Biofertilizing bacterial strain

Abstract

The present invention relates to the field of crop fertilization, and particularly to a biofertilizing bacterial strain. In particular, the present invention relates to the bacterial strain deposited on Oct. 24, 2018, at the Collection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCM I-5372, and to the uses of this strain. The invention also relates to a composition comprising the above-mentioned bacterial strain and to a fertilization process comprising the application of this composition to a plant or to a soil.

Claims

1. A fertilization process comprising a step in which the bacterial strain deposited on Oct. 24, 2018 at the Collection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCM I-5372, or a composition comprising said bacterial strain, is applied to a plant or to a soil.

2. The fertilization process according to claim 1, wherein said plant is selected from field crop plants, vegetable crop plants and plants grown in viticulture.

3. The fertilization process according to claim 1, wherein the application is made at sowing time, by application to the soil or by coating the seeds.

4. The fertilization process according to claim 1, wherein the application is made by inoculation of growing substrates intended for soilless cultivation.

5. The fertilization process according to claim 1, wherein said composition is in powder, granule, cream or liquid form.

6. The fertilization process according to claim 1, wherein said composition is in liquid form.

7. The fertilization process according to claim 1, wherein said composition comprises the bacterial strain as a suspension in a solution containing water and/or mineral oils and/or organic oils.

8. A method for biofertilizing a soil, comprising a step of applying the bacterial strain deposited on Oct. 24, 2018, at the Collection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCM I-5372 to a soil.

9. A method for stimulating a plant growth, comprising a step of applying the bacterial strain deposited on Oct. 24, 2018, at the Collection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCM I-5372 to a plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 represents the protease activity of the soil after inoculation with the strain in accordance with the invention;

(2) FIG. 2 represents the aminopeptidase activity of the soil after inoculation with the strain in accordance with the invention:

(3) FIG. 3 represents the nitrate content of the soil after inoculation with the strain in accordance with the invention;

EXAMPLES

Example 1: Analysis of the Biofertilizer Properties of the Strain of Interest on Soil Microcosm (Controlled Conditions)

(4) Materials and methods: A model soil was taken from an agricultural plot at the ENSAIA experimental farm. The soil (silty clay; OM 4.5%; total N 0.3%; C/N 13.8; pH.sub.water 6.5) was sieved to 5 mm and then stored at room temperature until use. Soil microcosms were made by placing 70 g of soil at 60% of its water holding capacity in 0.5 L Le Parfait jars. The microcosms were pre-incubated for 2 weeks at 20 C. and then inoculated with 5 mL of 7*10.sup.9 CFU bacterial suspension (i.e., 10.sup.8 CFU/g fresh soil) of the selected bacterial strain. Control microcosms were treated with 5 mL of sterile saline. The soil microcosms, at 80% water holding capacity, were then incubated at 20 C. in the dark. Soil samples were taken at 0), 3, 7, 14 and 28 days after inoculation. Four replicates per treatment and sampling time were performed. At each sampling time, a set of variables were measured within 48 h after sampling, namely (i) soil microbial enzymatic activities in relation to N, S and P dynamics, (ii) measurements of water-soluble C and N contents, (iii) microbial C and N biomass, (iv) mineral N, S and P contents and (v) soil amino acid contents. Soil samples were immediately frozen at 20 C. for DNA extractions and 16S rDNA gene abundance measurements.

(5) Potential arylsulfatase activity in the soil was measured according to the protocol of Tabatabai and Bremner (1970). Protease potential activity was measured according to the protocol of Ladd and Butler (1972). Potential phosphatase activity was measured according to the protocol of Dick et al. (1996). Finally, potential leucine-aminopeptidase activity was measured according to the protocol of Spungin and Blumberg (1989).

(6) To estimate the soluble organic N and mineral N contents of the soils, 10 g of fresh soil was extracted with 50 mL of 1 M KCL for 45 min using a revolution mixer. Extracts were filtered through Whatman No. 42 filter (Harper, 1924) and stored at 20 C. until analysis. The mineral N contents (NO.sub.3.sup. and NH.sub.4.sup.+) of the extracts were determined using a SAN++ CFA molecular absorption spectrophotometer (Skalar Analytical, Breda, The Netherlands) by the INRA, Nancy, analysis unit. Amino acids were quantified according to the protocol of Darrouzet-Nardi et al. (2013). Soluble C and N were extracted with hot water as described by Vong et al. (2007). The C and N content of the hot water extracts was determined with a TOC-V CHS (Shimadzu, Kyoto, Japan).

(7) SO.sub.4.sup.2 were extracted from the soil by revolution mixing of 10 g of soil in 50 mL of 16 mM KH.sub.2PO.sub.4 for 45 min (Walker and Doornenbal, 1972) and then filtered a first time on Whatman No. 42 paper and a second time on a 0.45 m pore size filter. SO.sub.4.sup.2 from the KH.sub.2PO.sub.4 extracts were subsequently determined by ion chromatography (Dionex).

(8) PO.sub.4.sup.2 were extracted by revolution mixing of 5 g of soil in 50 mL of 0.5 M NaHCO.sub.3 for 45 min and then filtered through Whatman No. 42 paper following the protocol of Hedley et al. (1982). PO.sub.4.sup.2 from the NaHCO.sub.3 extracts were subsequently determined according to the protocol of Irving and Mclaughlin (1990).

(9) Microbial C and N biomasses were measured by the fumigation-extraction method (Vance et al., 1987). C and N concentrations of fumigated and unfumigated 0.5 M K.sub.2SO.sub.4 extracts filtered on Whatman No. 42 were determined with a TOC-V CHS (Shimadzu, Kyoto, Japan). Microbial biomasses were determined by difference between fumigated and unfumigated extracts and division by an extraction coefficient of 0.45 for microbial C biomass (Vance et al., 1987) and 0.54 for microbial N biomass (Brookes et al., 1985).

(10) Results: Under controlled conditions, the Pseudomonas-affiliated strain (P strain) stimulates certain soil microbial activities involved in the mineralization of organic matter, without changing the size of the soil microbial biomass. Thus, microbial activities related to nitrogen decomposition are significantly higher in inoculated soils compared with uninoculated soils. Over 28 days of incubation, protease activity, which is involved in the breakdown of protein, the main form of organic N in soils, was on average 40% higher in soils inoculated with the P strain compared with uninoculated soils (FIG. 1). Leucine aminopeptidase activity, which degrades peptides released by proteases into amino acids, was increased by 22% to 32% in soils inoculated with the P strain from 14 days after inoculation (FIG. 2). Arylsulfatase activities that mineralize sulfate esters, a major and among the most labile form of organic S in soils, are also stimulated in inoculated soils, weakly 3 days after inoculation (+15% activity in soils inoculated with the P strain) and strongly 14 days after inoculation (+189% activity in soils inoculated with the P strain). Finally, phosphatase activities were only weakly and transiently stimulated 28 days after inoculation of the soils with the P strain (+8% compared with uninoculated soil).

(11) The P strain improves the availability of mineral N in the form of soil nitrate, which is the main source of N for plant nutrition. Nitrate levels in soils inoculated with the P strain were increased by 15 to 20% from 7 to 28 days after inoculation, i.e., an average increase of +11.5 mg nitrate/kg soil (FIG. 3). This represents a potential of about 45 N units/ha.

Example 2: Analysis of the Phytostimulant Properties of the Strain of Interest (Controlled Conditions)

(12) Materials and methods: A first experiment under gnotobiotic conditions was set up to study the effect of the bacterial P strain on the growth and root architecture of maize seedlings (Pioneer Hi Bred). The seeds used were surface sterilized. To this end, the seeds were successively placed in the presence of 70% ethanol for 5 min and 5% sodium hypochlorite for 10 min. The seeds were then rinsed 5 times with sterile distilled water. 100 L of the last rinse water was spread on NB agar medium to verify the efficiency of the surface sterilization.

(13) Roughly 25 sterilized seeds were then germinated in 13.5 cm diameter Petri dishes on sterile Whatman No. 3 filter paper moistened with 10 mL sterile distilled water. The Petri dishes were placed in the dark for 3 days at 28 C. Four pre-germinated seeds were then selected and arranged in an equatorial line in a 13.5 cm diameter Petri dish. Each seed was inoculated with 100 L of bacterial suspension (10.sup.9 bacteria/seed) of the selected strain. The bacterial inoculum was prepared from a culture at 10.sup.9 CFU/mL. One mL of culture was centrifuged at 10 000 rpm for 10 min and then the pellet was taken up in 100 L of sterile saline. Control pre-germinated seeds received 100 L of sterile saline. In total, 4 replicates were performed per treatment. The Petri dishes were placed in a climatic chamber with a photoperiod of 16 h, a temperature of 23 C. day and 18 C. night for 6 days. After incubation, root parts were collected. A set of root traits were analyzed. To this end, the root system was spread in a thin layer of water, in a Plexiglas tank (1.5*20)*30 cm), using fine tweezers. The root system was then scanned (Expression 1640XL scanner, Epson) and the images obtained were analyzed with WinRhizo software (Rgent Instruments Inc., Quebec, Canada). The total length (cm), the length of fine (diameter <2 mm) and coarse (diameter >2 mm) roots, the total surface area (cm.sup.2), the surface area of fine and coarse roots and the average diameter (mm) of the roots were estimated. After analysis, the roots were gently dried on filter paper and weighed to estimate the fresh mass. They were then placed in an oven at 80 C. for 48 h to determine the dry mass.

(14) A second experiment aimed at evaluating the effect of the P strain on the growth, physiology and nutritional status of maize during the early stages of development, as well as on the biological functioning and the content of mineral elements in the maize rhizosphere. The soil (silty clay; OM 1.6%; total N 0.12%; C/N 7.8; pH.sub.water 7.5) was collected from an agricultural plot (Saint Martin des Champs, 77), air-dried and sieved to 5 mm. Before use, the soil was mixed with 10% (m/m) aquarium sand to facilitate subsequent root collection.

(15) The inoculation of maize with the bacterial strains of interest was performed at sowing time, on sterilized and germinated seeds with 1 mL of an inoculum at a concentration of 10.sup.8 CFU/mL. Sterilized, germinated but uninoculated seeds were used as a control. One seed was placed at a depth of about 2 cm in a PVC tube (5*20 cm) containing 330 g of soil-sand mixture. This substrate was maintained at 80% of its water holding capacity by weighing the tubes 5 times a week and watering, if necessary. At the 1-2 leaf, 3-4 leaf, 5 leaf and 5-6 leaf stages, 4 tubes of each treatment modality were randomly selected and opened with a circular saw. After opening the tubes, the aerial parts were separated from the soil-root system and collected. The roots were separated from the soil, collected with tweezers and then washed with tap water. The aerial and root parts of the plants and the soil were stored at 4 C. until all variables were analyzed. An aliquot of soil was frozen at 20 C. for subsequent molecular analyses.

(16) At the plant level, fresh mass and dry mass of aerial and root parts were measured. Root architecture was also analyzed as previously described (experiment under gnotobiotic conditions). At the level of maize rhizosphere soil, the variables of microbial abundance, microbial enzymatic activities and mineral element content were measured as described in the context of the soil microcosm experiment.

(17) Results: Under gnotobiotic conditions (first experiment), the results obtained show a significant effect of the inoculation of the Pseudomonas-affiliated P strain on different root variables. Thus, 6 days after inoculation, the fresh root biomass of inoculated seedlings tended to be 1.5 times higher than that of control plants. These effects on root biomass resulted mainly from a significant increase in the length and surface area of fine roots (+64% and +58%, respectively), which are the roots considered to be mainly involved in plant nutrient uptake (Eissenstat, 1992).

(18) In the second experiment, we analyzed the effect of the P strain on the growth and nutritional status of maize grown under controlled conditions up to the 6-leaf stage as well as the biological functioning and availability of mineral elements in the rhizosphere of this maize.

(19) At the plant level, while the strain does not influence the biomass of the aerial parts of the maize or their height, it does alter root growth. Thus, the fresh root biomass of plants inoculated with the P strain tended to be higher than that of non-inoculated plants at the 1-2 leaf stage. Changes in root architecture of plants inoculated with the Pseudomonas-affiliated strain (P strain) are observed at the 1-2 leaf stage. Thus, the surface area (+15%, p=0.03) of fine roots (diameter <2 mm) was greater for inoculated plants compared with the control. These results tend to confirm the results obtained under gnotobiotic conditions.

(20) Concerning the effects of P strain inoculation on rhizosphere soil functioning, measurements of microbial abundance, microbial enzymatic activities involved in N, S and P dynamics and mineral element contents were performed. The main effects of P strain inoculation on soil enzyme activities related to N, S and P cycles were observed at the maize 3-4 leaf stage. Thus, the protease activity in the maize plant rhizosphere inoculated with Pseudomonas-affiliated P bacteria was on average 92% higher (p=0.02) than in the control soil. In these same soils, leucine aminopeptidase activities were also significantly higher than in control soils (+57%). Arylsulfatase activity involved in S mineralization was significantly increased in the rhizosphere of plants inoculated with the P strain (+101%) compared with the control at the 3-4 leaf stage. At the 5-6 leaf stage, this activity remained higher in inoculated soils (+21%, for the P strain).

Example 3: Use of Molecular Markers to Discriminate the P Strain

(21) The possibility of discriminating bacterial strains with RAPD-type markers was validated by comparing the profile of the P strain with other strains, in particular from the genus Pseudomonas (which is highly abundant in natural environments).

(22) The strains tested are listed in the table below:

(23) TABLE-US-00001 TABLE 1 List of strains tested Strain code Strain name Achromo Achromobacter sp. Ps.chloro Pseudomonas chlororaphis Ps.sp Pseudomonas sp. Ps.putida Pseudomonas putida Enterob Enterobacter ludwigii Ps-1 Pseudomonas moraviensis (P strain) Ps-2 Pseudomonas moraviensis (P strain) Micro-1 Microbacterium resistens Micro-2 Microbacterium resistens

(24) Two random amplified polymorphic DNA (RAPD) markers were tested: M13 5-GAGGGTGGCGGTTCT-3 (SEQ ID NO: 1) and RAPD2 5-AGCAGCGTGG-3 (SEQ ID NO: 2). Amplification reactions were performed in a final volume of 12.5 L in the presence of 15 ng DNA and 25 M primers. Amplicons were separated on a 2% agarose gel to reveal the profiles.

(25) Both markers are discriminating even against bacteria of the same genus (i.e., Pseudomonas). A better repeatability is observed for the M13 marker.

Example 4: Production of the P Strain

(26) The scale-up was performed in an Applikon bioreactor in a final volume of 4 L. The medium used was composed of 10 g/L whole milk powder and 5 g/L yeast extract. The tests will make it possible to determine the kLa (oxygen transfer coefficient), a parameter for scaling up (50 and then 500 L bioreactors). The optimal temperature is 30 C. (see table below).

(27) TABLE-US-00002 TABLE 2 Conditions for producing the P strain Parameter Aeration Stirring pH Temperature P. moraviensis 2 vvm 150 rpm 7.3-7.5 30 C. P strain

(28) Tests conducted in 5 L fermenters produced microbial biomass of the P strain at an average of 3.93.sup.E+09 CFU/mL.

Example 5: Drying of the P Strain

(29) The P strain shows very good survival to the freeze-drying process for the conditions with cryoprotectant, even more marked for the condition with sucrose. The freeze-drying schedules are presented in Table 3. In addition, freezing the samples for freeze-drying had no impact on the viability of the bacteria. The aw of the samples is at most about 0.150 and ensures good microbiological stability (viability and shelf life). The total duration of freeze-drying is 47 hours.

(30) TABLE-US-00003 TABLE 3 Freeze-drying schedules Final temperature Step of the shelves Ramp Pressure duration Step ( C.) (min) (bar) (min) Freezing 45 Before Atmo 120 loading Primary 30 60 50 990 Drying 10 150 50 1350 10 120 50 300 Secondary 20 90 50 60 Drying

(31) With cryoprotectant, the loss of viability is negligible. Thus, a positive effect of cryoprotectant (5% sucrose, Table) is observed.

(32) TABLE-US-00004 TABLE 4 Strain viability Viability Viability before Viability (CFU/mL) Viability freeze- after Viability Loss of before before drying freeze- after freeze- viability freeze- freeze-drying (CFU/g drying drying (log Condition drying (logCFU/mL) DM) (CFU/mL) (logCFU/mL) reduction) The P strain 3.57E+10 10.55 6.93E+10 1.24E+09 9.09 1.46 without cryoprotectant The P strain 4.47E+10 10.65 8.04E+11 2.27E+10 10.36 0.29 with 5% sucrose