CA2144739A1 - Microgranules useful in combination with bacterial inoculums; process for preparing the same and their use in agriculture - Google Patents

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

~ 1 ~ 4 ~ 3 ~

MICROGRANULES FOR USE IN COMBINATION WITH
BACTERIAL INOCULUMS, THEIR OBTAINMENT AND THEIR
APPLICATION IN AGRICULTURE
The invention relates to the field of crops in which seeds, plants and all other known culture media. Under another aspect the invention is part of the general technique of soil treatment in Agriculture. It is applicable with a lot benefits to legume crops.
In the prior art, we have already proposed to use microbial inocula in Agriculture. The purpose of this technique is to provide when placing seeds in the soil or plants, useful or essential bacteria for plant growth. Inocula used for this purpose can be cultures liquid microbials, or on a solid support. he can also microbial cultures dehydrated, in the form of a gel or lyophilisate.
The inocula currently being used and to be approved are pure cultures, free of contaminant in order to facilitate quality control, improve survival and prevent any introduction other than the microbe authorized.
We know several different techniques the use of such inocula. For example, in the legumes, a first technique consists to bring the inoculum at the time of sowing by setting in contact with the seed. A variant consists in putting using pre-inoculated seeds. Another technique provides for inoculation of the seedbed:
the inoculum is then brought into the seed line, either in liquid form or in the form of W094 / 06733 ~ ~ 9 PCT / FR93 / 00901 microgranules, these being practically applied to using known devices for this purpose for distribute phytosanitary products. This last technique is often preferred by farmers because the quantities of product to handle are lower than in the case of inoculation of seeds. For example, just use around 10 kg per hectare of microgranules instead of 80 to 150 kg seeds in the case of seed inoculation.
The results have been recorded in practice also show that the effectiveness of the technique inoculation of the seedbed as good.
As an illustration, we currently know a technique which consists in mixing at the time of dry use of a certain amount of inoculum bacterial on solid support, such as peat, with original mineral microgranules industrial, such as clays, carbonates or others, the size of which is suitable for spreading.
In practice, the particle size is approximately 0.2 to 0.8mm. Such a technique has the advantage of using inexpensive microgranules, in combination with small amounts of inocula, which may be easily kept apart. In such a technique, we don't really care about fixing the inoculum on the microgranules, these not being used actually dilute the inoculum. The nature of such microgranules can be very varied and apart from clay and mineral materials, de ~ a cited, we can mention peat, sand, logs polyethylene and other similar susceptible materials to be spread by agricultural machinery.
Another known technique is to inoculate in advance and during the production of the granules of diverse nature, so as to achieve ~ ~ 14 ~ 739 microgranules ready to use. We thus know granules containing the microorganism (s) a inoculate, based on clay, peat, alginates, vermiculite, perlite, various gels polysaccharides, silica, calcium sulfate. Have have also been described microgranules comprising constituent associations, for example from the calcite coated with peat, polysaccharide gels and silica, peat-clay and sulfate mixtures calcium, clays and alginates, among others.
This technique of making in advance of inoculated microgranules is attractive but it is very difficult to manufacture these products in conditions sterile and ensure low temperature storage taking into account the volumes involved, the quantity of product to be used being of the order of 10 ~ g per hectare for example. By the way, if we introduce also nutrients with the support of inoculated microgranules there is an increased risk of development of contagious germs in the environment thus enriched.
The following patents and patent applications illustrate this state of the art.
The patent US-5055293 (ARONSON) relates to the biological control of insect larvae infecting corn.
Granules of inert clay, or another mineral or organic material acceptable for use agricultural, covered with a layer of Bacillus bacteria laterosporus are described there.
They can be sprayed with a solution of cells or spores that may contain materials nutrients and excipients, then be dried.
This document does not mention the deposit of microorganisms, just before application in the field, 21 ~ L7 ~ 4 on granules already containing materials nutritious.
European patent application EP-A-0295968 (PIONEER FRANCE MAIS) aims to protect microorganisms contained in compositions fertilizer. Microorganisms are microencapsulated in a polysaccharide matrix, then the inoculum thus prepared is mixed with the fertilizer in the form solid or in liquid form immediately before spreading. Note that it is only a question mineral fertilizers for plant growth and no addition of nutritive substrates to obtain a growth in the soil of a microorganism. Furthermore, microorganisms are premicroencapsulated before they fertilizer mixture.
European application EP-A-0203708 (AG ~ ACE ~ US) relates to a process for adsorption of bacteria in phase stationary by mixing bacteria suspensions 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) has for object a method of production of a mycelial fertilizer in which incubates thermoactinomycetes 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.
Microorganisms are therefore not added just before the field application of the granules.
The present invention belongs to the art general microbial inoculation using microgranules to be inoculated extemporaneously. She has W094 / 06733 ~ 14 ~ 7 3 ~ PCT / FR93 / 0090l mainly for the purpose of using microgranules which, after burial in the soil, allow improve survival and get multiplication inoculum microorganisms.
In a first aspect, the invention relates microgranules comprising a solid support and less a substance capable of promoting growth germs from a microbial inoculum with which micro-granules are brought into contact at the time of employment.
The microgranules according to the invention are not inoculated in advance. They are implemented, at the time employment, together with the microbial inoculum.
In another aspect, the invention also has for object a process for improving the yield of agricultural crops, in which an inoculum is used containing at least one microbial culture and microgranules, said process being characterized in that the microgranules include at least one substance capable of promoting the growth of inoculum germs.
The process of the invention therefore consists in use microgranules containing a su ~ stance, in particular a nutritious substrate, capable of promoting the growth of germs in the inoculum, said microgranules being prepared in advance and capable of be stored in a dry state, and inoculate them at the time employment 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, it is can use any type of microgranules obtained by grinding or granulation, whether mineral or organic microgranules, or mixtures of such granules. Among the granules

2 ~ ~ 73 ~ 6 of mineral origin, we can cite clays industrial and clay mixtures, which gave good practical results. As for microgranules of organic origin, various substrates can be mentioned granules or crushed, based on peat, compost, residues plants and others. The technical literature contains many examples of usable microgranules as well as indications on the grain size desirable. Choose dimensions of grains suitable for spreading with machines usual sgricoles, and we know that one dimension appropriate for the size of the microgranules is 0.2 to 0.8 mm, for information only and not limiting.
According to the essential characteristic of the invention, the microgranules in question are enriched in advance, by contacting the microgranules proper and of the enrichment substrate, the ~ uel consists of at least one substance capable of promote the growth of inoculum germs microbial.
In practice, the enrichment of microgranules in advance can be achieved by imbibition, immersion or spraying of microgranules and subsequent drying, by granulation a suspension of solid material and the substrate enrichment or by mixing the microgranules with finely ground enrichment substrates. According to this embodiment, the microgranules prepared a the advance can be stored dry, so as not to allow the development of microorganisms during storage.
Regarding substances or substrates enrichment, we can use any product capable of promoting the survival and multiplication of germ to be inoculated, either directly by allowing or stimulating its growth, either by limiting the growth of competing soil germs. We can in particular use carbon-based substrates such as glucose, glycerol and any other source of carbon suitable for the growth of most micro-organizations or on the contrary call on carbon-specific substrates of the germ to favor.
It is also possible to use other products, notably nitrogen or phosphorus and more generally any nutrient that can provide the growth of the germ to be inoculated. But, according to the inventive concept now described, we can also use biocides compatible with the germ a inoculate but likely to inhibit the growth of competitors or slow it down, 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 be especially chosen from antibiotics, fungicides and more generally any product phytosanitary.
It was previously stated that in the process of the invention an inoculum could be used containing at least one microbial culture. Fact, inoculation may use one or more germs, which can be brought successively or in mixed. The microgranules are inoculated by user, at the time of use or shortly before this one. All types of inoculum can be used, whatever their support and packaging, for example inoculums on a support solid, such as peat, liquid inoculums, gel inoculums or lyophilized inoculums.
It's inoculation techniques are known to WO 94/06733 ~ ~ ~ PC ~ r / FR93 / 00901 2 ~ 8 skilled in the art and need not be described further in detail.
The invention can be applied with one or more several microorganisms likely to be used in the inoculation of seeds, plants, growing media and soils. It's about especially of all usable microorganisms to directly promote plant growth, such as those belonging to the Rhizobium species, Bradyrhizobium, Azospirillum, as well as ecto- and endomycorrhizogenic fungi. Results evidence was obtained with the microorganism Bradyrhizobium japonicum. Also enter the framework of the invention the usable microorganisms in biological control of plant diseases and pests, such as bacteria bacteria and phytopathogenic fungi (PGPR
: Plant Growth Promoting Rhizobacteria, Pseudomonas, Agrobacterium, Bacillus and other similar genera), mushroom antagonistic mushrooms phytopathogenes (Fusarium, Trichoderma, and others similar species) and bacteria and fungi entomopathogenes (Bacillus thuringiensis, Beauveria, and other similar species). We can still cite microorganisms usable in depollution and soil bioremediation.
The invention provides advantages over traditional use of inoculum. We know in effect that the effectiveness of microbial inoculations in agriculture directly depends on the number of living and effective germs brought. he is also known that the immediate losses to inoculation are generally very important and are due mainly to the mortality of germs brought by desiccation. So we are currently trying to W094 / 06733 ~ 1 ~ 4 7 ~ ~ PCT / FR93 / 00901 9 ~,.

find processes that increase the possible dose of microorganisms to inoculate. But he - must also limit losses of microorganisms during of use in the fields, while taking into account physical limits, the possible quantities to be inoculated being limited by the weight of seeds as well as by available equipment, as well as economical (production costs, storage). For this reason, we has already proposed in the prior art of realize the growth of the microorganism inoculated in soils, but only after inoculation. Some attempts have been made to add substrates nutritious directly in the soil, for example a carbon substrate. But then you have to add to the ground, for example in the case of the treatment of a bed of seeds, between 20 and 200 kg of carbonaceous substrate per hectare, which corresponds to totally unrealistic in field crops.
The invention provides growth germs brought into the soil by increasing their number, which translates to either an improved effect inoculation, either by a safety effect in case where difficult conditions are present during the inoculation treatment, which may be the case for partially deficient inoculum or mortality abnormal at inoculation. Thanks to the invention, the dose of microorganisms made available to the seed or plant can be grown or maintained in excellent safety conditions.
We already said before that we knew in the prior art the use of microgranules, and more particularly of pre-inoculated microgranules i.e. inoculated with advance during manufacture. Compared to a such a technique of pre-inoculated microgranules, the -WO 94/06733 ~ ~ ~ 10 PC ~ r / FR93 / 00901 The present invention provides numerous advantages, in especially the following:
- The invention allows the use of commercial microgranules, generally inexpensive, - the enrichment in nutritive substrate can be performed without sterility requirement and is therefore cheaper, - zero or very reduced risks of development contaminating germs in the microgranules dry storage), while such risks appear on pre-inoculated microgranules, - storage possible at ordinary temperature on long-lasting, the properties of the microgranule not related to the number of germs it contains and their survival, while we know the effect adverse temperature for the survival of a inoculum and pre-inoculated microgranules, - cold storage possible of the inoculum microbial in small packaging separate from microgranules, which can be packaged bulky, - varied possibilities to play on nature and the concentration of the substrates, the adaptation of the nature and specificity of the seed substrate a inoculate, - possibility of adding biocides disadvantaging competitors, without the risk of toxicity by prolonged contact between the inoculum and the microgranulated.
The invention will now be illustrated without being in no way limited by examples of implementation in the laboratory and in the field.
Figure l shows the results with granules enriched with Bradyrhizobium japonicum. The development of microorganisms (in W094 / 06733 1i PCT / FR93 / 00901 ordered) is followed as a function of time (in days).
Figures 2 and 3 illustrate the growth of Pseudomonas C7Rl2 strain populations under liquid form (L), in normal clay form (A) and in enriched clay form (Ae), respectively in soil natural (Figure 2) and in disinfected soil (Figure 3).
Figures 4 and 5 show the evolution of number of healthy flax plants (ordinate) in according to the number of days (on the abscissa) respectively in natural soil (figure 4) and in soil disinfected (Figure 5) treated by different formulations of microorganisms.
EXAMPLE 1: Microqranules inoculated with BradYrhizobium japonicum.
We will first expose all the means and modes of carrying out the experiment and then results of it.
STRAINS
All greenhouse and field trials are made with the commercial strain G49 (IARI SBl6, New Delhi) from Bradyrhizodium ~ aponicum. Culture chosen is that of soy.
We know that, for this plant, nodulation (number of knots, weight of knots) 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, we used a strain of B. japonicum resistant to antibiotics. At laboratory, was isolated on Petri dish containing antibiotics a spontaneous resistant G49 mutant rifampicin (100 mg / ml) and kanamycin (200 mg / ml), the stability of which has been checked and which has been called G49RfKm.
This new strain was deposited at the 73 ~ 12 CNCM collection of the Institut Pasteur under the N I-1264 on ll September 1992.
MICROGRANULES
These are commercial products responding to following criteria:
- density high enough to allow a 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 towards bacteria inoculated, - mixing with an easy-to-make inoculum, retaining enough peat, and giving a product homogeneous.
Granules with porosity were used sufficient and not destructuring in water, so to keep all their properties during the phases impregnation (by the enrichment solution) and drying. This procedure does not affect the ease distribution of microgranules by microgranulator.
Two types of microgranules meet these requirements have been implemented, one called K, based of cristoballite, and the other M, form of calcined montmorillonite.
Table 1 gives the characteristics essential of these microgranules.

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Essential characteristics of microqranules K and M used in the experiments KM
Composition cristoballite te (silica) calcined illite <10% (clay) Water content at pF 2.5 (point of soil drying) 0.64 0.39 at pF 4.2 (point of wilt) 0.48 0.21 pH 5.5 7.8 Granulometry :
> 0.8 mm 1.0 ~ 1.4%
0.8 - 0.25 mm 98% 98%
<0.25 mm 1% 0.2%
Density:
humidity 0% 0.57 1.0 humidity ~ t 13% 1.0 humidity 30% 0.70 Number of particles per kg 13 to 17X106 5 to 8X106 30 Destructuring by water no no ENRICHMENT SUBSTRATES
They are formed essentially from a source of carbon, a source of nitrogen, and minerals.
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 microgranules. But it must be high enough to, on the one hand, lead to complete dissolution of the products, on the other hand -40 allow homogenization of the mixture with microgranules before its complete absorption.
-This solution is provided on dry microgranules, the whole being stirred immediately to obtain a uniform mixture. She 7 3 ~ ~
WO 94/06733 14 PC ~ r / FR93 / 00901 is absorbed by the porosity.
A subsequent drying allows to leave the dry nutrient substrate in this porosity, allowing long storage, even in non-sterile.
The concentrations used for experiments vary from 16 to 96g of materials organic (expressed as organic carbon), per kg dry microgranules.
ASSOCIATED FUNGICIDES
After a test on their effectiveness against soil fungi and their harmlessness to inoculated strains, retained products are added in the enrichment solution, before their incorporation into microgranules.
SOILS AND CROP SUPPORTS USED
Field trials Most of the work was carried out on ground clay from the INRA domain - Dijon, and part secondary concerned sandy soil in the region. These soils were initially devoid of B. japonicum.
The essential characteristics are given in table 2.

2 ~ L7 ~

Table 2: Characteristics of the soils used for field trials CharacteristicsClay soil 5 sandy Grain size in g / kg clay 382 75 silt 562 227 sand 56 698 Equivalent humidity in p 100 28.1 9.9 Organic carbon method Anne 16.06.9 15 in g / k Organic matter in g / kg 27.5 11.9 Total organic nitrogen in g / kg 1,560.67 (Kjeldahl method) water pH 7.0 7.3 Total limestone 0 0 25 Cation exchange capacity Metson in meq p 100 21.7 4.3 Laboratory tests.
They were made for their almost totality with the same clay soil as that used for field trials. This soil is devoid of B. japonicum.
PLANT
All the tests were carried out with the variety of Maple Arrow soybeans, indeterminate type and belonging to the OO precocity group.
INOCULUMS
When using the normal G49 strain, inoculum, peat or liquid, is a product commercial ("Biodoz", from the LIPHA Company).
The resistant G49 inocolum (G49RfKm) is produced in the laboratory.
A flask containing a culture medium liquid, based on malt extract, is inoculated sterile from an agar tube containing the W094 / 06733 214 ~ ~ 3 9 16 PCT / FR93 / 00901 strain. It is then placed on a table agitation (200 rpm) at 28 C for 6 to 7 days. The culture obtained then constitutes the liquid inoculum uses, which can be stored for several months at 4 C.
For the production of peat inoculum, we start a sterile peat of the same origin as that of the commercial inoculum. In a bottle containing 30g of this product, at 12% humidity, 12 ml are added liquid inoculum. After homogenization and maturation 2 to 3 seconds at room temperature, obtains a product which has substantially the same properties than commercial peat: humidity 55-60%, richness of around 109 germs per gram of peat.
MICROGRANULE INOCULATION TECHNIQUES
A proportion corresponding to that of field inoculation, i.e. 4 ~ inoculum pure compared to microgranules.
Taking into account the quantities necessary for the introduction into the soil of manipulation and precision expected, we have worked on batches of 25 g of microgranules (measured dry and without enrichment), in vials of 250 ml.
For use with 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 next few minutes.
When used with an inoculum liquid, the microgranules, dry at the start, receive an inoculum previously diluted to provide simultaneously 1 ml of a pure inoculum for 25 g of microgranules, but also the amount of water required to be at the desired humidity (7.5 ml for K and 3.25 W094 / 06733 21 ~ ~ 7 ~ 9 PCT / FR93 / 00901 17 ~

ml for M in most experiments). After agitation for homogenization, the microgranules are - use quickly.
Introduction techniques in soils and incubation After sieving at 4 mm or 2 mm, the soils are introduced into glass bottles, either at a 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, in general at the level of 80 ~ of the capacity of water retention.
In these bottles, are introduced 300 mg and 120 mg of microgranules respectively, this which represents approximately 4500 and 1800 respectively microgranules for K, 2100 and 800 for M.
The bottles are then shaken manually by so as to distribute the microgranules throughout the volume of soil.
The inoculated vials are incubated by closing with a plastic film, intended to prevent loss of water while allowing exchanges respiratory. They are then placed in the dark, generally at 20- C, but also at 15- C or 28- C for some tests.
Counts are carried out on the whole of a bottle after several days of incubation.
Petri dish counts The bulk of the measures consist of population counts of B. japonicum capable to form colonies on Petri dishes containing a gelose culture medium. These counts are carried out at several stages of inoculation:
- inocula themselves, before use, on ~ uantites generally of 1 ml or 1 g, - microgranules after inoculation, in WO 94/06733 PC ~ r / FR93 / 00901 w14 ~ 7 ~ 18 working either on the whole of a lot or on aliquots, - soils having received inoculated microgranules, by working on the entire contents of a bottle.
5The products to be counted for their richness in B. japonicum are introduced into 100 ml of a extraction solution and placed on a table stirring (200 rpm) for 30 minutes.
Successive dilutions are then performed at 1/10, and the bacterial suspensions obtained are spread over 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 medium gelose from a Petri dish. This is then put incubate at 28- C, then the colonies present are numbers at 5 or 6 days, hence a count in cfu (units forming a colony). A calculation from dilutions used to determine the richness of the product to be analyzed.
In the presence of soil, the previous device is unusable, and counts are made by the method called "double layer". Low 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% gelose solution by supercooling (43 C). After homogenization, this mixture is spread on a box of Petri dish containing the gelose culture medium, as well as the antibiotics and fungicides to which the strain G49RfKm is resistant.
After incubation for 7 days at 28 ° C., the colonies on the boxes are numbered, the dilutions being made so as to count between 30 and 300 colonies per dish.
35All the counts are made with 3 ~ 1 ~ 4739 WO 94/06733 PC ~ r / FR93 / 00901 1 ~, ,, repetitions for each treatment, and results are given with the calculated standard deviations.
- Field trials Tests implemented on soils previously described are 4-block devices complete.
Sowing is carried out at the end of April-beginning of May, the objective being to bring 600,000 seeds per hectare (Maple Arrow variety) inoculated at the dose agronomic, i.e. 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, according to the needs of the culture.
RESULTS
The results of the experiments are reported below.
Figure 1 summarizes the results obtained during laboratory tests, comparing microgranules enriched (E) or not enriched (M), inoculated with inocolum liguide and incubated in unsterilized soil, kept moist (H) or subjected to desiccation (S).
Number of B. japonicum germs recovered after liquid inoculation on microgranules and introduction into the soil, as a function of time and according to the desiccation of the soil during incubation.
The legends of the figure are: N = microgranules not enriched, E = enriched microgranules GLY3C, H = soil kept moist (82% of the retention capacity in water) during the 21 days, S = soil subjected to a natural drying after 7 days of incubation.
From the results illustrated in the figure 1, we see that:

W094 / 06733 ~ 1 ~ 4 7 3 9 20 PCT / FR93 / 0090l - after 7 days, the enriched microgranules allow to obtain a multiplication of B.
japonicum by a factor greater than 10 compared to unenriched microgranules, - after 21 days, the enriched microgranules keep the number of B. japonicum at a level 10 times higher than that obtained with microgranules not enriched when the soil is kept moist and has a level more than 100 times higher when the ground dried.
Tables 3 to 7 summarize the results obtained during field trials. For each table, data in the same column followed by the same letter are not statistically different as of 5% threshold following the Newman and Keuls test.
In Tables 3 and 4, the first results obtained during field trials in 1988 and 1989 respectively. They show an effect significant enrichment on nodulation.
Tables 5, 6 and 7 relate to tests performed in 1990 and 1991 with various conditions of processing, as indicated at the bottom of each board.
In Table 5, the treatments corresponding to the invention (enriched microgranules inoculated with a peat inoculum) are the treatments

3, 6, 8, 10.
We can see an increase in the number of plant nodules at stage V3, general for all the treatments, compared to the microgranular control not enriched (treatment 2). This increase is significant only for treatments 6 and 10.
Similarly, an increase in the nitrogen level is obtained.
seeds 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 by peat inoculum (2 and 6), or by inoculum liquid (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 enrichment).
With a liquid inoculum (10 compared to 9), we gets a significant increase in the nodulation at V3 and R4, as well as an increase significant of 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, but significant increases in nitrogen seeds. Note that this last effect is obtained with different carbon substrates.
With the liquid inoculum (10 compared to 9), we gets a significant significant increase in nodulation (V3 and R4) and the nitrogen level of seeds.
EXAMPLE 2 Microqranules inoculated with Fusarium or Pseudomonas.
Among the cryptogamic malsdies which are affected vegetables and plants horticultural, vascular fusarium wilt, tracheomycosis due to pathogenic forms of the Fusarium fungus oxysporum, is one of the most problematic because it to date no chemical control means exist specific.

W094 / 06733 ~ J l ~ 47 3 22 PCT / FR93 / 0090l On the other hand, biological control using competition between a non-pathogenic form and the specific pathogenic form of the same species Fusarium oxysporum gives satisfactory results under greenhouse growing conditions, either artificial substrate, either in disinfected soil. The principle consists in introducing the inoculum not pathogen in the substrate at the time of sowing or transplanting. In addition, the antagonistic effect of non-pathogens 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 form of formulation which consists to give a competitive advantage to the inoculum by incorporation of a source of nutrients in the formulation support. Population dynamics bacteria of the genus Pseudomonas Sp. were measured both in natural soil and in disinfected soil.
The activity of inoculated microorganisms has also been tested in greenhouse biotests using the host plant-pathogen flax couple F.oxysporum f.sp. lini.
MATERIAL AND METHODS
1- Stumps and plants.
Fusarium oxysporum strain Fo47B10, mutant spontaneous benomyl-resistant strain Fo47 used in biological control (n- from collection CNCM I-1363).
Fusarium oxysporum f.sp. lini, Foln 3 strain, (collection number CNCM I-1362) pathogen of flax.
Mutant fluorescent pseudomonas strain C7R12 spontaneous rifampycin-resistant layer C7 used in biological control (collection number CNCM I-35 1361).

Linum usitatissimum flax, Opaline variety, susceptible to vascular fusarium wilt.
2- The ground.
Silty clay soil collected in the region of Dijon, air dried and sieved to 2mm. (composition:
sand 15 ~, silt 47%, clay 35%, C 1.2%, CaC03 0%, pH 6.9).
The soil is distributed in containers in aluminum at the rate of 140 g of dry soil / container. The disinfected soil was passed 3 times 1 hour a the autoclave at 120 C at 24 hour intervals. Humidity soil is adjusted to pF3 (20% dry soil) during inoculation.
3. Inocula 3.1- Preparation of fungal inocula For biotests.
Mushrooms are grown 5 ~ bears on liquid malt medium stirred at 25-C (Difco lOg malt, water demineralized 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-Fusarium strain pathogen Fo47B10 is prepared as an inoculum liquid (suspension of spores in sterile water).
Inoculum of the pathogenic Fusarium strain Foln3 is prepared as a talc formulation:
final pellet is taken up in a minimum volume of water (approximately 20% of the initial volume of the crop) and is incorporated with virgin talc (1 pellet volume -2 talc volumes). The mixture is left to dry for 48 hours in an oven ventilated at 20 C. The talc is then sprayed and sieved at 200 ym. It is kept at 4 C.
Its title is measured before any use. Talc inoculated is possibly diluted in virgin talc W094 / 06733 ~ 24 PCT / FR93 / 0090l 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 has 120 g of glycerol / kg of clay. Bacteria produced on King B solid medium (Peptone Difco 20g, glycerin 12.6g, K2HPO4 1.3g, MgSO47H2O 1.5g, Agar 15g, water 1000 ml) are collected in water sterile and washed 3 times by centrifugation (20 min, 4000g). They are incorporated into the clay at the time of their introduction into the ground.
Witnesses are made by inoculation direct suspension of bacteria washed in a volume allowing humidity to be adjusted to the value of pF3.

4- The analyzes Each container constitutes a unit experimental. 3 experimental units constituting 3 independent rehearsals are performed for a treatment given. The containers incubate at 25-C. The population dynamics are monitored for 20 to 30 days.
At regular time intervals, 5g of soil per experimental unit are withdrawn, suspended in 100 ml of water and stirred vigorously for 15 min on an agitator constituting the mother suspensions.
A series of 1/10 dilution suspensions is made from each of these mothers suspensions.
Fungal counts are made by incorporation of 1 ml of the suspension at dilution considered in agar + benomyl malt medium supercooling (lOg malt, 15g agar, citric acid after autoclaving 250 mg, Benomyl after autoclaving 10 mg in the form of benlate with 50% of active matter, water 21 4 ~

demineralized 1000 ml). 5 boxes per level of dilution are seeded.
Bacterial counts are performed by spreading with beads of 100 ~ 1 of the suspension at dilution level considered on King B agar medium add 75 mg / l rifampycin. Three boxes per dilution level are seeded.
The dishes are incubated at 25 ° C. and are read after 24h for bacteria and 48 to 72h for mushrooms.

5- Biotests Treatments identical to those provided for population dynamics are realized in a way concomitant to these and incubate in same conditions. Containers are also open frequently than those intended for the study of dynamic. Biotests are performed after the populations have reached a plateau in this case, after 30 days of incubation.
The principle of biotests consists in introducing in the soil containing either the Fusarium strain Fo47B10, either the Pseudomonas C7R12 strain or two, a strain of Fusarium oxysporum pathogenic to flax, the Foln3 strain is to grow flax on this ground. The antagonistic activity of Fo47B10 and C7R12 is measures relative to the number of healthy plants remaining.
3g of talc providing 104 propagules of Foln3 / g of soil are mixed with the soil of each container (mixture made with Turbula type T2C, 20 '). For the co-inoculation experiments, the Fo47B10 strain is provided in the form of a suspension of spores after incorporating the clay inoculated by Pseudomonas, in a volume that allows to adjust humidity has the value of pF3.

W 0 94/06733 ~ ~ A 4 ~ 3 9 26 PC ~ r / FR93 / 00901 The theoretical inoculum densities are 105, 104 and 107 CEU / g of dry soil for respectively Fo47B10, Foln3 and C7R12, whatever the mode of formulation. Clay formulations, enriched clay and liquid are coded A, Ae, and L respectively in the text and in the figures. The floor is then distributed in the 12 wells of a row of a plate of polystyrene with 20 rows. The volume of a well is approximately 10 ml. The plates are placed in air-conditioned room under the following conditions:
photoperiod 3 p.m. - 9 a.m., 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 20-C
night and 26 C during the day for the remaining 8 weeks. The waterings are carried out twice a day with water from tap and once a week with solution nutritious.
Scoring: Scoring is done every three days from the 3rd week after the date for setting up the biotest in an air-conditioned room. The plants with 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 DYNAMIOUES:
1.1 Population dynamics of C7R12 in soil natural.
The results are shown in Figure 2 in which L, A and Ae mean respectively liquid culture, clay and enriched clay.
The inoculum provides is in theory 107 bacteria / g dry soil. The liquid inoculum allows 70% immediate germ recovery, and 7 ~ ~

Pseudomonas populations remain at a density close to the initial value, i.e. 3.2 107.
Inocolum A authorizes an immediate recovery of 1.3% germs and stabilizes in the soil at this level. On the other hand, if the inoculum Ae authorizes only one 0.02% immediate germ recovery, the population inoculated according to this formulation goes from 1.3 105 to 8.5 107 in 4 days and stabilizes at a level higher than the population obtained with the liquid inoculum.
1.2 Dynamic of C7R12 in soil disinfected.
Again, the recovery percentage varies with the type of formulation. It is 42% for the liquid formulation versus 0.6% for formulation Ae. In all cases, the population density of Pseudomonas increases sharply. The affected plateaus by the treatments L and A are close to and close to 108 by excess. This plateau is higher in the case of treatment Ae where the density attained is greater than 109 (2.3 109) bacteria / g dry soil.
The results are shown in Figure 3.
II. A ~ llvll ~ ANTAGONIST
2.1- C7R12 in natural soil.
The results are shown in Figure 4. The Fo47blO strain alone causes significant delay in the onset of symptoms. Co-inoculation with Pseudomonas slightly increases this delay and results in a percentage of survival at the end of the test greater than that obtained with Fo47B10 alone, but it it is not possible to differentiate the two formulations A and Ae.
2.2- C7R12 in disinfected soil.
As shown in Figure 5, the strain Fo47B10 only delays the onset of symptoms, but ~ 1,447 ~ 28 in a smaller proportion than that observed in the natural soil. Co-inoculation with Pseudomonas Formula A increases this delay. The biggest efficiency is however obtained with the formulation Ae.
DISCUSSION - CONCLUSION
We see that the intake of nitrogen nutrients and carbons in the formulation support (Ae) allows very significant population growth introduced bacteria, both in natural soil only in disinfected soil. If the growth of bacteria introduced into disinfected soil is not a phenomenon again, we can see that the capacity biotic environment was increased by the contribution enriched clay, while the trays obtained with the other two modes of formulation are confused. However, the growth of a population bacterial and its establishment at a high density (> 107 bacteria / g dry soil) have only rarely been described in natural soil.
We observe a better protection efficiency of Fusarium-Pseudomonas co-inoculation in s ~ l disinfects via the formulation Ae which is explained, by a higher density of the bacterial population.
In natural soil, this effect is less marked because the clean efficacy of Fusarium important in this case can hardly be improved but the trend is going however in the sense of a beneficial effect of the formulation Ae.

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Claims (15)

1. Method for improving the performance of agricultural crops, in which an inoculum is used containing at least one microbial culture and microgranules, said method being characterized in that that we use microgranules prepared in advance, said microgranules containing a substance , in particular a nutrient substrate, capable of promoting the growth of the 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, obtained by grinding or granulation. 3. Method according to claim 2, characterized in that original granules are used mineral, such as industrial clays and clay mixtures. 4. Method according to claim 2, characterized in that microgranules are used of organic origin, for example based on peat, compost, plant residues and others. 5. Process according to any of the claims 1 to 4, characterized in that the microgranules have a grain size suitable for spreading with the usual 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 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 the enrichment substrate or by mixing the microgranules with the substrates finely ground enrichment. 7. Process according to any of claims 1 to 6, characterized in that as enrichment substance or substrate, one uses any product capable of promoting the survival and multiplication of the germ to be inoculated either directly, by allowing or stimulating its growth, either by limiting the growth of competing soil germs. 8. Method according to claim 7, characterized in that carbonaceous substrates are used such as glucose, glycerol and any other source of carbon suitable for the growth of microorganisms or specific carbonaceous substrates of the germ to to favor. 9. Method according to claim 7, characterized in that other products are used, in particular nitrogen or phosphorus and, in a way general, any nutrient likely to allow the growth of the germ to be inoculated. 10. Method according to claim 7, characterized in that as substance or substrate enrichment, compatible biocides are used with the germ to be inoculated, but likely to inhibit competitors' growth or to slow it down, such as only active products in the soil against bacteria, fungi and protozoa, these products which may in particular be chosen from antibiotics and fungicides. 11. Process according to any of the claims 1 to 10, characterized in that the inoculation uses one or more germs which can be introduced successively or in a mixture. 12. Process according to any of the claims 1 to 13, characterized in that one uses inocula on a solid support, such as peat, liquid inoculums or gel inoculums as well as freeze-dried inoculums. 13. Process according to any of the claims 1 to 12, characterized in that, as of microorganisms for inoculation, a or more of the microorganisms intended to promote directly plant growth, such as Rhizobium, Bradyrhizobium, Azospirillum, as well as ecto and endomycorrhizal fungi, as well as micro-organisms used in biological control against plant diseases and pests, as bacteria antagonistic to bacteria and phytopathogenic fungi (PGPR: Plant Growth Promoting Rhizobacteria, Pseudomonas, Agrobacterium, Bacillus and other similar genera), fungi antagonists of phytopathogenic fungi (Fusarium, Trichoderma, and other similar species) and entomopathogenic bacteria and fungi (Bacillus thuringiensis, Beauveria, and other species similar) and micro-organisms usable in soil depollution and bioremediation. 14. Microgranules with a solid carrier and at least one substance capable of promoting the sprout growth of a microbial inoculum with which the microgranules are brought into contact when employment. 15. Microgranules according to claim 14, prepared in advance and stored separately, in particular at the dry state.