 independent repetitions are performed for a given treatment. The containers incubate at 25ºC. Population dynamics are monitored for 20 to 30 days.

At regular time intervals, 5 g of soil per experimental unit are taken, suspended in 100 ml of water and stirred vigorously for 15 min on an agitator constituting the mother suspensions. A series of suspensions diluted 1/10 is produced from each of these mother suspensions.

The fungal counts are carried out by incorporating 1 ml of the suspension at the dilution level considered in malt agar + benomyl medium in supercooling (malt 10g, agar 15g, citric acid after autoclaving 250 mg, Benomyl after autoclaving 10 mg in benlate form 50% active ingredient, water demineralized 1000 ml). 5 boxes per dilution level are seeded.

The bacterial counts are carried out by spreading with 100 μl beads of the suspension at the dilution level considered on King B agar medium supplemented with 75 mg / 1 of rifampycin. Three dishes per dilution level are seeded.

The dishes are incubated at 25 ° C and are read after 24 hours for bacteria and 48 to 72 hours for fungi.

5- Biotests

Treatments identical to those planned for population dynamics are carried out concomitantly with the latter and incubate under the same conditions. The containers are opened as frequently as those intended for the study of dynamics. Biotests are carried out after the populations have reached a plateau in this case, after 30 days of incubation.

The principle of the biotests consists in introducing into the soil containing either the Fusarium Fo47B10 strain, or the Pseudomonas C7R12 strain or both, a Fusarium oxysporum pathogenic strain of flax, the Foln3 strain is to cultivate flax on this soil. The antagonistic activity of Fo47B10 and C7R12 is measured relative to the number of healthy plants remaining.

3g of talc providing 10 propagules of Foln3 / g of soil are mixed with the soil of each container (mixture produced with Turbula type T2C, 20 '). For the co-inoculation experiments, the strain Fo47B10 is brought in the form of a suspension of spores after the incorporation of the clay inoculated by Pseudomonas, in a volume which makes it possible to adjust the humidity to the value of pF3. The theoretical inoculum densities are 10 ⁵ , 10 ⁴ and 10 ⁷ CFU / g of dry soil for respectively

Fo47B10, Foln3 and C7R12, whatever the mode of formulation. The clay, enriched clay and liquid formulations are coded A, Ae, and L respectively in the text and in the figures. The soil is then distributed in the 12 wells of a row of a polystyrene plate comprising 20 rows. The volume of a well is approximately 10 ml. The plates are placed in an air-conditioned room under the following conditions: photoperiod 3 pm to 9 am, light intensity 13,000 lux, humidity 80% at night and 60% during the day, temperature 15 ° C at night and 17 ° C during the day for 15 days then 20ºC at night and 26 ° C during the day for the remaining 8 weeks. Watering is carried out twice a day with tap water and once a week with nutritive solution.

Notation: Notations are made every three days from the 3rd week after the date of installation of the biotest in an air-conditioned room. Plants showing symptoms of Fusarium wilt are cut (isolation is performed for verification) and the number of healthy plants remaining is recorded and expressed as a percentage relative to the initial number of plants.

RESULTS

1. POPULATION DYNAMICS:

1.1 Population dynamics of C7R12 in natural soil.

The results are shown in FIG. 2 in which L, A and Ae respectively mean liquid culture, clay and enriched clay.

The inoculum supplied is in theory 10 ⁷ bacteria / g of dry soil. The liquid inoculum allows immediate germ recovery of 70%, and the Pseudomonas populations remain at a density close to the initial value, namely 3.2 10 ⁷ . Inocolum A allows immediate germ recovery of 1.3% and stabilizes in the soil at this level. On the other hand, if the inoculum Ae authorizes an immediate recovery of the germs of 0.02%, the population inoculated according to this formulation goes from 1.3 10 ⁵ to 8.5 10 in 4 days and stabilizes at a level higher than the population obtained with the liquid inoculum.

1.2 Population dynamics of C7R12 in disinfected soil.

Again, the recovery percentage varies with the type of formulation. It is 42% for the liquid formulation against 0.6% for the Ae formulation. In all cases, the density of the Pseudomonas population increases sharply. The plateaus reached by treatments L and A are close to and close to 10 ° by excess. This plateau is higher in the case of the Ae treatment where the density reached is greater than 10 ⁹ (2.3 10 ⁹ ) bacteria / g dry soil.

The results are shown in Figure 3.

II. ANTAGONIST ACTIVITY

2.1- C7R12 in natural soil.

The results are shown in Figure 4. The Fo47bl0 strain alone causes a significant delay in the onset of symptoms. Co-inoculation with Pseudomonas slightly increases this delay and leads to a higher percentage of survival at the end of the test than that obtained with Fo47B10 alone, but it is not possible to differentiate between the two formulations A and Ae.

2.2- C7R12 in disinfected soil.

As shown in Figure 5, the Fo47B10 strain only delays the onset of symptoms, but in a proportion less than that observed in the case of natural soil. Co-inoculation with Pseudomonas formulated A increases this delay. The greatest efficiency is however obtained with the formulation Ae.

DISCUSSION - CONCLUSION

It is noted that the addition of nitrogenous and carbonaceous nutrients in the formulation support (Ae) allows a very significant growth of the bacterial populations introduced, as well in natural soil as in disinfected soil. If the growth of bacteria introduced into disinfected soil is not a new phenomenon, we can nevertheless observe that the biotic capacity of the medium has been increased by the supply of enriched clay, whereas the plates obtained with the two other modes of formulation are confused. On the other hand, the growth of a bacterial population and its establishment at a high density (> 10 'bacteria / g dry soil) have only rarely been described in natural soil.

We observe a better protective efficacy of Fusarium-Pseudomonas co-inoculation in disinfected soil via the Ae formulation which is explained by a higher density of the bacterial population. In natural soil, this effect is less marked because the high efficiency of Fusarium, which is significant in this case, can hardly be improved, but the trend is however in the direction of a beneficial effect of the formulation Ae.

Table 5: THICK MICROGRANULES TEST 1990

Number of M. S. of Number of M. S. of M. S. to R4 nodules nodos nodosities nodosities of the parts by plant by plant by plant by plant aerial to V3 to V3 to R4 to R4 in g by

Treatments in mg in mg plant

1 0.68 c 6.42 b 5.07 c 40.00 b 6.78 b

2 10.60 b 114.38 a 46.23 a 373.59 a 11.12 a

3 14.63 ab 127.28 a 48.32 a 383.11 a 10.31 a

4 14.38 ab 181.48 a 47.15 a 372.44 a 10.69 a

0 16.05 ab 160.98 a 45.40 a 334.83 a 9.60 a

O 18.73 to 126.38 to 50.45 to 410.08 to 10.95 a

7 12.23 ab 107.35 a 41.83 a 369.57 a 1 1.03 a

8 16.45 ab 135.55 a 51.88 a 367.20 a 10.40 a c 16.98 ab 149.27 a 52.28 a 331.00 a 9.37 a

10 18.15 a 141.75 a 52.35 a 355.33 a 10.23 a

1 1 17.02 ab 123.55 a 42.85 a 327.44 a 9.59 a

12 2.47 c 44.50 bc 18.70 b 252.64 bc 9.84 a N.K. test S S S S S

CV: 21.2% 48.5% 17.8% 1 1.6% 10.9%

TABLE 5 (CONTINUED)

treatment rate quantity yield rate quantity of nitrogen nitrogen net of nitrogen nitrogen of the parts of the parts in q / ha of aerial aerials at 14% seeds seeds at R4 to R4 in qx / ha in g / plant

1 29.6 5.62 1.66 2 34.2 a 6.53 c 2.23 a

3 32.7 a 6.62 abc 2.17 ab 4 32.3 a 6.70 abc 2.16 ab 5 33.7 a 6.56 bc 2.20 a

O 31.3 a 6.66 abc 2.08 ab

7 32.6 to 6.76 ab 2.20 a

8 31.2 a 6.77 ab 2.1 1 ab

9 33.3 a 6.77 ab 2.26 a

10 30.3 a 6.84 a 2.07 ab

11 34.2 a 6.78 ab 2.32 a

12 31.1 a 6.28 d 1.95 b N.K. test NS S S

CV: 5.1% 1.6% 4.8%

TABLE 5 (CONTINUED)

Treatments:

1 - uninoculated witness

2 - witness M not enriched

3 - M * enriched with glucose 2C

4 - M enriched with glucose 2C + benomyl

5 - M benomyl

6 - M enriched with 2C glycerol

7 - M enriched with 2C glycerol + benomyl

8 - M enriched with glucose 6C

9 - M enriched glucose 6C + benomyl

10- M enriched with 6C glycerol

1 1 - M enriched with 6C glycerol + benomyl

12- Commercial microgranulate

* M = Microgranules marketed by AGC, Microbiodivision (Rothamsted Herts,

Britain)

Claims

1. A method for improving the yield of agricultural crops, in which an inoculum containing at least one microbial culture and microgranules is used, said method being characterized in that microgranules prepared in advance are used, said microgranules containing a substance , in particular a nutritive substrate, capable of promoting the growth of germs of the inoculum and said microgranules being inoculated at the time of use with a microbial inoculum.

2. Method according to claim 1, characterized in that microgranules of mineral or organic nature or mixtures of such granules are used, obtained by grinding or granulation.

3. Method according to claim 2, characterized in that granules of mineral origin are used, such as industrial clays and clay mixtures.

4. Method according to claim 2, characterized in that microgranules of organic origin are used, for example based on peat, compost, plant residues and others.

5. Method according to any one of claims 1 to 4, characterized in that the microgranules have a grain size suitable for spreading with conventional agricultural machinery, in particular a particle size of the order of 0.2 to 0, 8 mm.

6. Method according to one of claims 1 to 5 characterized in that the enrichment of the microgranules in advance is carried out by inhibition, immersion or spraying of the microgranules and subsequent drying, by granulation of a suspension of solid material and enrichment substrate or by mixing the microgranules with the substrates finely ground enrichment.

7. Method according to any one of claims 1 to 6, characterized in that as enrichment substance or substrate, any product capable of promoting the survival and the multiplication of the germ to be inoculated is used either directly, by allowing or stimulating its growth, that is by limiting the growth of competing germs in the soil.

8. Method according to claim 7, characterized in that carbon substrates such as glucose, glycerol and any other source of carbon suitable for the growth of micro-organics or else carbon substrates specific for the germ to be favored are used.

9. Method according to claim 7, characterized in that other products are used, in particular nitrogenous or phosphorous and, in general, any nutrient capable of allowing the growth of the germ to be inoculated.

10. Method according to claim 7, characterized in that as enrichment substance or substrate, biocides are used compatible with the germ to be inoculated, but capable of inhibiting the growth of competitors or slowing it down, such as products active in the soil against bacteria, fungi and protozoa, these products can in particular be chosen from antibiotics and fungicides.

11. Method according to any one of claims 1 to 10, characterized in that the inoculation implements one or more germs which can be introduced successively or as a mixture.

12. Method according to any one of claims 1 to 13, characterized in that 'inocula on a solid support are used, such as peat, liquid inocula or gel inocula as well as lyophilized inocula.

13. Method according to any one of claims 1 to 12, characterized in that, as microorganisms for inoculation, one or more of the microorganisms intended to directly promote the growth of plants are used, such as Rhizobium, Bradyrhizobium, Azospirillum, as well as ecto and endomycorrhizogenic fungi, as well as microorganisms usable in biological control against plant diseases and pests, such as bacteria antagonists of bacteria and phytopathogenic fungi (PGPR: Plant Growth Promoting Rhizobacteria , Pseudomonas, Agrobacterium, Bacillus and other similar genera), antagonistic fungi of phytopathogenic fungi (Fusarium, Trichoderma, and other similar species) and entomopathogenic bacteria and fungi (Bacillus thuringiensis, Beauveria, and other similar species) and microorganisms usable in depollution and bioremediation of soils.

14. Microgranules comprising a solid support and at least one substance capable of promoting the growth of germs in a microbial inoculum with which the microgranules are brought into contact at the time of use.

15. Microgranules according to claim 14, prepared in advance and stored separately, in particular in the dry state.