Novel species of Leucobacter and its applications

Abstract

The present invention discloses a new species of Leucobacter chunongensis and its applications. The present invention provides Leucobacter chunongensis CN001, which has the registration number of CGMCC No. 25924 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The strain CN001 of the present invention can produce IAA, and it is a new species of the genus Leucobacter with the potential to promote plant growth, and has broad application prospects in aspects such as promoting plant growth and enhancing the stress resistance characteristics of crops.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to the novel species of Leucobacter initium and its applications. Background Art

[0002] The genus Leucobacter is a major branch of the family Microbacteriaceae within the phylum Actinobacteria. In 1996, Takeuchi et al. proposed the establishment of the genus Leucobacter (Takeuchi, M., Weiss, N., Schumann, P., and Yokota, A. 1996. Leucobacter komagatae gen. nov., sp. nov., a new aerobic Gram-positive, nonsporulating rod with 2,4-diaminobutyric acid in the cell wall. Int. J. Syst. Bacteriol. 46, 967–971.). Currently, there are 33 validly described species in this genus (https: / / lpsn.dsmz.de / genus / leucobacter) (Parte et al., 2020). The common taxonomic characteristics of the strains in this genus are: Gram-positive bacilli, aerobic, non-motile cells, non-spore-forming, the main respiratory quinones contain MK-10 and MK-11, the characteristic amino acid in the cell wall is LL-DAP, and the dominant fatty acids include iso-C16:0 and anteiso-C15:0. The genomic GC content is 59.9–72 mol% (Chun, B.H., Lee, H.J., Jeong, S.E., Schumann, P., and Jeon, C.O. 2017. Leucobacter ruminantium sp. nov., isolated from the bovine rumen. Int. J. Syst. Evol. Microbiol. 67, 2634–2639. Benga, L., C., Schumann, P., Verbarg, S., Bunk, B., Engelhardt, E., Benten, W.P.M., and Sager, M. 2019. Leucobacter muris sp. nov., isolated from the nose of a laboratory mouse. Int. J. Syst. Evol. Microbiol. 69, 2095–2100.). Leucobacter is a widespread bacterium in the biosphere, and different species of Leucobacter have been found in the following habitats respectively: different types of crop cultivated land soil (Sturm G, Jacobs J, Sproer C, Schumann P, Gescher J. Leucobacter chromiiresistens sp. nov., an achromate-resistant strain. Int J Syst Evol Microbiol 2011;61:956-960; Hyun DW, Sung H, Kim PS, Yun JH, Bae JW. Leucobacter coleopterorum sp. nov., Leucobacter insecticola sp. nov., and Leucobacter viscericola sp. nov., isolated from the intestine of the diving beetles, Cybister brevis and Cybister lewisianus, and emended description of the genus Leucobacter. J Microbiol 2021;59:360-368; Zhu J, Che J, Jiang X, Ma M, Guan D, Li L, Cao F, Zhao B, Kang Y, Zhao J, et al. Leucobacter chinensis sp. nov., with plant growth-promoting potential isolated from field soil after seven-years continuous maize cropping. Int J Syst Evol Microbiol 2022;72:5417.), wastewater (Schumann and Pukall, 2017; Sun et al., 2018), seafood (Shin NR, Kim MS, Jung MJ, Roh SW, Nam YD, Park EJ, Bae JW. Leucobacter celer sp. nov., isolated from Korean fermented seafood. Int J Syst Evol Microbiol 2011;61:2353 - 2357; Yun, J.H., Roh, S.W., Kim, M.S., Jung, M.J., Park, E.J., Shin, K.S., Nam, Y.D., and Bae, J.W. 2011. Leucobacter salsicius sp. nov., from a salt - fermented food. Int. J. Syst. Evol. Microbiol. 61, 502–506.) and chromium - enriched environments (Morais, P.V., Paulo, C., Francisco, R., Branco, R., Paula Chung, A., and da Costa, M.S. 2006. Leucobacter lutis sp. nov., and Leucobacter alluvii sp. nov., two new species of the genus Leucobacter isolated under chromium stress. Syst Appl Microbiol 29, 414–421.). Several new species of Leucobacter have been isolated and cultured from environments with high heavy metal chromium content, and these strains have been found to have strong tolerance to heavy metals or the ability to absorb heavy metals (Morais, P.V., Paulo, C., Francisco, R., Branco, R., Paula Chung, A., and da Costa, M.S. 2006. Leucobacter lutis sp. nov., and Leucobacter alluvii sp. nov., two new species of the genus Leucobacter isolated under chromium stress. Syst Appl Microbiol 29, 414–421. Yun, J.H., Roh, S.W., Kim, M.S., Jung, M.J., Park, E.J., Shin, K.S., Nam, Y.D., and Bae, J.W. 2011. Leucobacter salsicius sp.Nov., from a salt-fermented food. Int. J. Syst. Evol. Microbiol. 61, 502–506; Yun, J. H., Cho, Y. J., Chun, J., Hyun, D. W., and Bae, J. W. 2014. Genome sequence of the chromate-resistant bacterium Leucobacter salsicius type strain M1-8T. Stand. Genomic Sci. 9, 495–504.). These strains obtained from heavy metal-enriched environments also provide ideal experimental materials for the research on the application of Leucobacter species. Summary of the Invention

[0003] The object of the present invention is to provide a new species of Leucobacter that can produce the plant growth hormone 3-Indoleacetic acid (IAA).

[0004] In the first aspect, the present invention claims to protect a new species of Leucobacter.

[0005] The new species of Leucobacter claimed to be protected by the present invention is specifically Leucobacter chunongensis CN001, and its registration number in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 25924.

[0006] The Leucobacter chunongensis CN001 is a Gram-positive bacterium. The cells are non-motile, short bacilli, and the cell size is (0.6-0.8) μm × (1.4-2.1) μm. When cultured on tryptic soy agar medium at 30 °C for 48 h, white to milky white colonies can be formed. The pH tolerance range of strain CN001 is 6.0-9.0, and the optimal growth pH value is 7.0-8.0. The optimal growth temperature is 28–32 °C; there is no growth at 4 or 42 °C. Oxidase is negative, and catalase reaction is positive. It can hydrolyze starch and urea and has the ability to reduce nitrate.

[0007] In the second aspect, the present invention claims to protect a culture.

[0008] The culture claimed to be protected by the present invention is the substance obtained by culturing Leucobacter chunongensis CN001 described in the first aspect above in a bacterial medium.

[0009] In the above-mentioned culture, the substance includes the Leucobacter chunongensis CN001 (the bacteria themselves) and the metabolites of the Leucobacter chunongensis CN001.

[0010] In the above-mentioned culture, the bacterial medium can be a solid medium or a liquid medium.

[0011] The term "culture" refers to the general name of a liquid or solid medium with a microbial population after artificial inoculation and culture. That is, a product obtained by growing and / or amplifying microorganisms, which can be a pure biological culture of microorganisms, or can contain a certain amount of medium, metabolites or other components produced during the culture process. The term "culture" also includes subcultures obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0012] In a specific embodiment of the present invention, the bacterial medium is specifically tryptone soy agar medium.

[0013] In a third aspect, the present invention claims protection for a metabolite.

[0014] The metabolite claimed to be protected by the present invention is the metabolite of Leucobacter chunongensis CN001 described in the first aspect above.

[0015] The term "metabolite" refers to primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and generate substances and energy for maintaining life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monomers of monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, etc., and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process by which microorganisms synthesize some substances with no clear function for the life activities of microorganisms using primary metabolites as precursors during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be classified into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0016] In a fourth aspect, the present invention claims protection for a bacterial agent.

[0017] The bacterial agent claimed to be protected by the present invention contains the Leucobacter chunongensis CN001 described in the first aspect above, the culture described in the second aspect above, and / or the metabolite described in the third aspect above.

[0018] The microbial agent is a microbial agent for producing indoleacetic acid.

[0019] In the above microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier can be a carrier commonly used in the pesticide field and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane and / or dodecane.

[0020] In the above microbial agent, the dosage form of the microbial agent can be various dosage forms, such as a liquid agent, an emulsion, a suspension agent, a powder agent, a granule agent, a wettable powder or a water dispersible granule.

[0021] As needed, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the microbial agent.

[0022] In the fifth aspect, the present invention claims the use of Leucobacter chunongensis CN001 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the microbial agent described in the fourth aspect above in any of the following:

[0023] (A1) Producing indoleacetic acid;

[0024] (A2) Preparing a product for producing indoleacetic acid.

[0025] In the sixth aspect, the present invention claims the use of Leucobacter chunongensis CN001 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the microbial agent described in the fourth aspect above in any of the following:

[0026] (B1) Promoting plant growth;

[0027] (B2) Enhancing the stress resistance of crops.

[0028] In the seventh aspect, the present invention claims a product for producing indoleacetic acid.

[0029] The product for producing indoleacetic acid protected by the present invention has an active ingredient which is Leucobacter chunongensis CN001 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0030] In the eighth aspect, the present invention claims to protect a method for promoting plant growth.

[0031] The method for promoting plant growth protected by the present invention may include the following steps: applying Leucobacter chunongensis CN001 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above to plants or plant growth substrates.

[0032] In the ninth aspect, the present invention claims to protect the application of Leucobacter chunongensis CN001 described in the first aspect above in preparing the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0033] Experimental results show that the physiological and biochemical characteristics, cytochemical classification data, 16S rRNA gene and phylogenetic analysis results of strain CN001 of the present invention indicate that strain CN001 of the present invention represents a new species of the genus Leucobacter, named Leucobacter chunongensis. At the same time, through the detection of the ability to produce IAA, it is also proved that the strain of the present invention can produce IAA, and it is a new species of the genus Leucobacter with the potential to promote plant growth, having broad application prospects in promoting plant growth, enhancing the stress resistance characteristics of crops, etc.

[0034] Depository Instructions

[0035] Taxonomic name: Leucobacter chunongensis;

[0036] Biological material referred to: CN001;

[0037] Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0038] Abbreviation of depository institution: CGMCC;

[0039] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0040] Date of deposit: October 17, 2022;

[0041] Accession number in the depositary institution: CGMCC No. 25924. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the polar lipid component of strain CN001.

[0043] Figure 2 is the phylogenetic tree of strain CN001 and its related bacteria constructed based on the 16S rRNA gene sequence.

[0044] Figure 3 is the IAA standard curve. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0046] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0047] Example 1 Isolation and Identification of Leucobacter chunongensis CN001

[0048] I. Isolation and Screening of Strain CN001

[0049] The strain CN001 of the present invention was isolated from the rhizosphere soil sample of Panax notoginseng in the Panax notoginseng planting area in Wenshan, Yunnan Province. The specific isolation operation is as follows:

[0050] Preparation of soil suspension: Add 2 g of soil sample to 18 mL of sterile mixed solution containing 0.1% sodium pyrophosphate and 0.85% sodium chloride, place it in a shaker at 28 °C, shake at 180 rpm for 40 min to make the soil particles fully suspended. Gradient dilute the formed soil suspension to 10 -4 for standby.

[0051] Isolation medium: Soluble starch 10 g·L -1 , dipotassium hydrogen phosphate 1 g·L -1 , magnesium sulfate heptahydrate 1 g·L -1 , ammonium sulfate 2 g·L -1 , calcium carbonate 2 g·L -1 , ferrous sulfate heptahydrate 0.001 g·L-1 , manganese chloride 0.001 g·L -1 , zinc sulfate 0.001 g·L -1 , sodium chloride 1 g·L -1 , glycerol 12.5 mL·L -1 , arginine 2 g·L -1 , marine trace salts 0.38 g·L -1 , agar 15 g·L -1 ; pH 7.

[0052] Isolation method: Spread the above-diluted soil suspension on the separation medium plate and culture it at 30 °C for 3 weeks. Pick well-grown single colonies and culture them on the PYG slant medium (formula: peptone 3 g·L -1 , yeast extract 5 g·L -1 , glycerol 10 g·L -1 , betaine 1.25 g·L -1 , sodium pyruvate 1.25 g·L -1 , agar 15 g·L -1 ; pH 7) to obtain pure cultures for subsequent research. At the same time, the obtained pure strains are cryopreserved in liquid nitrogen and at -80 °C using 20% (v / v) glycerol as a cryoprotectant. A strain with the highest similarity to the 16S rRNA gene of the genus Leucobacter is obtained. The similarity of the obtained strain to the 16S rRNA genes of Leucobacter tardus K 70 / 01(T) and Leucobacter muris 924 / 12(T) is 95.9% and 95.7% respectively. That is, the similarity to the 16S rRNA genes of all known valid species of strains is less than 96%. The strain number is CN001.

[0053] II. Identification of Strain CN001

[0054] Strain CN001 grows on tryptic soy broth medium (Solarbio) at 30 °C for morphological, physiological and biochemical, cytochemical and genetic level studies, and other special cases will be described.

[0055] 1. Observation of cell morphology and detection of physiological and biochemical characteristics of Strain CN001

[0056] After the strain CN001 was cultured on tryptic soy agar medium at 30 °C for 48 h, the cell morphology was observed using a transmission electron microscope (JEOL, JEM-1400). The growth temperature detection range of the strain CN001 was 4, 10, 28, 30, 32, 37, 42, and 45 °C; the growth salt concentration (NaCl) detection range was 0, 1, 3, 5, 7, 10% (g / 100 mL); the growth pH detection range was 8 gradients between pH 4 - 11 (4, 5, 6, 7, 8, 9, 10, 11). The physiological and biochemical characteristics of the strain were detected using API50CH, API ZYM, and BiOLOG GEN III carbon source detection kits. Other strain physiological characteristics, including Gram staining properties, oxygen demand, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly referred to the "Actinomycete System Identification Manual" (Xu L H (2007). Actinomycete systematics: principles, methods and practices. Beijing: Science Press, 93 - 108.).

[0057] The identification results showed that the strain CN001 was a Gram-positive bacterium, the cells were non-motile, short bacilli, and the cell size was (0.6 - 0.8) μm × (1.4 - 2.1) μm. The strain was cultured on tryptic soy agar medium at 30 °C for 48 h and could form white to milky white colonies. The pH tolerance range of the strain CN001 was 6.0 - 9.0, and the optimal growth pH value was 7.0 - 8.0. The optimal growth temperature was 28 - 32 °C; there was no growth at 4 or 42 °C. The oxidase was negative, and the catalase reaction was positive. It could hydrolyze starch and urea and had the ability of nitrate reduction. The physiological and biochemical characteristics of the strain CN001 and its related bacteria are shown in Table 1.

[0058] Table 1. Physiological and biochemical characteristics of the strain CN001 and its related bacterium Leucobacter tardus K 70 / 01(T)

[0059]

[0060]

[0061] Note: +, positive; W, weakly positive; -, negative.

[0062] 2. Detection of the cytochemical characteristics of the strain CN001

[0063] The fatty acids, quinone types, polar lipids, characteristic amino acids, sugar components and other cytochemical components of strain CN001 were detected by GC (gas chromatography), HPLC (high performance liquid chromatography) and TLC (thin layer chromatography) (Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI inc; 1990. Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M et al. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 1984; 2: 233–241.). The fatty acid composition of strain CN001 is shown in Table 2, and the results show that the main fatty acids of strain CN001 are antesio-C 15:0 and iso-C 16:0 ; in the cell membrane of strain CN001D, the dominant menaquinone is MK-10, and small amounts of MK-7 and MK-11 were also detected; the polar lipid components mainly contain diphosphatidylglycerol (DPG), and the polar lipid components include phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phospholipids (PL), and contain a small amount of aminolipids (AL), as Figure 1 shown.

[0064] Table 2. Cellular fatty acid composition of strain CN001

[0065]

[0066]

[0067] 3. Determination of the phylogenetic position of strain CN001

[0068] The genomic DNA of strain CN001 was extracted for sequencing, and the 16S rRNA gene sequence (SEQ ID No. 1) was aligned online in the internationally authoritative bacterial taxonomy analysis database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results showed that the strain CN001 of the present invention had the highest similarity with the species of the genus Geodermatophilus. The 16S rRNA gene sequence of the strain CN001 of the present invention had the highest similarity with the strains of the genus Leucobacter in the database, and the similarity value was approximately 94.5-95.9%. See Table 3 for details. These similarity values were all lower than the boundary of 98.65% for differentiating prokaryotic microbial species (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014;64:346–351.). The strain with the highest similarity was Leucobacter tardus DSM19811 T (95.9%). The 16S rRNA gene sequences of all valid species strains of the genus Leucobacter and some species strains of the genera adjacent to the genus Leucobacter in the Microbacteriaceae were retrieved to construct a phylogenetic tree ( Figure 2 ). It was shown that the strain CN001 was a member of the genus Leucobacter, and the strain CN001 occupied an evolutionary position of a new species within the genus Leucobacter.

[0069] Table 3. Comparison results of the 16S rRNA gene sequence of strain CN001 with the 16S rRNA gene information of related bacteria

[0070]

[0071]

[0072] 4. Detection of the Activity of Strain CN001 in Producing Indoleacetic Acid (IAA)

[0073] The ability of strain CN001 to produce indoleacetic acid was determined by colorimetry (Bric et al., 199Bric, J.M., Bostock, R.M., and Silverstone, S.E. (1991). Rapid in situ assay for indoleacetic Acid production by bacteria immobilized on a nitrocellulose membrane. Appl. Environ. Microbiol. 57, 535 - 538. doi:10.1128 / aem.57.2.535 - 538.1991). Standard curves were made with IAA concentrations of 0, 2, 4, 6, 8, 10, 12 mg / L respectively, and the absorbance values of OD 540 nm were measured ( Figure 3 , y = 0.0244x + 0.0482, r 2 = 0.9998). The cells of the strain in the logarithmic growth phase were transferred to a culture medium containing 3 mmol / L L - tryptophan and 1% (w / v) tryptone, and incubated at 30 °C for 72 h. The absorbance value of OD 540 nm of the culture medium was measured. According to the standard curve, the concentration of indoleacetic acid in the culture medium of strain CN001 was calculated to be 3.85 ± 0.12 mg / L (the average value ± standard deviation obtained from three independent experiments). It was confirmed that strain CN001 has a strong ability to produce indoleacetic acid.

[0074] Based on the physiological and biochemical characteristics, cytochemical classification data, 16S rRNA gene and phylogenetic analysis results of strain CN001, the strain CN001 of the present invention represents a new species of the genus Leucobacter, named Leucobacter chunongensis. At the same time, through the detection of the IAA - producing ability, it was also proved that the strain of the present invention can produce IAA, and it is a new species of the genus Leucobacter with the potential to promote plant growth, and has broad application prospects in promoting plant growth and enhancing the stress resistance characteristics of crops, etc.

[0075] Leucobacter chunongensis CN001 was deposited at the China General Microbiological Culture Collection Center on October 17, 2022, and its deposit number is CGMCC No. 25294.

[0076] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, in accordance with the principle of the present invention, this application intends to cover any variations, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Bacillus amyloliquefaciens Leucobacter chunongensis ), strain CN001, with the accession number CGMCC No. 25924 deposited at the China General Microbiological Culture Collection Center.

2. The culture of Leucobacter chunongensis as claimed in claim 1 ( Leucobacter chunongensis ), CN001, is a substance obtained by culturing Leucobacter chunongensis as claimed in claim 1 ( Leucobacter chunongensis ), CN001, in a bacterial culture medium, and the substance includes Leucobacter chunongensis CN001 and metabolites of Leucobacter chunongensis CN001.

3. Bacterial agent, characterized in that: The microbial agent contains the *Leucobacter chuniiae* described in claim 1 ( Leucobacter chunongensis ) CN001 or the culture described in claim 2.

4. The microbial agent according to claim 3, characterized in that: The bacterial agent is a bacterial agent for producing indoleacetic acid.

5. Use of the Bacillus amyloliquefaciens ( Leucobacter chunongensis ) CN001, or the culture according to claim 2, or the bacterial agent according to claim 3 or 4 in any of the following: (A1) Produce indoleacetic acid; (A2) Prepare a product for producing indoleacetic acid.

6. Use of the Leucobacter initialis ( Leucobacter chunongensis ) CN001 described in claim 1, or the culture described in claim 2, or the bacterial agent described in claim 3 or 4 in promoting plant growth.

7. A product for producing indoleacetic acid, the active ingredient of which is the Leucobacter chuniiformis Leucobacter chunongensis ) CN001 or the culture as claimed in claim 2 or the bacterial agent as claimed in claim 3 or 4.

8. A method for promoting plant growth, comprising the following steps: applying the Bacillus amyloliquefaciens CN001 as described in claim 1 Leucobacter chunongensis ), or the culture as described in claim 2, or the microbial inoculum as described in claim 3 or 4 to plants or plant growth substrates.

9. Use of the Leucobacter chungiensis ( Leucobacter chunongensis ) CN001 in the preparation of the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

Images