WO1998030077A1

WO1998030077A1 - Ready-for-use seeds treated with bacteria and method for obtaining them

Info

Publication number: WO1998030077A1
Application number: PCT/FR1998/000041
Authority: WO
Inventors: Bernard Digat
Original assignee: Institut National De La Recherche Agronomique (Inra)
Priority date: 1997-01-10
Filing date: 1998-01-09
Publication date: 1998-07-16
Also published as: FR2758231B1; FR2758231A1; AU5870198A

Abstract

The invention concerns seeds coated with a film-forming polymer, biodegradable, water-retaining and adhering to the seeds, compatible with the contained bacteria. The polymer further comprises at least an agent for rapidly increasing the intracellular osmotic pressure of the bacteria, thus preserving their viability until the seeds are sown.

Description

"Ready-to-use bacteria seeds and process for obtaining them"

The present invention relates to ready-to-use bacteria seeds. It further relates to a process for obtaining such seeds.

Several genera and bacterial species produce beneficial effects on plants and their environment. Thus, the symbiotic bacteria of legumes of the genera Rhizobium and Bradyrhizobium fix nitrogen from the air and, by restoring it to plants, improve their growth. Similarly, certain non-symbiotic bacteria, such as Pseudomonas fluorescens and Ps. Putida, avirulent mutants of Pseudomonas (such as Ps.solanacearum), Agrobacterium antagonists and A. tumefaciens, Bacillus, Azospirillum promote the growth of plants. and protect them against fungal and bacterial parasites directly by mechanisms of antagonism or indirectly by inducing systemic resistance (DIGAT, 1994, CRAcad.Agric. Fr. 80.2: 113-122). Finally, other bacteria such as Bacillus thuringiensis secrete insecticidal toxins allowing a biological fight against insects.

Plants in the broad sense such as seeds, tubers, bulbs, rhizomes and cuttings are favorable targets for bacteria because they represent individualized propagating material that can be effectively colonized by beneficial bacterial populations.

So far, the most commonly used method for bacteria is to immerse the plant material in an aqueous bacterial suspension just before planting. However, seeds bacterized by this process must be sown immediately because the bacterial cells can only survive a few minutes to a few hours in a dehydrated state.

In order to prolong the survival of microorganisms, it has been proposed to constitute inoculants, or bacterial inoculates by fixing the bacteria on supports such as particles of silica and silicates, sand, clay, peat, carbon. porous or salt minerals such as calcium sulphate, phosphate or carbonate (SMITH, 1992, Can.J. Microbiol. 38, 485-492).

The survival of bacteria in and / or on these supports is relatively satisfactory when they are wet (water activity AW> 0.9). However, when these inoculants are mixed with the seeds, it is found that, as before, the majority of the bacterial population does not survive more than a few hours on the surface of the seeds (BROCKWELL and BOTTOMLEY, 1995, Soil Biol. Biochem, 27 , 4-5: 683-697) whose AW is frequently less than 0.5. Consequently, the mixing of these inoculants thus conditioned with the seeds must be carried out just before sowing. This results in major drawbacks and in particular an impossibility of keeping the seeds thus bacteriaized.

In this area of so-called indirect bacterization, an improvement has been made thanks to the use of microgranules based on clay, peat, or alginate possibly enriched with a carbon source (patents FR-2,519,022 and FR-2,695,929). These means make it possible to increase bacterial survival somewhat, but the microgranules must still be inoculated by the farmer at the time of their use in the field, that is to say just before sowing, and be placed close to the seeds. in the seed line.

Furthermore, in order to improve the adhesion of bacteria to the seed and their resistance to dehydration, it has been proposed to mix various adhesives and film-forming agents, such as artificial polymers (polyvinyl, polyacrylamide) or natural polymers such as polysaccharides ( cellulose and its derivatives, alginates and its derivatives, xanthan) with bacteria. Most of these film-forming agents having a fairly high water retention capacity, this property has been used to improve the survival of bacteria. Attempts have also been made to improve the tolerance of certain bacteria to dehydration by cultivating them in media containing mannitol, sorbitol, fructose, dextrin or starch (European patent O.O83.267).

However, these last two solutions do not lead to a sufficient increase in the survival of the bacteria. The beneficial effect on bacteria of various osmoregulatory molecules has also been described, in general, for example in the article by CSONKA (1989, Microbiol. Rev., 53, 121-147). The use of non-crosslinked polysaccharides to stabilize microorganisms for the inoculation of seeds is described in US Pat. No. 5,292,507. This patent mentions that the polysaccharide solutions can contain osmoregulatory agents, such as buffers. Such agents are not such as to enable them to perform an osmoprotection function. PCT application WO 92/08 355 also relates to inoculated seeds. The bacteria are mixed with an inert clay in powder form, and the mixture is allowed to dry. According to a first embodiment, an osmoprotective is added when the bacteria are mixed with the clay. Slow drying is then necessary. According to another embodiment, the osmoprotective is produced by bacteria, which requires an even slower drying. This slowness in drying results in an imbibition of the seed, which reduces its germination power.

It emerges from the analysis of the state of the art that no industrially applicable means were known making it possible to obtain seeds having on the surface a film of bacteria and which can be stored during the periods between their manufacture and the sowing, without losing their beneficial properties. The applicant has endeavored to solve this problem and has shown that seeds coated with a polymer containing a certain type of agent increasing the intracellular osmotic pressure of bacteria, keep viable bacteria until sown.

The subject of the present invention is therefore ready-to-use bactericidal seeds, coated with a biodegradable film-forming polymer, hydroretentant and adhering to the seed, said polymer comprising the bacteria and in addition at least one osmoprotective agent or increasing the intracellular osmotic pressure of the bacteria.

In the context of the present invention, the term "film coating" should be understood in its usual sense of the art seeds, that is to say as signifying a thin layer, of thickness generally between approximately 10 and 100 μm preferably between 20 and 50 μm, deposited on the seed. In a “film-coated” seed, the external shape is little modified compared to the original seed, which is not the case of so-called “coated” seeds, in which the coating layer containing the seed has a thickness greater than 1000 μm and changes the external shape of the seed, which then becomes approximately spherical. Preferably, the agent increasing the intracellular osmotic pressure of the bacteria is an amino acid, such as L-glutamic acid, L-proline, betaine, L-serine, or one of their salts or l 'one of their derivatives. It is preferably L-glutamic acid. This agent can be used alone or in admixture with other aforementioned agents having the same function.

The bacteria likely to be included in the film coating are advantageously bacteria producing beneficial effects on the seedlings, or on their environment, and in particular the symbiotic or non-symbiotic bacteria mentioned above, such as bacteria of the genera Rhizobium, Bradyrhizobium, Pseudomonas, Agrobacterium, Bacillus and Azospirillum. These bacteria can have various effects on plants and their environment. They can fix nitrogen in the air, protect plants from fungal and bacterial parasites, or even secrete insecticidal toxins.

The polymer forming the film is advantageously a biodegradable film-forming polymer. It can in particular be a chitosan, an alginate, a cellulose or one of their derivatives. It is chosen according to the type of exopolysaccharide (EPS) of the bacteria that one wishes to use.

Thus, for bacteria such as Pseudomonas and Bacillus, a cationic biopolymer such as chitosan or better chitosan-glutamate sterilized by gamma rays is chosen. The latter polymer alone forms a microcapsule visible under the electron microscope around the bacterial cells and quickly gives bacteria the ability to better resist dehydration. It contains excess glutamate ions in the form of salts (sodium, potassium, magnesium) which accumulate very quickly in bacterial cells. For other bacteria, such as Rhizobium and

Bradyrhizobium, an anionic biopolymer is preferably chosen, such as a soluble alginate salt to which is added L-glutamic acid or one of its salts or another amino acid (L-proline, betaine, L- serine) or one of its salts. For Pseudomonas, Bacillus, Rhizobium and

Bradyrhizobium, it is also possible to choose a cellulose or one of its derivatives to which is added L-glutamic acid or one of its salts or another amino acid (L-proline, betaine, L-serine) or one of the salts of these acids. Those skilled in the art can, from a simple test, determine which polymer should preferably be used for a given type of bacteria.

Such a test may consist firstly in mixing the polymer and the bacteria and then in observing the evolution of the solution. If the bacteria aggregate by flocculating, the polymer is considered not to be compatible. On the contrary, if no flocculation is observed, it is considered that there may be compatibility. However, to precisely check the compatibility of the polymer with bacteria, it is necessary to carry out tests according to standardized methods well known to those skilled in the art in order to determine the minimum inhibitory concentration (MIC) or the minimum bactericidal concentration (CMB) of the polymer.

Advantageously, the coating formed around the seed constitutes a film or coating having a thickness generally between approximately 10 and 100 μm, preferably between 20 and 50 μm.

In the coated seeds according to the invention, the concentrations of agent increasing the intracellular pressure of the bacteria, in particular L-glutamic acid, can vary between wide limits within the coating polymer. Some indications Illustrative are given in the following examples. For routine prior testing, those skilled in the art can optimize the concentration per seed, knowing the approximate number of seeds per kg for a given species and the volume of formulation required to film a given weight of seed.

Within the coating formulation, the agents increasing the intracellular pressure of the bacteria are advantageously at a maximum concentration. Preferably, L-glutamic acid is present in the film-coating polymer at a concentration greater than about 8 g / l.

The proline is advantageously included at a concentration greater than approximately 150 g / l, the betaine at a concentration greater than approximately 1600 g / l and the serine at a concentration greater than approximately 50 g / l. In the coated seeds according to the invention, the amounts of bacteria per seed vary according to the nature of the bacteria and the surface and the volume of the seed. Routine tests are within the reach of those skilled in the art to determine the optimal number of bacteria actually present in the film surrounding the seed.

The present invention has the advantage of combining:

- on the one hand the formation of a microcapsule around the bacterial cells slowing down the exchange of water and gas with the external environment, and - on the other hand the triggering of a rapid rise in the intracellular osmotic pressure in the cells bacterial.

The bacteria seeds can be obtained by a manufacturing process comprising the following steps:

- mixing of the polymer and the osmoprotective agent, then mixing with the bacteria,

- application to seeds of the formulation thus obtained, and by lamination and

- seed drying. Advantageously, the application of the formulation and the drying are carried out in a fluidized bed processor or in a film-coating turbine.

According to a particular mode of implementation, the bacterial strains are kept in the lyophilized state and must be reactivated in a rich nutritive medium before inoculating the definitive culture medium suitable for the bacterial species. In a large capacity fermenter, the culture medium is continuously stirred to optimize oxygenation. The temperature and pH conditions are determined for each bacterial species. The bacterial inoculum is formed when the end of the stationary phase is reached: its final concentration must be greater than 10 ¹⁰ cfu / ml.

The sterilized biopolymer is dissolved in sterile water at its specific concentration in the presence of the agent or agents increasing the osmotic pressure, for example glutamic acid at the minimum concentration of 8 g / l. It is necessary to adjust the pH from around 6.2 to 6.5 (depending on the polymer used) preferably using KOH 10N, avoiding any precipitation of the polymer. Finally, the concentrated bacterial suspension containing the osmoprotective agent (s) at the same concentration as in the polymer is gently dissolved in the polymer in a volume ratio close to 1: 1. It is left to stand for at least 1 hour at laboratory temperature.

For the seed coating, an industrial turbine, or better a fluidized bed processor, or any other device suitable for this operation is used. During these operations it is necessary to take care of the adjustment of the temperature, which must not be higher than 30 ° C, to avoid any clogging of filter or sticking of seeds between them. Drying should be as quick as possible.

The present invention is illustrated without however being limited by the examples which follow, with reference to the appended drawings in which:

FIG. 1 represents the survival of Pseudomonas fluorescens G92 bacteria on sunflower seeds stored at 12 ° C. as a function of the number of days, the seeds having been film-coated in a fluidized bed. FIG. 2 represents the survival of Pseudomonas putida TL5 bacteria on sunflower seeds stored at 12 ° C., the seeds having been film-coated in a fluidized bed and in a turbine respectively. FIGS. 3A, 3B and 3C show the quantities of glutamate, determined by enzymatic method, under different conditions, FIG. 3A representing the evolution of the average quantity of L-glutamic acid, in or on 10 ¹⁰ bacterial cells Pseudomonas putida TL5, as a function of the time of incubation in the osmoprotector (1, 30, and 60 minutes), FIG. 3B representing the average percentage of L-glutamic acid and its salts (glutamates) in the formulation at pH 6.08 at two concentrations of chitosan (3 and 6%), FIG. 3C representing the amount of L-glutamic acid and its salts (glutamates) present on film-coated sunflower seeds, after four months of storage, respectively with and without Pseudomonas fluorescens G 92 bacteria, FIG. 4 illustrates the survival of Bradyrhizobium japonicum G49 on soybeans stored at 12 ° C. and coated with a sodium alginate polymer (Manucol) and a derivative respectively. é cellulosique (Sepiret).

Figures 5A and 5B illustrate the survival of bacteria

Pseudomonas putida TL5, in the presence and absence of L-glutamic acid, respectively in sodium alginate (Manucol) and in a cellulosic polymer (Sepiret). The number of bacteria is expressed in cfu / ml.

EXAMPLE 1 Bacterization of seeds by Pseudomonas

In order to constitute the inoculum, the bacteria are grown in LPG medium (yeast extract 5 g / l, peptone 5 g / l, glucose 10 g / l) or in King's medium B (KING, WARD and RANEY, 1954, J. Lab. Clin. Med., 44: 301-307) or in the midst of MISAGHI (MISAGHI and GROGAN, 1969, Phytopathology 59: 1436-1450) at 26-27 ° C for 60 to 72 hours with continuous stirring and pH regulated at 6.5-6.8. When the end of the stationary phase has been reached, the bacterial suspension is concentrated so as to obtain a useful concentration of approximately 10 ¹¹ cfu / ml.

A solution of sterilized chitosan-glutamate (purity> 95%) is prepared in sterile water at a concentration of 6%. The pH of the biopolymer solution is adjusted to 6.2 using 10N KOH. The concentrated bacterial aqueous suspension is gently mixed with the biopolymer in a 1: 1 volume ratio. It is left to stand for at least 1 hour at laboratory temperature.

The water activity (AW) or the equilibrium relative humidity (HRE) of a sample of seeds to be bacteria are measured, the optimum of AW at the end of the filming operation being around 0 , 50. The seeds are placed in an industrial turbine of the DUMOULIN type or in a fluidized bed processor equipped with a WURSTER tank. The following parameters are set:

^* air temperature at the inlet: 30 ° C,

^* outlet air temperature: 20 ° C, ^* spraying pressure: 0.5 bar (fluidized bed) or 2 bars

(turbine),

^* diameter of the spray nozzles: 1.0 to 1.2 mm,

^* ventilation pressure: -115 Pa (fluidized bed) or -200 Pa (turbine), ^* average pump flow: 10 ml / minute.

The volume of formulation required varies depending on the type of seed. Thus 200 to 300 liters of formulation are necessary to film 1 ton of sunflower seeds.

When the filming is finished, one counts on the medium B of King agar of the surviving population on the seed. This survival of the bacterial population is monitored until final marketing so as to guarantee the user a quantity of surviving bacteria estimated sufficient to produce the desired biological effect. Germination tests are carried out according to the standards of the ISTA (International Seed Testing Association). According to this example, at 12 ° C., the survival of Pseudomonas fluorescens G92 on the surface of sunflower seeds, film-coated using the fluidized bed technology according to the process described above, is followed for more than 550 days (FIG. 1). We observe that at this date the population loss was only 1.5 log cfu.

On the other hand, the coating of sunflower seeds using the same formulation containing Pseudomonas putida TL5 at 3 x 10 ¹⁰ cfu / ml is carried out simultaneously in a fluidized bed processor (brand GLATT) and in a turbine (brand FROM THE MILL). The survival of the bacterial population fixed on the seeds is followed for 70 days (Figure 2). We observe that at this date the loss of population was only slightly greater than 1 log cfu.

The amount of L-glutamic acid and glutamates was further measured under various conditions and at different times as described by Bernt and Bergmeyer (1965, Bergmeyer, Methods of Enzymatic Analysis, 384-388, 2nd printing). FIG. 3A illustrates the evolution of the amount of L-glutamic acid absorbed or adsorbed by 10 ¹⁰ bacterial cells as a function of the incubation time in the solution. FIG. 3B represents the variation in the quantity of glutamic acid and of glutamates in the formulation as a function of the concentration of chitosan. FIG. 3C is a comparison of the evolution of the amounts of glutamate respectively on film-coated seeds without and with bacteria after 4 months of storage.

EXAMPLE 2

Bacterization of legume seeds by Rhizobium and Bradyrhizobium

In order to constitute the inoculum, the bacteria are cultivated: for Rhizobium (example; Rhizobium meliloti strain 2011) in the YEM medium (VINCENT, 1970, A manual for practical study of root-nodule bacteria, IBP Handbook n ° 15, Blackwell Scientific Publications, Oxford, 169 pp.) For 6 days at 28 ° C in continuous stirring, pH regulated at 7 and for Bradyrhizobium (example: B japonicum strain G49, IARI SB16, New Delhi) in the medium of Burton (BURTON, 1976, Symbiotic nitrogen fixatio, ed. PS Nutman, Cambridge University Press, 175-189) for 8 days at 30 ° C in continuous stirring, pH regulated at 6.5.

The end of the stationary phases being reached, the bacterial suspensions are concentrated so as to obtain a useful concentration of approximately 10 ¹¹ cfu / ml.

For the formulation, either sterile sodium alginate (Manucol marketed by Kelco International) at a concentration of 1.0% is used, or a cellulose derivative (Sepiret O7G marketed by Seppic) at 8% in which l sterilized L-glutamic acid (Sigma, purity 99-100%) at the final concentration of 0.86%. The pH of the solution is adjusted to 6.4 using 10N KOH. The concentrated bacterial aqueous suspension containing glutamic acid 0.86% is gently mixed with this polymer solution in a 1: 1 ratio by volume. It is left to stand for at least 1 hour at laboratory temperature.

The same steps are carried out as in Example 1 for the coating.

The volume of formulation required varies depending on the type of seed. For example, to film 1 ton of soybean seed, approximately 1000 liters of formulation is required.

When the filming is finished, we proceed to count, respectively on YEM medium or BURTON agar medium, the surviving population on the semen and we carry out nodulation tests (under controlled conditions) by noting the number of nodules appeared on the plants. Germination tests are carried out according to the standards of the ISTA (International Seed Testing Association).

According to this example, at 12 ° C., the survival of Bradyrhizobium japonicum G49 on the surface of soybeans film-coated using a fluidized bed processor according to the method described above was followed for 70 days (Figure 4). We observe that date loss dice ^¬ population is approximately 4 log in the two polymers (Manucol and Sepiret) used for the formulation. Each soybean seed must carry 10 ⁶ bacteria to induce sufficient nodulation, it is estimated that this quantity is available until the 70th day. EXAMPLE 3:

Influence of the presence of glutamic acid in the coating on the survival of bacteria.

Comparative tests were carried out on sterile inert supports represented by “Teflon” pellets with a diameter of 10 mm in order to determine the influence of L-glutamic acid on the survival of the bacteria. Ten microliters of a mixture of alginate (Manucol) and of Pseudomonas putida TL5 bacteria at 10 ⁴ C cfu / ml, with or without L-glutamic acid (8g / l) were deposited on each tablet. The tablets were placed in boxes of Nunclon delta sterilized with gamma rays at the rate of 12 tablets per treatment (3 repetitions of 4). The inoculated pellets were subjected to 2 h and 4 h of dehydration by ventilation of dry air at 25 ° C. Then the number of bacteria (cfu / ml) was determined by the conventional method of dilutions on King's medium B.

The results are shown in Figure 5A.

They clearly show that the addition of L-glutamic acid to the alginate allows the survival of bacteria after 4 h of dehydration, while in the absence of L-glutamic acid the bacteria do not survive.

Comparable experiments were carried out with the same bacterial strain at 10 ⁶ cfu / ml, in a cellulose polymer (Sepiret 07 G). The survival of the bacteria was estimated after 1 h and 2 h of dehydration under the same conditions as for FIG. 5A.

The conclusion is similar for this polymer, the L-glutamic acid exerting a very marked beneficial effect on the survival of the bacteria, as shown in FIG. 5B.

Claims

1. Ready-to-use bacterized semen coated with a biodegradable film-forming polymer, hydroretentant and adhering to the semen, comprising the bacteria, said polymer further comprising at least one osmoprotective agent or increasing the intracellular osmotic pressure of the bacteria.

2. Seed according to claim 1 characterized in that said agent is an amino acid.

3. Seed according to one of claims 1 and 2, characterized in that said agent is L-glutamic acid, L-proline, betaine, L-serine or one of their salts or one of their derivatives, or a mixture of said acids, salts or derivatives.

4. Seed according to one of claims 1 to 3 characterized in that said agent is L-glutamic acid, one of its salts or one of its derivatives, and in that it is included in a concentration higher than about 8 g / l.

5. Seed according to one of claims 1 to 4, characterized in that the bacteria are of the genus Rhizobium, Bradyrhizobium,

Pseudomonas, Agrobacterium, Bacillus or Azospirillum.

6. Seed according to one of claims 1 to 5 characterized in that said polymer is a chitosan, an alginate, a cellulose, or one of their derivatives.

7. Seed according to one of claims 1 to 6 characterized in that the film surrounding the seed has a thickness of between approximately 10 and 100 μm, preferably between approximately 20 and 50 μm.

8. A process for the manufacture of bacteria seeds according to one of claims 1 to 7 comprising the following steps: - mixing of the polymer and the osmoprotective agent, then mixing with the bacteria,

- application to the seeds by coating the formulation thus obtained,

- seed drying.

9. Method according to claim 8 characterized in that the application and drying are carried out in a fluidized bed processor or in an industrial turbine.