FR2695929A1 - Enriched microgranules, usable in combination with microbial inocula, their production and their application 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

 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 solid support. They can also be dehydrated microbial cultures, in the form of a gel or a 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 to this effect 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 inoculate 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 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 amount 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, one does not really worry about the fixation of the inoculum on the microgranules, these in fact only serving to dilute the inoculum. The nature of such microgranules can be very varied and apart from the clay and mineral materials already mentioned, we can mention peat, sand, polyethylene balls and other similar materials likely to be spread by agricultural machinery.

 Another known technique consists in inoculating in advance and during the production 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. Also described have been microgranules comprising combinations of constituents, for example calcite coated with peat, polysaccharide and silica gels, peat-clay and calcium sulfate mixtures, clays and alginates, among others. making 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 of the order 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 present invention belongs to the general technique of microbial inoculation using microgranules to be inoculated extemporaneously. Its main purpose is to use 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 process 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 able to be prepared in advance and 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.

 According to a variant, the method of the invention does not use such microgranules prepared in advance but consists in adding to known microgranules, at the time of use, together or not with the microbial inoculum, at least one substance, for example a nutritive substrate, capable of promoting the growth of germs in the 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, either in advance or at the time of use, by bringing the microgranules themselves into contact with the enrichment substrate, which consists of at least a substance capable of promoting the growth of germs in the bacterial 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.

 In another embodiment, the enrichment of the microgranules is only carried out at the time of use, by any of the techniques already mentioned (imbibition, immersion or spraying or even mixing with dry matter). The enrichment substrate can then be used alone or as a mixture with the microbial inoculum.

 With regard to enrichment substances or substrates, any product capable of promoting the survival and multiplication of the germ to be inoculated can be used, either directly by allowing or stimulating its growth, or by limiting the growth of competing germs in the soil. 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 micro-organisms, or on the contrary to use carbon-based substrates specific for the germ to be favored. It is also possible to use other products, in particular nitrogenous or phosphorus-containing and more generally any nutrient capable of allowing the growth of the germ to be inoculated. However, according to the inventive concept presently described, it is also possible to use biocides compatible with the germ. to inoculate but capable of inhibiting or slowing the growth of competitors, such as active products 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 previously 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 inocuums
In the event that the enrichment of the microgranules is carried out at the time of use, inoculation can be carried out before or after the enrichment or alternatively in mixture with the enrichment products. These inoculation techniques are known from 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 are in particular all the microorganisms which can be used to directly promote the growth of plants, such as those belonging to the genera Rhizobium, Bradyrhizobium, Azospirillum, as well as ecto- and endomycorrhizogenic fungi. Successful results have been obtained with the microorganism
Bradyrhizobium japonicum. Also included 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 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. Efforts are therefore being made at present 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 physical limits, the possible quantities to be inoculated being limited by the weight of seeds as well as by the available equipment, as well as economic ( production costs, storage). For this reason, it has already been proposed in the prior art to achieve 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 present invention provides many advantages, in particular the following
the invention makes it possible to use commercially available 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,
- possible storage 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 inocuîum 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, all of which use the bacterium
Bradyrhizobium japonicum designated by the abbreviation B.

japonicum, the chosen crop being that of soybeans. It is known that, for this plant, the nodulation (number of nodules, weight of the nodules) increases when the dose of B. japonicum provided increases. Furthermore, the yield is linked to nodulation.

 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 trials are carried out with the commercial strain G49 (IARI SB16, New Delhi) from B.

 j aponicum.

 To follow the evolution of populations introduced into the soil, a strain of B. japonicum resistant to antibiotics was used. 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 has been deposited in the collection
CNCM of the Institut Pasteur under N I-1264 on September 11, 1992.

MICROGRANULES
These are commercial products meeting the following criteria
- density high enough to allow good descent into the microgranulator,
- grain 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 batten, in order to retain all their properties during the phases of impregnation (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.

Table 1: Essential characteristics of iicrogranules K and M used in the experiments
KN
Cristoballite montmorillonite composition
(silica) calcined
illite <10: (clay)
Water content
a pF 2.5 (point of) soil drying) 0.64 0.39
at pF 4.2 (wilting point) 0.48 0.21 pH 5.5 7.8 Grain size:
> 0.8 mm 1.0 t 1.4%
0.8 - 0.25 mm 98% 98%
<0.25mm 1% 0.2%
Density ::
humidity O% 0.57 1.0
humidity 13% 1.0
humidity 30 t 0.70
Number of particles per kg 13 to 17x106 5 to 8x106
Water destructuring no no
ENRICHMENT SUBSTRATES
They are essentially formed from a carbon source, a nitrogen source, and mineral elements.

 The products selected for enrichment are dissolved in a batten. 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. It 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 96 g 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 area, and a secondary part concerned sandy soil from the same region. These soils were initially devoid of B. japonicum.

 The essential characteristics are given in table 2.

Table 2: Characteristics of the soils used
for field trials
Characteristics Clay soil Sandy soil
Grain size in g / kg
clay 382 75
lijon 562 227
sand 56 698
Equivalent humidity in p 100 28.1 9.9
Organic carbon method Anne in g / kg 16.0 6.9
Organic dobbies in g / kg 27.5 11.9
Total organic nitrogen in g / kg 1.56 0.67 (Kjeldahl method) pH water 7.0 7.3
Total limestone 0 0
Cation exchange capacity
Hetson in meq p 100 21.7 4.3
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 resistant G49 inoculum (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 28 C for 6 to 7 days. The culture obtained then constitutes the liquid inoculum used, which can be stored for several months at 4 C.

 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 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 109 germs 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 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, or of 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 the loss of water while allowing respiratory exchanges. They are then placed in the dark, generally at 20 C, but also at 15 C or at 28 C for certain tests.

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

Petri dish counts
Most of the measurements consist of population counts of B. japonicum capable of forming colonies on Petri dishes containing 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, working either on the whole of a batch or on aliquots,
- soils having received inoculated microgranules, working on the entire contents of a bottle.

 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 28 C, then the colonies present are counted at 5 or 6 douros, hence a count in uf c (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 (43 ° 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 280 ° 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 performed with 3 repetitions for each treatment, and the results are given with the calculated standard deviations.

Field trials
The tests set up on the previously described soils are complete block devices.

The sowing is carried out at the end of April-beginning of May, the objective being to bring 600,000 seeds per hectare (Maple variety
Arrow) inoculated at the agronomic dose, 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, depending on the needs of the crop.

RESULTS
The results of the experiments are reported below.

 The single Figure of the appended drawing summarizes the results obtained during laboratory tests, comparing enriched (E) or unenriched (M) microgranules, inoculated with a liquid inoculum and incubated in unsterilized soil, kept moist (H) or subject to desiccation (S). Number of B. japonicum germs recovered after inoculation in liquid on micrograulics 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 = 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 the Figure, 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.

Table 3: Results of the 1988 tests
inoculation of soybeans with enriched microgranules Inoculation Inoculation Inoculation Inoculation Inoculation
seeds by microgranules by microgranules liquid liquid
enriched (106 live germs (107 live germs
per seed) per seed
Nodules per plant at V3 10.2el 14.6 from 23.6 bc 24.7bc 37.9a
MS of nodules at V3 26.8 from 32.9 bc from 42.5ab 32.5 bc from 45.1 a (in mg per plant)
Nodules per plant at R4 14.0c 22.1b 25.1b 29.7b 38.7a
DM of nodes at R4 240a 261a 299a 301a 270a (in mg per plant)
Nitrogen content of 2.45bc 2.45bc 2.68ab 2.57abc 2.75a plants with R4 (% of seohe matter)
MS of plants at R4 4520bc 5049abc 5571ab 5635ab 4929abc (in mg per plant)
Yield 33.39ab 38.10ab 39.01a 36.38ab 36.35ab (in q / ha at 14% humidity) Table 4: 1989 test results
inoculation of soybeans with enriched microgranules Inoculation Inoculation Inoculation Inoculation Inoculation
seeds by microgranules by microgranules liquid liquid
enriched + fungicide (106 live germs
by seed)
Nodules per plant at V3 15.7 bcde 20.7 bcd 24.3 b 31.7 a 15.2 bcde
MS of nodules at V3 18.8 abc 334 a 272 ab 280 ab 137 bc (in mg per plant)
Nodules per plant at R4 21.4 bc 26.5 bc 33.1 b 52.8 a 29.7 bc
MS of nodules at R4 1430 1654 1641 1764 1610 (in mg per plant)
Nitrogen content of 2.88 abc 2.65 bcd 2.91 ab 3.00 a 2.78 abcd plants at R4 (% of dry matter)
MS of plants with R4 5140 4800 5530 5320 5510 5510 (in mg per plant)
Yield 45.7 ab 41.9 b 46.1 a 46.0 a 45.4 ab (in q / ha at 14% humidity) * Fungicide: benomyl (for the definition of benomyl, see Merk Index-Edition 1989, compound 1053, page 162)
Table 5: THICK MICROGRANULES TEST 1990
Number of MS from Number of MS from MS to R4
knots knots knots knots of the parts
per plant per plant per plant per aerial plant
to V3 to V3 to R4 to R4 in g by
Treatments in mg in mg plant
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 14.63ab 127.28 a 48.32 a 383.11 a 10.31 a
4 14.38ab 181.48a 47.15a 372.44a 10.69a ::
5 16.05 ab 160.98 a 45.40 a 334.83 a 9.60 a
6 18.73 to 126.38 to 50.45 to 410.08 to 10.95 a
7 12.23ab 107.35a 41.83a 369.57a 11.03a
8 16.45ab 135.55a 51.88a 367.20a 10.40a
9 16.98ab 149.27a 52.28a 331.00a 9.37a
10 18.15 a 141.75 a 52.35 a 355.33a 10.23a
11 17.02ab 123.55a 42.85a 327.44a 9.59a
12 2.47 c 44.50 bc 18.70 b 252.64 bc 9.84 a
NKS SSSS test
CV: 21.2% 48.5% 17.8% 11.6% 10.9% treatment rate quantity return rate quantity
nitrogen nitrogen net nitrogen nitrogen
parts of parts in q / ha des des
aerial aerial 14% seeds seeds
to 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 32.7a 6.62abc 2.17ab
4 32.3a 6.70abc 2.16ab
5 33.7 a 6.56 bc 2.20 a
6 31.3a 6.66abc 2.08ab
7 32.6 to 6.76 ab 2.20 a
8 31.2 a 6.77 ab 2.11 ab
9 33.3 a 6.77ab 2.26 a
10 30.3a 6.84a 2.07ab
11 34.2a 6.78ab 2.32a
12 31.1 a 6.28 d 1.95 b NKNS SS test
CV: 51% 1.6% 4.8% Salaries:
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
11- M enriched with 6C glycerol + benomyl
12- Commercial microgranulate
Table 6: 1991 THICK MICROGRANULES TEST
Number of MS from Number of MS from MS to R4 Color
knots knots knots knots parts of the
per plant per plant per plant per plant aerial plants
to V3 to V3 to R4 to R4 in g by to R4
Treatments in mg in mg plant
1 17.7 bcd 19.0abc 29.7 cd 259abc 7.24 5.0
2 19.1 bcd 15.4 bc 33.8 c 243 bc 8.26 5.0
3 20.1 bc. 19.0 abc 32.6 cd 283 ab 8.60 4.9
4 15.8 cde 16.1 bc 32.3 cd 245 bc 7.83 4.9
5 23.5 bc 19.7abc 30.6 cd 286 ab 7.67 5.0
6 27.3 b 23.8 ab 45.3 ab 285 ab 8.11 5.0
7 6.7 e 8.1 c 12.6 e 183 c 7.94 4.0
8 9.7 from 10.2 c 19.5 from 207 bc 8.04 4.6
9 9.8 from 12.5 bc 19.8 from 225 bc 7.76 4.5
10 38.0a 28.0a 53.6a 336a 8.68 5
11 9.5 from 10.3 c 26.7 cd 251 bc 7.30 4.6
14 0 0 0.1 3 6.51 2.8
NKS SSS NS test
CV: 24.8% 32.4% 19.3% 14.1% 11.3%
processing rate quantity yield rate quantity
nitrogen nitrogen net nitrogen nitrogen
parts of parts in q / ha des des
aerial aerial 14% seeds seeds
to R4 to R4 in qx / ha
in g / plant
1 2.74 ab 195.7 ab 36.01 6.49 b 2.34 ab
2 2.75 ab 226.3 ab 36.95 6.54 b 2.42 ab
3 2.81 ab 242.7 a 36.59 6.53 b 2.39 ab
4 2.80 ab 219.1 ab 35.58 6.52 b 2.32 ab
5 2.67 ab 205.4 ab 37.16 6.54 b 2.43 ab
6 2.94 a 238.7 ab 35.15 6.56 b 2.31 ab
7 2.28 c 181.2 b 34.98 6.26 d 2.19 b
8 2.48 bc 199.3 ab 36.43 6.39 bcd 2.33 ab
9 2.49 bc 193.2ab 34.68 6.30 cd 2.18 b
10 2.88 ab 249.6 to 37.36 6.72 to 2.51 a
11 2.70 ab 197.1 ab 35.76 6.43 bc 2.30ab
14 1.65 108.0 19.50 5.14 1.00 test NK SS NS SS
CV: 6.4% 11.3% 5.0% 1.3% 5.0%
Treatments: 1 - K not enriched 2 - K enriched GLYCEROL 3C 3 - K enriched SODIUM GLUCONATE 3C 4 - K enriched GALACTOSE 3C 5 - K enriched MANNITOL 3C 6 - K enriched GLYCEROL 3C + quintozene 7 - K not enriched, poor peat 108 8 - K enriched GLYCEROL 3C, poor peat 108 9 - K not enriched, diluted liquid inoculum 10 - K enriched GLYCEROL 3C, diluted liquid inoculum 11 - LIPHA (standard inoculation control) 14 - not inoculated (not taken into account in the analysis variance)
The microgranule K is used at a humidity of 20% and, unless otherwise specified, it is inoculated with a BIODOZ LIPHA inoculum.

Table 7: 1991 PAGNY MICROGRANULES TEST
Number of MS from Number of MS from MS to R4 Color
knots knots knots knots parts of the
per plant per plant per plant per plant aerial plants
to V3 to V3 to R4 to R4 in g by to R4
Treatments in mg in mg plant
1 21.5 bc 13.6 39.9 ab 170.6 6.12 3.5
2 32.9 ab 12.0 58.3 a 187.6 5.91 4.0
3 24.4 bc 12.2 50.2 ab 167.5 5.76 4.0
4 28.6abc 10.8 54.6a 187.1 5.75 4.0
5 21.5bc 12.7 39.3ab 162.8 5.56 4.0
6 28.3abc 16.9 55.7a 195.1 5.38 4.0
7 21.6bc 10.3 45.8ab 167.2 5.67 3.9
8 26.1 abc 15.5 49.9 ab 178.3 5.76 4.0
9 18.4 bc 11.9 40.4ab 179.3 6.09 4.0
10 38.4 to 16.9 56.0 to 175.1 5.32 4.0
11 23.1 bc 14.1 37.7 ab 175.8 5.80 4.0
12 16.0 c 11.2 33.1 b 138.5 5.48 4.0
13 17.9 bc 11.8 44.4ab 165.2 5.31 4.0
14 0 0 0.1 0.7 5.56 3.0
test NKS NS S NS NS
CV: 26.4% 23.2% 18.0% 15.6% 13.1%
processing rate quantity yield rate quantity
nitrogen nitrogen net nitrogen nitrogen
parts of parts in q / ha des des
aerial aerial 14% seeds seeds
to R4 to R4 in qx / ha
in g / plant
1 3.18 194.6 29.8 6.56 c 1.95
2 3.24 191.3 29.4 6.87 ab 2.02
3 3.20 184.1 29.7 6.84 ab 2.03
4 3.24 185.6 29.6 6.83 ab 2.02
5 3.14 175.0 28.8 6.85 ab 1.97
6 3.29 176.3 29.0 6.93 to 2.01
7 3.26 184.0 28.6 6.71 abc 1.92
8 3.22 185.6 30.4 6.73 abc 2.05
9 3.15 191.9 29.7 6.66 bc 1.98
10 3.26 173.0 29.3 6.91 to 2.02
11 3.10 179.8 30.5 6.60 c 2.01
12 3.10 169.7 29.1 6.50 c 1.89
13 3.24 171.8 29.5 6.57 c 1.93
14 2.31 127.8 15.5 4.65 0.72
test NK NS NS NS S NS
CV: 3.3% 13.0% 6.6% 1.6% 6.7%
Treatments: 1 - K not enriched 2 - K enriched GLYCEROL 3C 3 - K enriched SODIUM GLUCONATE 3C 4 - K enriched GALACTOSE 3C 5 - K enriched MANNITOL 3C 6 - K enriched GLYCEROL 3C + trifusol 7 - K enriched GLYCEROL 1C 8 - K enriched GLYCEROL 1C, diluted liquid inoculum mixed with enrichment during inoculation 9 - K not enriched, diluted liquid inoculum 10 - K enr GLYCEROL 1C (prior enrichment) diluted liquid inoculum 11 - LIPHA (classic inoculation witness) 12 - SFM not enriched 13 - SFM enriched SODIUM GLUCONATE 3C dry 14 - not inoculated (not taken into account in the analysis of variance)
The microgranulate K is used at 20% humidity and unless otherwise specified, it is inoculated with an inoculum
BIODOZ LIPHA.

Claims (19)

 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 the microgranules comprise at least one substance capable of promoting the growth of germs of the 'inoculum.  2. Method according to claim 1, 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, said microgranules being inoculated at the time of use with a microbial inoculum.  3. Method according to claim 1, characterized in that one adds to microgranules, at the time of use, together or not with a microbial inoculum, at least one substance, for example a nutritive substrate, capable of promoting growth inoculum germs.  4. Method according to any one of claims 1 to 3, characterized in that microgranules of mineral or organic nature or mixtures of such granules are used, obtained by grinding or granulation.  5. Method according to claim 4, characterized in that granules of mineral origin are used, such as industrial clays and clay mixtures.  6. Method according to claim 4, characterized in that microgranules of organic origin are used, for example based on peat, compost, plant residues and the like.  7. Method according to any one of claims 1 to 6, 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.  8. Method according to claim 2, characterized in that the enrichment of the microgranules in advance is 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.  9. Method according to claim 3, characterized in that the enrichment of the microgranules is carried out at the time of use by imbibition, immersion or spraying or else mixing in the dry state with the enrichment substrate, the latter being able to be used alone or mixed with the microbial inoculum.  10. Method according to any one of claims 1 to 9, 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, either by limiting the growth of competitive soil germs.  11. Method according to claim 10, characterized in that carbon substrates such as glucose, glycerol and any other source of carbon suitable for the growth of microorganisms or else carbon substrates specific for the germ to be favored are used.  12. Method according to claim 10, characterized in that other products are used, in particular nitrogen or phosphorus and, in general, any nutrient capable of allowing the growth of the germ to be inoculated.  13. Method according to claim 10, characterized in that as enrichment substance or substrate, biocides are used which are compatible with the germ to be inoculated, but which are capable of inhibiting or slowing the growth of competitors, such as products active in the soil against bacteria, fungi and protozoa, these products can in particular be chosen from antibiotics and fungicides.  14. Method according to any one of claims 1 to 13, characterized in that the inoculation implements one or more germs which can be supplied successively or as a mixture.  15. Method according to any one of claims 1 to 1t, characterized in that inoculums on solid support are used, such as peat, liquid inoculums or inoculums in gel as well as lyophilized inoculums.  16. Method according to claim 3, characterized in that the inoculation is carried out before or after the enrichment or alternatively in mixture with the enrichment products.  17. Method according to any one of claims 1 to 16, 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 antagonistic bacteria and phytopathogenic fungi (P.G.P.R.:: 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 usable microorganisms in remediation and bioremediation of soils.  18. 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.  19. Microgranules according to claim 18, prepared in advance and stored separately, in particular in the dry state.