Salt-tolerant growth-promoting bacillus cereus and application thereof

Abstract

The invention discloses a salt-tolerant growth-promoting bacillus cereus strain and an application thereof. In particular discloses bacillus cereus T146, and the preservation number of the bacillus cereus T146 is CGMCC (China General Microbiological Culture Collection Center) No.30429. The bacillus cereus T146 disclosed by the invention has good biofilm production, siderophore secretion and nitrogen fixation capabilities, and can remarkably increase the root length, the overground dry weight, the plant height, the number of lateral roots and the average density of the lateral roots of wheat under the condition of salt stress. According to the invention, a low-cost, efficient and applicable culture medium aiming at the bacillus cereus T146 is determined, a stable and controllable fermentation system is established, the preparation method of the microbial inoculum with high mature spore number is obtained by optimizing a spray drying process, and the active spore number of the microbial inoculum can reach 1.3 * 10 < 11 > CFU / g. The bacillus cereus T146 has good development and application prospects in the fields of microbial agents, microbial fertilizers, soil improvement and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and relates to a salt-tolerant growth-promoting Bacillus cereus and an application thereof, and specifically relates to the excavation of salt-tolerant rhizosphere growth-promoting bacteria and the development of a microbial agent prepared using the same. Background Art

[0002] Bacillus cereus ( Bacillus cereus ) can form stable, stress-resistant dormant spores to withstand harsh external environments. In recent years, it has been widely prepared into microbial agents for use in plant fertilizers, growth regulators, animal feed, and ecological environment improvement products. Due to its growth-promoting, disease-preventing, and stress-resistant properties, Bacillus cereus is increasingly used as a plant growth-promoting rhizobacteria (PGPR) in regulating plant growth.

[0003] Under the dual pressures of shortage of available arable land resources and continuous population growth, developing and utilizing saline-alkali land to increase the arable land area is a solution to increase crop yields. However, saline-alkali soil usually has low fertility, which is not conducive to the growth of crops. In order to improve the utilization rate of saline-alkali soil and agricultural production efficiency, chemical fertilizers were used in large quantities in agricultural production in the past. However, with the long-term irrational application of agricultural chemicals, a series of problems have been brought about, which has further reduced the fertility of the originally poor saline-alkali soil, aggravated pests and diseases, and destroyed the ecological balance of soil and water bodies.

[0004] In order to solve the above problems that are not conducive to the sustainable development of agriculture, the development and utilization of environmentally friendly plant fertilizers is an important strategy for the biological improvement of saline-alkali land. Summary of the invention

[0005] The purpose of the present invention is to provide a salt-tolerant growth-promoting Bacillus cereus and its application in plant growth and / or soil improvement. The technical problem to be solved is not limited to the technical subject as described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] To achieve the above object, the present invention first provides a strain of Bacillus cereus, wherein the Bacillus cereus is Bacillus cereus T146, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.30429.

[0007] The present invention also provides a culture containing the Bacillus cereus T146.

[0008] The present invention also provides a bacterial agent containing the Bacillus cereus T146 and / or the culture.

[0009] Furthermore, the bacterial agent can be used to promote plant growth, promote plant growth under salt stress conditions, improve plant salt tolerance and / or improve salinized soil.

[0010] Furthermore, the bacterial agent may also include a carrier.

[0011] The carrier may be a solid carrier or a liquid carrier. The solid carrier includes, but is not limited to, inorganic carriers (such as kaolin, light calcium carbonate, diatomaceous earth, medical stone, calcite, zeolite, silica, montmorillonite, white carbon black, talc, vermiculite, fine sand and clay, etc.), organic carriers (such as cellulose, lignin, starch, rice flour, soybean flour, casein, glucose and gelatin, etc.) and polymer materials (such as polyvinyl alcohol and polyethylene glycol, etc.). The liquid carrier includes, but is not limited to, vegetable oil, mineral oil and water.

[0012] Furthermore, the bacterial agent may also include auxiliary agents, such as wetting agents (including but not limited to glycerol, trehalose, sodium dodecylbenzene sulfonate, sorbitan monolaurate (Span 20) and Tween 60), dispersants (including but not limited to sodium lignin sulfonate, calcium lignin sulfonate, sodium methylene bisnaphthalene sulfonate (NNO), sodium methylene bismethylnaphthalene sulfonate (MF), sodium salt of alkylnaphthalene sulfonic acid condensate (DNA), non-ionic surfactants and water-soluble polymers), stabilizers (including but not limited to inorganic salts, glucose, sucrose, mannitol and sorbitol), permeants (including but not limited to polyethers and alkylphenol polyoxyethylene ether phosphates), spreading agents (including but not limited to sodium oleate, polyvinyl alcohol, soap and bamboo chrysanthemum) and defoaming agents (including but not limited to higher alcohols such as silicone, C8-10 fatty alcohols, polyalkylene glycols and octadecyl alcohol), etc.

[0013] The formulation of the bacterial agent can be liquid (such as aqueous solution, aqueous emulsion, microemulsion, suspension and emulsifiable concentrate, etc.), powder (such as wettable powder, soluble powder and water dispersible granules, etc.) or granule.

[0014] The active ingredient of the bacterial agent may be Bacillus cereus T146 and / or the culture.

[0015] Furthermore, the content of Bacillus cereus T146 in the bacterial agent can be 1×10 10 ~2×10 12 CFU / g.

[0016] The 1×10 10 ~2×10 12 CFU / g can be 1×10 11 ~2×10 11 CFU / g.

[0017] Furthermore, the bacterial agent may be a microbial agent.

[0018] The present invention also provides the use of the Bacillus cereus T146, the culture or the bacterial agent in any of the following: A1) Use in promoting plant growth or in preparing products for promoting plant growth; A2) Use for promoting plant growth under salt stress conditions or for preparing a product for promoting plant growth under salt stress conditions; A3) Use in improving the salt tolerance of plants or in preparing products for improving the salt tolerance of plants; A4) Use in soil improvement or in the preparation of products for soil improvement; A5) Use in improving saline-alkali soil or preparing products for improving saline-alkali soil.

[0019] Furthermore, the product can be a plant growth promoter, a plant growth regulator, a plant rooting agent, a plant salt-resistant agent, a plant salt-tolerant growth promoter, a microbial fertilizer, a fertilizer enhancer, a soil conditioner or a saline-alkali soil conditioner.

[0020] The promoting of plant growth, promoting plant growth under salt stress conditions or improving plant salt tolerance can be achieved by applying the Bacillus cereus T146, the culture or the bacterial agent of the present invention to the plants and / or the soil where the plants are planted.

[0021] The improved soil or improved salinized soil can be achieved by applying the Bacillus cereus T146, the culture or the bacterial agent described in the present invention to the soil to be improved, or by planting plants in the soil to be improved and applying the Bacillus cereus T146, the culture or the bacterial agent described in the present invention to the plants and / or the soil in which the plants are planted.

[0022] The Bacillus cereus T146 of the present invention has a good function of secreting siderophores. Siderophores are small molecule iron ion chelates secreted by microorganisms, which help plants absorb and utilize iron in the soil. Iron plays a very important role in the metabolic process of microorganisms. By secreting siderophores, oxidized iron and Fe in the soil can be absorbed and utilized by plants. 3+ Converted into Fe that can be directly absorbed and utilized by plants 2+ , which can not only promote plant growth, but also chelate many harmful metal ions in the soil, such as Cr 3+ 、Al 3+ , Cu 2+ 、Eu 3+ , Pb 2+ In addition, the plants can absorb and remove ions in the soil, reduce the pH value of the soil, and ultimately achieve soil desalination and alkalinity reduction.

[0023] Further, the manner of applying may include spraying, atomizing, atomizing, spraying, flooding, pouring, washing and / or rinsing but is not limited thereto.

[0024] The present invention also provides a product, which includes the Bacillus cereus T146, the culture or the bacterial agent; the product can be a plant growth promoter, a plant growth regulator, a plant rooting agent, a plant salt-resistant agent, a plant salt-tolerant growth promoter, a microbial fertilizer, a fertilizer synergist, a soil improver or a saline-alkali soil improver but is not limited thereto.

[0025] The present invention also provides a method for preparing the bacterial agent, which comprises: culturing the Bacillus cereus T146 in a culture medium to obtain a culture, and post-treating the culture to obtain the bacterial agent.

[0026] In the above method, the post-treatment may be spray drying, the drying aid used in the spray drying may be β-cyclodextrin, the inlet temperature of the spray drying may be 145-155°C, and the air flow rate may be 65-75 m 3 / h.

[0027] Furthermore, the inlet temperature of the spray drying can be 150°C and the air flow rate can be 70 m 3 / h.

[0028] Furthermore, the amount of β-cyclodextrin added to the fermentation broth may be 18%-22% (mass percentage), specifically 20% (mass percentage).

[0029] Furthermore, the feed rate of the spray drying may be 8-12 mL / min, specifically 10 mL / min.

[0030] Furthermore, the outlet temperature of the spray drying may be 80-120°C, specifically 100°C.

[0031] In the above method, culturing the Bacillus cereus T146 in a culture medium may include the following steps: B1) inoculating the Bacillus cereus T146 into a seed culture medium for seed culture to obtain a seed solution; B2) inoculating the seed solution into a fermentation medium at an inoculum volume ratio of 10% for fermentation culture; The fermentation medium may be composed of: 4-6 g / L soybean meal, 8-12 g / L corn meal, 4-6 g / L sodium chloride, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L manganese sulfate, 1.0-3.0 mL / L defoamer, and the balance is water.

[0032] Furthermore, the composition of the fermentation medium may be: 5 g / L soybean meal, 10 g / L corn meal, 5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, 0.4 g / L manganese sulfate, 2.0 mL / L defoaming agent, and the balance is water.

[0033] In the above method, the fermentation culture temperature can be 33-37°C, the rotation speed can be 280-320 rpm, the pressure can be 0.08-0.12 MPa, the ventilation volume can be 2.8-3.2 L / min, and the fermentation time can be 24-96 h.

[0034] In the above method, the fermentation culture temperature can be 35°C, the rotation speed can be 300 rpm, the pressure can be 0.1 MPa, the ventilation volume can be 3 L / min, and the fermentation time can be 48-72 h.

[0035] Furthermore, the parameters of the fermentation culture also include: a pH value of 6-8, specifically 7.

[0036] Furthermore, the volume of the fermentation culture (i.e. the volume percentage of the fermentation medium in the fermenter) may be 65%-75%, specifically 70%.

[0037] Furthermore, after 16 hours of the fermentation culture, feeding can be carried out at a flow rate of 8 mL / h, and the composition of the feed medium can be: 18-22 g / L soybean meal, 13-17 g / L corn meal, 8-12 g / L sodium chloride, 5-7 g / L potassium dihydrogen phosphate, 5-7 g / L manganese sulfate, and the balance is water.

[0038] Furthermore, the composition of the feed medium may be: 20 g / L soybean meal, 15 g / L corn meal, 10 g / L sodium chloride, 6 g / L potassium dihydrogen phosphate, 6 g / L manganese sulfate, and the balance is water.

[0039] Furthermore, the seed culture in B1) includes inoculating the Bacillus cereus T146 into a seed culture medium for primary and secondary expansion culture to obtain a seed solution.

[0040] Furthermore, the seed culture medium may be composed of: 2 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water. The pH value of the seed culture medium may be 6.8.

[0041] Furthermore, the seed culture conditions may be a temperature of 37° C. and a rotation speed of 200 rpm.

[0042] Furthermore, the primary and secondary expansion cultures may include the following steps: 1) Primary shake flask seed culture: Inoculate Bacillus cereus T146 into the seed culture medium at 37°C, 200 rpm, 50 mL of liquid, and culture time for 48 h.

[0043] 2) Secondary shake flask seed culture: set the inoculation volume to 3% (volume percentage), temperature to 37°C, rotation speed to 200 rpm, liquid volume to 250 mL, and culture time to 48 h.

[0044] The promoting of plant growth or promoting plant growth under salt stress conditions described herein may be embodied in at least one of the following aspects: (1) Promote the growth of plant roots; (2) Promote the increase of plant aboveground dry weight; (3) Promote the increase in the number of plant lateral roots; (4) Promote the increase of plant height; (5) Promote the increase of plant lateral root density.

[0045] The plants described herein include monocots and dicots.

[0046] Furthermore, the plants described herein include plants of the Poaceae family.

[0047] After extensive and in-depth research, the inventors of the present application discovered a salt-tolerant growth-promoting Bacillus cereus, named T146. The Bacillus cereus T146 has good biofilm production, siderophore secretion and nitrogen fixation abilities. Experiments have shown that the Bacillus cereus T146 screened and obtained by the present invention is an excellent salt-tolerant growth-promoting bacterium, which can increase the root length of wheat by 32.15% and the aboveground dry weight by 211.52% in salt-stressed soil; under hydroponic conditions of salt stress, the plant height of wheat can be increased by 35.77%, the root length by 33.22%, the number of lateral roots of wheat by 144.44%, and the average lateral root density of wheat by 112.79%. The present invention determines a low-cost, economical and efficient culture medium suitable for strain propagation and spore formation for Bacillus cereus T146; establishes a stable and controllable process fermentation system by measuring parameters such as temperature, dissolved oxygen and feed; obtains a preparation method of a high-mature spore count inoculant by optimizing the spray drying process of the inoculant, and the number of active spores of the Bacillus cereus T146 inoculant can reach 1.3×10 11 CFU / g.

[0048] In summary, the Bacillus cereus T146 of the present invention has good effects of promoting plant growth and improving plant salt resistance, and has good development and application prospects in the fields of microbial agents, microbial fertilizers, soil improvement, etc.

[0049] Definition of terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0050] The term "culture" generally refers to a liquid or solid product (all materials in the culture container) that has a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0051] The term "culture" may also be referred to herein as "microorganism culture", which generally refers to the growth and reproduction of microorganisms under suitable culture medium and culture conditions. The "culture" may be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and batch fed culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium may be appropriately adjusted according to actual conditions.

[0052] The term "microbial agent" generally refers to a microbial preparation made from one or several microorganisms with specific functions.

[0053] The term "carrier" in this article generally refers to the medium substance added when preparing the bacterial agent, which can provide nutrition, protection and delivery for the bacterial agent.

[0054] The term "adjuvant" in this article generally refers to any auxiliary substance added during the preparation of a bacterial agent to improve its physical and / or chemical properties, or to enhance its quality and / or stability, or to enhance its biological activity and / or persistence.

[0055] The term "culture medium" generally refers to a mixture of various nutrients prepared in a certain proportion that are required for the growth and reproduction of microorganisms and the biosynthesis of various metabolites.

[0056] The term "seed medium" generally refers to any medium used to grow microorganisms to produce or accumulate microorganisms for inoculating a bioreactor, such as a fermentor.

[0057] The term "fermentation medium" generally refers to a culture medium used in a bioreactor (eg, a fermentor) that contains nutrients to support the growth, reproduction, and / or product synthesis of microorganisms.

[0058] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to." The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of.

[0059] Collection Instructions Bacteria name: Bacillus cereus Latin name: Bacillus cereus Classification name: Bacillus cereus Bacillus cereus Strain ID: T146 Depository: China National Microbiological Culture Collection Administration General Microbiology Center Abbreviation of depository unit: CGMCC Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Storage date: April 24, 2024 The registration number of the Collection Center is: CGMCC No.30429. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The morphology and classification of Bacillus cereus T146. Figure 1 A in the middle shows the morphology of a single colony of Bacillus cereus T146; Figure 1 Middle B is the activity staining image of Bacillus cereus T146; Figure 1 Middle C is the phylogenetic tree analysis of Bacillus cereus T146.

[0061] Figure 2 This is a biofilm production test for Bacillus cereus T146. Figure 2 Middle A is the staining image of Bacillus cereus T146 biofilm; Figure 2 Middle B is the quantitative statistical diagram of Bacillus cereus T146 biofilm.

[0062] Figure 3 It is a test for the secretion of siderophore by Bacillus cereus T146. Figure 3 A in the middle is the siderophore test diagram of Bacillus cereus T146; Figure 3 Middle B is the quantitative statistical graph of siderophore of Bacillus cereus T146.

[0063] Figure 4 This is a test chart of the nitrogen fixation ability of Bacillus cereus T146.

[0064] Figure 5 The wheat growth promotion phenotype and statistical analysis of Bacillus cereus T146. Figure 5 A in the middle is the growth phenotype of wheat after Bacillus cereus T146 acted on wheat; Figure 5Middle B is the analysis of the growth-promoting ability of Bacillus cereus T146 under salt stress (T test, *P < 0.05).

[0065] Figure 6 The phenotype and statistical analysis of wheat hydroponic growth promotion by Bacillus cereus T146. Figure 6 A in the middle is the growth phenotype of wheat after Bacillus cereus T146 acted on wheat; Figure 6 Middle B is the analysis of wheat root morphology; Figure 6 Middle C shows the effects of Bacillus cereus T146 on wheat plant height, root length, lateral root number and lateral root density. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0067] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0068] The culture medium used in the following examples is: The composition of LB liquid medium is: tryptone, 10.0 g / L; yeast extract, 5.0 g / L; NaCl, 10.0 g / L; pH adjusted to 7.0, sterilized at 121°C for 15 min. LB solid medium (LB plate medium) is a solid medium obtained by adding 15.0 g / L agar to LB liquid medium.

[0069] The composition of CAS medium is: chrome azuro blue S, 60.5 mg / L; hexadecyltrimethylammonium bromide, 72.9 mg / L; ferric chloride hexahydrate, 2.645 mg / L; sodium dihydrogen phosphate dihydrate, 295.25 mg / L; disodium hydrogen phosphate dodecahydrate, 1213.5 mg / L; ammonium chloride, 125 mg / L; potassium dihydrogen phosphate, 37.5 mg / L; sodium chloride, 62.5 mg / L; agar, 9000 mg / L; pH value 6.8, sterilization at 116℃ for 30 min.

[0070] The composition of the nitrogen source-free culture medium is: potassium dihydrogen phosphate, 0.2 g / L; magnesium sulfate, 0.2 g / L; sodium chloride, 0.2 g / L; calcium carbonate 5.0 g / L; mannitol 10.0 g / L; calcium sulfate 0.1 g / L; agar 15.0 g / L; pH is adjusted to 7.0 and sterilized at 121℃ for 15 min.

[0071] The composition of Hoagland's medium is: potassium nitrate, 606.6 mg / L; ammonium dihydrogen phosphate, 230.16 mg / L; magnesium sulfate heptahydrate, 246.48 mg / L; potassium chloride, 3.73 mg / L; boric acid, 1.55 mg / L; manganese sulfate, 0.34 mg / L; zinc sulfate, 0.58 mg / L; sodium molybdate, 0.11 mg / L; sodium iron EDTA, 11.17 mg / L; calcium nitrate 945g mg / L, pH adjusted to 6.0, sterilized at 116℃ for 30 min.

[0072] Example 1. Isolation and identification of Bacillus cereus T146 The saline-alkali soil from the Yellow River Delta in Dongying was diluted in a gradient manner using the soil dilution method. Then, the soil dilution solution of appropriate concentration was inoculated into LB plate culture medium. After culturing at 30°C in a constant temperature incubator for 1-2 days, single bacterial colonies of different morphologies were isolated. After initial and rescreening of salt tolerance using LB culture medium containing 5% NaCl, strain T146 was selected for identification.

[0073] Primary screening: Streak the monoclonal colonies of each candidate strain on LB plate medium containing 5% NaCl for growth.

[0074] Second screening: The strains with good growth status obtained after the initial screening were inoculated into ordinary LB liquid medium and LB liquid medium containing 5% NaCl at an inoculum of 1%, respectively, and cultured at 30°C and 200 rpm. The OD was measured every 4 h. 600 nm The salt stress tolerance of the strain was then evaluated.

[0075] The initial screening results showed that strain T146 grew normally.

[0076] Rescreening results: Comparing the growth curves of strain T146 in the two culture media, it was found that salt stress did not inhibit the growth of the strain. At 48 h, the OD of T146 in ordinary LB liquid medium was 600 nm The average value was 1.67, and the OD of T146 in LB liquid medium containing 5% NaCl was 600 nm The average value was 1.69. Strain T146 was a salt-tolerant strain, significantly superior to other strains, and was selected for further identification and functional verification.

[0077] (1) Morphological characteristics identification: The strain T146 was streaked onto LB plate medium and cultured at 37°C for 24 h before observing the morphology of single colonies ( Figure 1 A). A single colony was picked and transferred into LB liquid culture medium, and then stained after shaking culture at 30°C and 180 rpm for 36 h. The bacterial cells and spore formation of strain T146 were observed under a microscope (10×100 times). Figure 1 Middle B).

[0078] The results of morphological identification showed that the colonies of strain T146 cultured on LB plates were white, waxy, nearly round, and had uneven edges; strain T146 was short rod-shaped and had spores.

[0079] (2) Identification and classification using 16S rRNA sequences The genomic DNA of Bacillus cereus T146 strain was extracted using the genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., and 16S rDNA was amplified by PCR using 27F and 1492R as primers. The primer sequences are as follows: Primer 27F: 5'-AGAGTTTGATCATGGCTCAG-3', Primer 1492R: 5′-GGCTACCTTGTTACGACTT-3′.

[0080] PCR reaction system (50 μL): 2 × Rapid Taq Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.) 25 μL, primer 27F (10 μmol / L) 2 μL, 1 μL 1492R (10 μmol / L), 1 μL genomic DNA of Bacillus cereus T146, ddH2O to 50 μL. PCR amplification program: 95℃ 3 min; 95℃ 15 s, 56℃ 30 s, 72℃ 30 s, 30 cycles; 72℃ 5 min.

[0081] The PCR amplification product was detected by 1% agarose gel electrophoresis and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing to obtain the 16S rRNA sequence of strain T146.

[0082] The 16S rDNA nucleotide sequence corresponding to the 16S rRNA of the T146 strain is as follows:

[0083] The sequencing results showed that the length of the 16S rRNA gene amplified fragment of strain T146 was 1429 bp. The sequence was compared with the 16S rDNA of strain T146 in GenBank, and the result showed that the homology was 100% between strain T146 and Bacillus cereus. The cluster evolution tree was constructed using the neighbor-joining method of MEGA 11.0 software ( Figure 1 C), strain T146 and Bacillus cereus ( Bacillus cereus ) were aggregated together, preliminarily indicating that it was Bacillus cereus.

[0084] (3) Physiological and biochemical identification of strains: The strain T146 was tested by crystal violet staining, CAS medium and nitrogen-free medium growth methods, respectively.

[0085] The crystal violet staining method was used to detect the biofilm formation ability of strain T146. In a sterile environment, the strain was inoculated into LB liquid culture medium at a volume of 1% (volume percentage), 1 mL was aspirated and placed in a 24-well plate at 37°C for 48 h, and then gently washed twice with sterile water, then stained with 0.1% crystal violet for 30 min, washed 5 times with sterile water, dried and completely dissolved with 1 mL of 95% anhydrous ethanol, and 200 μL of the dissolved solution was read and recorded at a wavelength of 595 nm. The data can characterize the biofilm formation ability. The results are shown in Figure 2 As shown in A, strain T146 can produce biofilm and has a strong ability to produce biofilm (e.g. Figure 2 (shown in B).

[0086] The liquid culture of strain T146 was resuspended in sterile water and 5 μL was spotted on CAS medium and inverted at 37°C. A transparent circle (such as Figure 3 The ratio (D / d) of the diameter of the orange transparent circle (D) and the diameter of the colony (d) can be used to characterize the ability of the strain to secrete siderophores. T146 has a strong ability to secrete siderophores (as shown in Figure 1). Figure 3 (shown in B).

[0087] The strain T146 was smeared on the nitrogen-free medium with a sterile toothpick and cultured at 37°C. The strain grew successfully (e.g. Figure 4 The strain has the ability to fix nitrogen.

[0088] Biochemical identification tests were performed using gelatin biochemical tubes (purchased from Ouk Biotech, catalog number OK3212), nitrite content detection kits (purchased from Solebo, catalog number BC1480), carbohydrate identification reagent strips (purchased from BioMérieux, catalog number API50CH), and catalase tests (3% hydrogen peroxide solution). It was found that strain T146 was a Gram-positive bacterium with all spores in the bacteria. It was positive for gelatin liquefaction, motility, nitrate reduction, anaerobic glucose fermentation, catalase, and mannitol.

[0089] The physiological and biochemical characteristics of strain T146 were compared with those of the model bacteria Bacillus cereus in the Manual of Identification of Common Bacteria (Dong Xiuzhu, Cai Miaoying. Manual of Identification of Common Bacteria. Beijing: Science Press, 2011.). Bacills cereus The physiological and biochemical characteristics are consistent.

[0090] Finally, strain T146 was identified as Bacillus cereus Bacillus cereus The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on April 24, 2024, with the deposit number CGMCCNo.30429.

[0091] Example 2: Growth-promoting effect of Bacillus cereus T146 on soil-cultured wheat under salt stress The wheat variety "Ningchun 4" seeds were surface sterilized with 2% NaClO solution for 10 min, then rinsed three times with sterile water and placed in the dark at 4°C for 48 h for vernalization. Then, the seeds were placed in pots for growth (9×9×10 cm square pots), with the substrate being nutrient soil: vermiculite = 1:1 (volume ratio).

[0092] Three treatment groups were set up, and the following substances were added to the matrix of each group for treatment: (1) Control: Hoagland's culture medium; (2) NaCl: Hoagland's culture medium + 400 mM NaCl; (3) NaCl+T146: Hoagland's culture medium + 400 mM NaCl + B. cereus T146, so that each gram of substrate in the flower pot contains 1×10 9 Bacteria.

[0093] Five wheat seeds were placed in each tray, and three replicates were placed in each experimental group. The seeds were grown in a light incubator at 25°C with a photoperiod of 12 h light / 12 h dark. The experimental period was 4 weeks. Plant samples were collected and the plant height, root length, aboveground fresh weight and dry weight ( Figure 5 Middle A).

[0094] The results showed that compared with the 400 mM NaCl salt stress treatment group, B. cereusThe root length of T146 wheat increased significantly by 32.15%, and the aboveground dry weight increased significantly by 211.52% ( Figure 5 Middle B).

[0095] The above results show that under salt stress, inoculation with Bacillus cereus T146 has a significant growth-promoting effect on plants compared with the uninoculated control.

[0096] Example 3. Growth-promoting effect of Bacillus cereus T146 on hydroponic wheat under salt stress The wheat variety "Ningchun No. 4" seeds were surface sterilized with 2% NaClO solution for 10 min, then rinsed three times with sterile water and placed in the dark at 4°C for vernalization for 48 h. Then, the seeds were placed in sterile gauze for growth, and seedlings with consistent growth were selected. Four hydroponic treatment groups were set up, and the following substances were added to each group for treatment: (1) Control: Hoagland's culture medium. (2) T146: Hoagland's culture medium + B. cereus T146. (3) NaCl: Hoagland's medium + 200 mM NaCl (first week), Hoagland's medium + 250 mM NaCl (second week). (4) NaCl+T146: Hoagland's medium + 200 mM NaCl+ B. cereus T146 (first week), Hoagland medium + 250 mM NaCl + B. cereus T146 (second week). Each experimental group had five replicates, with four plants in each replicate. They were grown in a light incubator at 25°C with a photoperiod of 12 h light / 12 h dark. Figure 6 (A) The experimental period was 2 weeks. Plant samples were collected and plant height, root length, lateral root number and lateral root density were measured.

[0097] from Figure 6 In B, it can be seen that under salt stress, B. cereus The number of lateral roots of wheat increased after T146; by measuring, e.g. Figure 6 As shown in C, under salt stress, the average plant height of the NaCl+T146 group was 8.71 cm, and the average plant height of the NaCl group was 6.41 cm. B. cereus T146 significantly increased the plant height of wheat by 35.77%; the average root length of the NaCl+T146 group was 7.53 cm, and the average plant height of the NaCl group was 5.65 cm. B. cereus T146 significantly increased the root length of wheat by 33.22%; the average number of lateral roots in the NaCl+T146 group was 22, and the average number of lateral roots in the NaCl group was 9. B. cereusT146 significantly increased the number of lateral roots of wheat by 144.44%; the average lateral root density of the NaCl+T146 group was 3.13, and the average lateral root density of the NaCl group was 1.74. B. cereus T146 significantly increased the average lateral root density of wheat by 112.79%.

[0098] In summary, under the hydroponic conditions of salt stress, Bacillus cereus T146 has a significant ability to promote plant growth. Therefore, the strain will be made into a bacterial agent in the future, in order to be applied in actual agricultural production.

[0099] Example 4. Preparation of Bacillus cereus T146 bacterial agent 1) Primary shake flask seed culture. Prepare seed culture medium (glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH adjusted to 6.8 with 0.1 mol / L sodium hydroxide or 0.1 mol / L hydrochloric acid). Pick a single clone of Bacillus cereus T146, activate it and inoculate it into the seed culture medium, at 37°C, 200 rpm, 50 mL of liquid, and culture for 48 h.

[0100] 2) Secondary shake flask seed culture: The inoculation volume was set to 3%, the temperature to 37°C, the rotation speed to 200 rpm, the liquid volume to 250 mL, and the culture time to 48 h.

[0101] 3) Optimization of fermentation tank scale-up process. The seed liquid was inoculated into the fermentation tank, and the 5 L, 15 L, 50 L, and 100 L fermentation tanks were scaled up step by step. In the 5 L fermentation tank culture stage, the fermentation conditions were optimized by single factor and orthogonal experiments to increase the number of viable bacteria and spore yield of Bacillus cereus T146. A screening experiment of carbon sources (bran, corn flour, glucose) and nitrogen sources (soybean meal powder, soybean cake powder, ammonium chloride, ammonium sulfate) was carried out, and a single-factor shake flask experiment was designed to investigate different temperatures (30, 35, 37, 39 and 41°C), inoculation size (3%, 5%, 7% and 10%), culture medium pH (6, 7, and 8), liquid volume (50%, 60%, 70% and 80%), rotation speed (200, 300, 400 and 500 rpm), pressure (0.025, 0.05, 0.1 and 0.15 MPa), and ventilation rate (1, 2, 3 and 4 L / min).

[0102] The experiment found that when corn flour was used as the carbon source and soybean meal meal as the nitrogen source, the fermentation medium components were: 5 g / L soybean meal meal, 10 g / L corn flour, 5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, 0.4 g / L manganese sulfate, 2.0 mL / L defoamer, fermentation temperature was 35℃, inoculation size was 10%, medium pH was 7, liquid volume was 70% (volume percentage), rotation speed was 300 rpm, pressure was 0.1 MPa, and ventilation volume was 3 L / min, the biomass and spore number of Bacillus cereus T146 were the highest, and the spore formation rate was 95%.

[0103] The total fermentation time was 48 h, and feed medium (soybean meal 20 g / L, corn meal 15 g / L, sodium chloride 10 g / L, potassium dihydrogen phosphate 6 g / L, manganese sulfate 6 g / L) was added at a flow rate of 8 mL / h after 16 h.

[0104] After determining the optimal values ​​of all conditions and parameters of fermentation culture, the fermentation parameters such as carbon source, nitrogen source, inoculation amount, culture medium pH, liquid volume, rotation speed, pressure and ventilation volume were appropriately adjusted during the step-by-step scale-up process of the fermenter. The effective fermentation volume was increased to 100 L. The experiment proved that the fermentation conditions were stable and a stable and controllable small-scale fermentation process was successfully established.

[0105] 4) Establish a spray drying process for the inoculant. Based on the optimized fermentation process, the fermentation broth in the 100 L fermenter was collected and the bacteria were retained after sterile filtration.

[0106] The spray drying conditions were optimized by single factor and orthogonal experiments to improve the potency retention rate and spore survival rate of the inoculant. At a feed rate of 10 mL / min and an outlet temperature of 100 °C, the drying aids (corn starch, β-cyclodextrin and soluble starch), the amount of drying aid added (mass percentage: 5%, 10%, 15% and 20%), the inlet temperature (150, 170, 190 and 210 °C), the air flow rate (30, 50 and 70 m 3 / h) parameters optimization screening.

[0107] The experiment found that with 20% β-cyclodextrin as a drying agent, an inlet temperature of 150°C, and an air flow rate of 70 m 3 / h, the highest number of active spores was 1.3×10 11 CFU / g.

[0108] In summary, the experiment obtained the optimal fermentation parameters and conditions for the preparation of high mature spore counts for the industrial production of salt-tolerant rhizosphere growth-promoting bacteria Bacillus cereus T146. Using corn flour, soybean meal and other raw materials as fermentation medium components is low-cost and economical, providing a versatile and simple method for large-scale production of Bacillus cereus with high titer retention rate and spore survival rate.

[0109] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A strain of Bacillus cereus, characterized in that The Bacillus cereus is Bacillus cereus Bacillus cereus T146, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.30429.

2. A culture containing the Bacillus cereus according to claim 1.

3. A bacterial agent containing the Bacillus cereus according to claim 1 and / or the culture according to claim 2.

4. Use of the Bacillus cereus according to claim 1, the culture according to claim 2 or the bacterial agent according to claim 3 in any of the following: A1) Use in promoting plant growth or in preparing products for promoting plant growth; A2) Use for promoting plant growth under salt stress conditions or for preparing a product for promoting plant growth under salt stress conditions; A3) Use in improving the salt tolerance of plants or in preparing products for improving the salt tolerance of plants; A4) Use in soil improvement or in the preparation of products for soil improvement; A5) Use in improving saline-alkali soil or preparing products for improving saline-alkali soil.

5. The product, characterized in that The product includes the Bacillus cereus described in claim 1, the culture described in claim 2 or the bacterial agent described in claim 3; the product is a plant growth promoter, a plant growth regulator, a plant rooting agent, a plant salt-resistant agent, a plant salt-tolerant growth promoter, a microbial fertilizer, a fertilizer synergist, a soil improver or a saline-alkali soil improver.

6. A method for preparing the bacterial agent according to claim 3, characterized in that: The method comprises: culturing the Bacillus cereus according to claim 1 in a culture medium to obtain a culture, and post-treating the culture to obtain the bacterial agent.

7. The method according to claim 6, characterized in that The post-treatment is spray drying, the drying aid used in the spray drying is β-cyclodextrin, the inlet temperature of the spray drying is 145-155°C, the air flow rate is 65-75 m 3 / h.

8. The method according to claim 6 or 7, characterized in that: The step of culturing the Bacillus cereus according to claim 1 in a culture medium comprises the following steps: B1) inoculating the Bacillus cereus according to claim 1 into a seed culture medium for seed culture to obtain a seed solution; B2) inoculating the seed solution into a fermentation medium at an inoculum volume ratio of 10% for fermentation culture; The fermentation medium is composed of: 4-6 g / L soybean meal powder, 8-12 g / L corn powder, 4-6 g / L sodium chloride, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L manganese sulfate, 1.0-3.0 mL / L defoaming agent, and the balance is water.

9. The method according to claim 8, characterized in that The fermentation culture temperature is 33-37° C., the rotation speed is 280-320 rpm, the pressure is 0.08-0.12 MPa, the ventilation volume is 2.8-3.2 L / min, and the fermentation time is 24-96 h.

10. The method according to claim 8 or 9, characterized in that: After 16 hours of fermentation, feeding was carried out at a flow rate of 8 mL / h. The composition of the feeding medium was: 18-22 g / L soybean meal, 13-17 g / L corn meal, 8-12 g / L sodium chloride, 5-7 g / L potassium dihydrogen phosphate, 5-7 g / L manganese sulfate, and the balance was water.

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