Cold-tolerant brevibacterium TRM86011 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a brevibacterium coldtolerant strain TRM86011 and application of the brevibacterium coldtolerant strain TRM86011. The invention provides a strain of cold-resistant brevibacterium which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 2023038. The strain has the ability to grow in a saline-alkali environment, and can also produce siderophore, IAA and other agricultural growth-promoting beneficial components. Through verification, the brevibacterium coldtolerant can promote growth of wheat, corn, cotton and tomatoes in a salt stress environment, and plant growth limitation caused by salt stress is reduced. On the basis, the brevibacterium coldtolerant has a good application prospect in preparation of microbial fertilizers or microbial agents suitable for saline-alkali areas and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and discloses a cold-resistant short bacillus strain TRM86011 and its applications. Background Technology

[0002] Plant growth-promoting bacteria (PGPRs), as microorganisms that promote plant growth through mechanisms such as nitrogen fixation, phosphorus solubilization, and synthesis of plant hormones (e.g., IAA), have attracted widespread attention. Especially in adverse environments such as saline-alkali lands, crop growth is severely inhibited, and traditional agricultural measures are insufficient to effectively improve soil conditions. Therefore, screening and developing salt-tolerant and highly efficient PGPRs is crucial. These bacteria directly promote plant growth through nitrogen fixation, phosphorus and potassium solubilization, and the secretion of plant hormones (e.g., IAA) and ACC deaminase. Simultaneously, they alleviate salt-alkali stress by synthesizing osmotic regulators (e.g., proline and betaine), antioxidant enzymes (e.g., SOD), and extracellular polysaccharides (EPS), improving the rhizosphere microenvironment, promoting root development, enhancing nutrient absorption, and increasing plant tolerance to adverse conditions. Salt-tolerant strains, in particular, can colonize and exert their growth-promoting effects in high-salt, high-pH environments, providing more solutions for saline-alkali land agriculture and ecological restoration. Summary of the Invention

[0003] To overcome the aforementioned problems in the prior art, this invention provides a cold-resistant short bacillus strain TRM86011 and its applications. Compared with the prior art, the advantage of this invention is that it provides a cold-resistant short bacillus strain with growth-promoting properties such as adaptability to saline-alkali environments, synthesis of IAA, and secretion of siderophores (or ferritins).

[0004] In the technical solution provided by this invention, "TRM86011", "psychrothermic short bacillus TRM86011", or "psychrothermic short bacillus" all refer to the psychrothermic short bacillus provided by this invention. Peribacillus frigoritolerans It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2023038.

[0005] On one hand, this invention relates to a cold-resistant short bacillus, the preservation information of which is as follows: Strain name: Brevibacterium frigoritolerans TRM86011; Classification and nomenclature: Psychrothermic short bacilli Brevibacterium frigoritolerans (now called) Peribacillus frigoritolerans ); Preservation date: January 6, 2023; Depository: China Center for Type Culture Collection; Accession number: CCTCC NO: M 2023038.

[0006] On the other hand, the present invention relates to a microbial inoculant, wherein the microbial inoculant is the supernatant, fermentation product, filtrate or extract of the aforementioned psychrophilic short bacilli and / or their cultures.

[0007] On the other hand, the present invention relates to the application of the aforementioned cold-resistant short bacilli or the aforementioned microbial agents in plant growth promotion.

[0008] Furthermore, in the applications provided by this invention, the plant growth promotion includes promoting plant growth in salt stress environments, alkali stress environments, or saline-alkali environments.

[0009] On the other hand, the present invention relates to a microbial fertilizer, wherein the active ingredients of the microbial fertilizer include the supernatant, fermentation product, filtrate or extract of the aforementioned psychrophilic short bacilli and / or their cultures.

[0010] On the other hand, the present invention relates to a method for promoting plant growth under adverse conditions, comprising: applying the cold-resistant short bacillus or the microbial fertilizer to the plants and / or their planting site; The adversity is a salt stress environment, an alkaline stress environment, or a saline-alkali environment.

[0011] Furthermore, in the method for promoting plant growth under adversity provided by the present invention, the psychrophilic short bacillus has the ability to grow under said adversity, and promotes plant growth under said adversity by secreting IAA, siderophores and extracellular polysaccharides, thereby helping the plant resist the stress of said adversity.

[0012] On the other hand, the present invention relates to a method for increasing the content of plant growth-promoting substances in soil, comprising: applying the cold-resistant short bacillus or the microbial fertilizer to the soil.

[0013] Furthermore, in the method for increasing the content of plant growth-promoting substances in soil provided by the present invention, the plant growth-promoting substances include at least one of IAA, siderophores, and extracellular polysaccharides.

[0014] Furthermore, in the method for increasing the content of plant growth-promoting substances in soil provided by the present invention, the soil includes one of saline soil, alkaline soil, or saline-alkali soil.

[0015] Combinations of any of the psychrophilic short bacilli or any active variants thereof provided herein can be formulated into pastes, wettable powders, lumps, dust, granules, slurries, dry powders, aqueous or oil-based liquid products, etc. Such formulations will contain the psychrophilic short bacilli or active variants thereof provided herein and / or compositions derived therefrom, as well as carriers and other reagents. The formulations can be used in a variety of methods disclosed elsewhere herein.

[0016] The various compositions and formulations disclosed herein may comprise combinations of psychrophilic short bacilli or their active variants; and / or may comprise a certain amount of a composition derived from any of the psychrophilic short bacilli or any of their active variants. Such amounts may include at least about 10 4 CFU / mL to approximately 10 11 CFU / mL, at least approximately 10 5 CFU / mL to approximately 10 11 CFU / mL, approximately 10 5 CFU / mL to approximately 10 10 CFU / mL, approximately 10 5 CFU / mL to approximately 10 12 CFU / mL, approximately 10 5 CFU / mL to approximately 10 6 CFU / mL, approximately 10 6 CFU / mL to approximately 10 7 CFU / mL, approximately 10 7 CFU / mL to approximately 10 8 CFU / mL, approximately 10 8 CFU / mL to approximately 10 9 CFU / mL, approximately 10 9 CFU / mL to approximately 10 10 CFU / mL, approximately 10 10 CFU / mL to approximately 10 11 CFU / mL or approximately 10 11 CFU / mL to approximately 10 12 The strain concentration is CFU / mL. In other embodiments, the strain concentration includes at least approximately 10. 4 CFU / mL, at least approximately 10 5 CFU / mL, at least approximately 10 6 CFU / mL, at least approximately 10 7 CFU / mL, at least approximately 10 8 CFU / mL, at least approximately 10 9 CFU / mL, at least approximately 10 10 CFU / mL, at least approximately 10 11 CFU / mL, at least approximately 10 12 CFU / mL. The strain at the above concentration can be formed in any type of formulation for any purpose, including, for example, liquid formulations, wettable powders, sprayed dry formulations, pastes, wettable granules, or freeze-dried formulations.

[0017] As used herein, "supernatant" refers to the liquid remaining after psychrophilic short bacilli have been grown in a liquid culture medium or harvested from a solid culture medium into another liquid and removed by centrifugation, filtration, sedimentation, or other means known in the art. In some embodiments, the supernatant may be diluted with another substance, such as water, a buffer, fresh culture medium, and / or a formulation. The diluted supernatant is still considered to be the supernatant of this invention.

[0018] As used herein, "filtrate" refers to the liquid from a fermentation culture of psychrophilic short bacilli in a liquid medium that passes through a membrane. The filtrate may contain a concentrated amount of the active compounds or metabolites compared to the concentration of the active compounds or metabolites in the fermentation culture or supernatant.

[0019] As used herein, "extract" refers to a liquid substance separated from the psychrophilic short-lived bacteria by centrifugation, filtration, evaporation, or other methods known in the art, either by means of a solvent (e.g., water, detergent, buffer, and / or organic solvent) or directly from a psychrophilic short-lived bacteria fermentation culture, or by such means. The extract may contain a concentrated or diluted amount of the active compound or metabolite compared to the concentration in the psychrophilic short-lived bacteria fermentation culture prior to extraction. Alternatively, the filtrate or extract may then be diluted with another composition, such as water, buffer, fresh culture medium, and / or formulation. Such diluted filtrate or extract is still considered the filtrate and extract of the present invention.

[0020] As used herein, “metabolite” or “metabolic product” refers to a compound, substance, or byproduct produced by the fermentation of psychrophilic short-lived bacteria. An effective compound or metabolite is a compound present in the supernatant, fermentation culture containing psychrophilic short-lived bacteria, or in the psychrophilic short-lived bacteria, which, when applied in an effective amount to the target plant or the space in which the target plant is located, can improve any target agronomic trait of the plant.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages.

[0022] This invention provides a cold-resistant short bacillus strain, its strain or modified strain, its active variants and / or compositions derived therefrom, which can be used with any plant species or its habitat to promote plant growth and / or enhance plant resistance to stress. Specifically, the cold-resistant short bacillus TRM86011 can grow in an environment with a NaCl concentration of 1-5% (w / v) or in a pH range of 7.0-12.0. Furthermore, the cold-resistant short bacillus TRM86011 can secrete IAA, siderophores and extracellular polysaccharides, with an IAA secretion capacity of 29.23 mg / L under L-tryptophan induction. Experiments have confirmed that the cold-resistant short bacillus TRM86011 can significantly promote the growth of wheat, corn, cotton and tomato under salt stress, and significantly improve or enhance plant height, root length, and fresh weight of stems and leaves. Based on the characteristics of the psychrophilic short bacillus TRM86011, which produces IAA, heptaphilin, extracellular polysaccharides, and is tolerant to saline-alkali conditions, this strain provides a valuable resource for the preparation of microbial fertilizers or inoculants, mitigating the environmental harm caused by excessive application of chemical fertilizers. Furthermore, it provides a safe and effective microbial resource for microbial fertilizers or inoculants suitable for saline-alkali areas. This strain has broad application prospects in saline-alkali agriculture, ecological restoration, and stress-affected crop breeding, and is expected to become an important tool for increasing crop yields and improving soil quality. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This shows the colony growth of the cold-resistant short bacillus TRM86011 on LB medium.

[0025] Figure 2 This is the phylogenetic tree of the cold-resistant short bacillus TRM86011.

[0026] Figure 3 This is a qualitative test to determine the ability of the cold-resistant short bacillus TRM86011 to secrete IAA.

[0027] Figure 4 It is the IAA standard curve.

[0028] Figure 5 This is a qualitative test to determine the ability of the cold-resistant short bacillus TRM86011 to secrete siderophores.

[0029] Figure 6 This shows the growth status of wheat seedlings in each group.

[0030] Figure 7 This is a bar chart showing the plant height, root length, fresh weight of stems and leaves, and fresh weight of roots of wheat seedlings in each group. In the chart, A represents the plant height and root length of wheat seedlings in each group, and B represents the fresh weight of stems and leaves and the fresh weight of roots of wheat seedlings in each group.

[0031] Figure 8 This shows the growth status of corn seedlings in each group.

[0032] Figure 9 This is a bar chart showing the plant height, root length, fresh weight of stems and leaves, and fresh weight of roots of corn seedlings in each group. In the chart, A represents the plant height and root length of corn seedlings in each group, and B represents the fresh weight of stems and leaves and the fresh weight of roots of corn seedlings in each group.

[0033] Figure 10 This shows the growth status of cotton seedlings in each group.

[0034] Figure 11 This is a bar chart showing the plant height, root length, stem and leaf fresh weight, and root fresh weight of cotton seedlings in each group. In the chart, A represents the plant height and root length of cotton seedlings in each group, and B represents the stem and leaf fresh weight and root fresh weight of cotton seedlings in each group.

[0035] Figure 12 This shows the growth status of tomato seedlings in each group.

[0036] Figure 13 This is a bar chart showing the plant height, root length, stem and leaf fresh weight, and root fresh weight of tomato seedlings in each group. In the chart, A represents the plant height and root length of tomato seedlings in each group, and B represents the stem and leaf fresh weight and root fresh weight of tomato seedlings in each group.

[0037] Explanation of figure labels: TRM86011 represents plants treated with a suspension of cold-resistant brevicorba (negative control); CK represents plants treated with sterile water (blank control); TRM86011-200 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 200 mmol / L compound saline-alkali solution (experimental group); CK-200 represents plants treated with sterile water and 200 mmol / L compound saline-alkali solution (positive control); TRM86011-400 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 400 mmol / L compound saline-alkali solution (experimental group); CK-400 represents plants treated with sterile water and 400 mmol / L compound saline-alkali solution (positive control). Detailed Implementation

[0038] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, % and ‰ in the following embodiments refer to mass percentage and mass thousandths of content.

[0039] Example 1 This embodiment describes the isolation, purification, and colony growth morphology of the cold-resistant short bacillus TRM86011.

[0040] LB solid medium: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, 1000mL H2O, pH=7.0~7.2, sterilized at 121℃ for 30min.

[0041] LB liquid medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1000mL H2O, pH=7.0~7.2, sterilized at 121℃ for 30min.

[0042] IAA fermentation medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1g L-tryptophan, 1000mL H2O, pH=7.0~7.2, sterilized at 121℃ for 30min.

[0043] CAS medium: C6H 12 O610 g, Ca3(PO4)25 g, MgCl25 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, agar powder (for solid culture medium) 16 g, pH 7.0±0.2, sterilize at 121℃ for 30 min; Strain source: The cold-resistant short bacillus TRM86011 was isolated from the Pamir Plateau meadow soil in Kashgar region, Xinjiang Uygur Autonomous Region.

[0044] Separation and purification method: Weigh 1g of the collected meadow soil sample and dissolve it in 9 mL of sterile water. Prepare a series of sample dilutions with sterile water, selecting a dilution gradient of 10. -4 and 10 -5 The diluted solution was plated onto solid LB medium containing NaCl and incubated in a 30°C incubator. Single colonies of different shapes and colors were picked and streaked multiple times. After 2-3 generations of subculture, the purified, cold-resistant short bacillus strain TRM86011 with consistent growth morphology was obtained.

[0045] Example 2 This example describes the classification and identification of the cold-resistant short bacillus TRM86011.

[0046] 1. Biological identification After culturing the psychrophilic short bacillus TRM86011 on LB agar plates at 35°C for 2 days, the initial growth stage showed semi-transparent or opaque white specks resembling water; later, the colonies became round, milky white, semi-transparent, with neat edges and extracellular polysaccharide aggregation on the cell surface. The colony growth pattern is as follows: Figure 1 As shown.

[0047] 2. System classification and identification Colony PCR was used for identification. Cold-resistant short bacilli TRM86011 were picked from LB agar plates using an inoculation loop. The bacterial block was diluted with 15 μL of sterile water, centrifuged at 12000 rpm for 5 min, the supernatant was discarded, and the bacterial suspension was resuspended in 15 μL of sterile water. Centrifugation was repeated once, and the suspension was incubated at 95℃ for 5 min, followed by placement on ice for 15–20 min. The treated bacterial suspension was used as a template for PCR identification. The target gene fragment was amplified using universal bacterial primers 27F and 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGCTACCTTGTTACGACTT-3'). The PCR reaction system was as follows: 1 μL each of 27F and 1492R primers (10 mmol / L), 3 μL of psychrophilic short bacillus TRM86011 bacterial suspension, 12.5 μL of 2×PCR mix, and 7.5 μL of sterile water, totaling 25 μL; a control system was used without bacterial suspension. The amplification program was: 95℃ for 3 min; 95℃ for 1 min, 56℃ for 1 min, 72℃ for 1 min, 25 cycles; 72℃ for 10 min. The PCR products were submitted for sequencing (Sangon Sequencing (Xi'an) Co., Ltd.), and the 16S rDNA of psychrophilic short bacillus TRM86011 was detected as shown in SEQ ID NO.1.

[0048] SEQ ID NO.1:

[0049] The obtained SEQ ID NO.1 was compared with the nucleic acid data in Ezbiocloud (https: / / www.ezbiocloud.net / identify). The results showed that the psychrophilic short bacillus TRM86011 is homologous to... Peribacillus frigoritolerans The homology of strain DSM 8801 was 99.37%. A phylogenetic tree was constructed using MOLECULAR EVOLUTIONARYGENETIC ANALYSIS software (MEGA 7.0). Figure 2 As shown, the psychrophilic short bacillus TRM86011 and Peribacillus frigoritolerans The sequence of DSM 8801 forms a stable evolutionary branch, therefore it was named a cold-resistant short bacterium. Peribacillus frigoritolerans )TRM86011.

[0050] Example 3 This embodiment describes the evaluation of the salt (NaCl) and alkali (NaOH) tolerance of the cold-resistant short bacillus TRM86011.

[0051] 1. Salt tolerance evaluation Single colonies of the psychrophilic short bacillus TRM86011 were transferred to LB agar plates and incubated at 35°C for 24 h. Single colonies were then transferred to LB agar plates containing 1%, 3%, 5%, 7%, 9%, and 11% (w / v) NaCl, and incubated at 35°C for 48 h. The results showed that the psychrophilic short bacillus TRM86011 could grow in LB medium with NaCl concentrations ranging from 1% to 5% (w / v).

[0052] 2. Alkali resistance evaluation Single colonies of the psychrophilic short bacillus TRM86011 were transferred to LB agar plates and incubated at 35°C for 24 h. The pH of the LB liquid medium was adjusted with 1M NaOH or 1M HCl, and single colonies were then transferred to LB agar plates with pH gradients of 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively, and incubated at 35°C for 48 h. The results showed that the psychrophilic short bacillus TRM86011 could grow within a pH range of 7.0–12.0.

[0053] Example 4 This embodiment describes the qualitative and quantitative determination of the ability of the cold-resistant short bacillus TRM86011 to secrete IAA.

[0054] Salkowski colorimetric reagent: Dissolve 12g FeCl3 in 300mL deionized water, slowly add 429.7mL concentrated H2SO4 (concentration ≥98%), cool and then bring the volume to 1L.

[0055] To prepare an IAA standard curve: Prepare 50 mL of a 100 µg / mL standard IAA solution, and dilute it to concentrations of 6.25, 12.5, 25, and 50 µg / mL, respectively. Mix the IAA solution with Salksowski colorimetric solution at a 1:1 volume ratio and react at room temperature in the dark for 30 min. Use a mixture of distilled water and an equal volume of Salksowski colorimetric solution as a control. Then, determine the OD value (OD) at 530 nm for each concentration using ultraviolet spectrophotometry. 530 (And plotted IAA concentration on the x-axis, OD...) 530 Plotting the vertical axis yields the IAA standard curve.

[0056] Quantitative determination of IAA concentration in bacterial culture: A single colony of the psychrophilic short bacillus TRM86011 was inoculated into LB liquid medium and cultured on a shaker at 30℃ and 150 rpm for 12 h as the seed culture. 100 µL of the seed culture was added to IAA fermentation medium and cultured on a shaker at 30℃ and 180 rpm for 3 days. The culture was then centrifuged at 12000 rpm for 2 min. 500 µL of the supernatant was mixed with an equal volume of Salksowski colorimetric solution and incubated at room temperature in the dark for 30 min. The OD value was then measured. 530 A mixture of fermentation medium and an equal volume of Salksowski colorimetric solution was used as a control group. The results were based on OD... 530 The corresponding IAA content is calculated using the standard curve.

[0057] Figure 3 This is a qualitative test to determine the IAA secretion capacity of the cold-resistant short bacillus TRM86011; Figure 4 This is the standard curve for quantitative determination of IAA. (From...) Figure 3 It can be seen that the psychrophilic short bacillus TRM86011 has the ability to produce IAA, and based on the absorbance value and Figure 4 Based on the standard curve, the IAA secretion capacity of the cold-resistant short bacillus TRM8601 under L-tryptophan induction was calculated to be 29.23 mg / L.

[0058] Example 5 This embodiment describes the qualitative determination of the ability of the cold-resistant short bacillus TRM86011 to secrete ferophiles.

[0059] A single colony of the cold-resistant short bacillus TRM86011 was picked and inoculated into LB liquid medium. The culture was incubated at 30°C and 150 rpm for 12 hours using a shaker as the seed culture. 10 μL of the bacterial suspension (OD200) was then pipetted onto the culture medium. 600 =1.5) Spot-connect the strain to the CAS function screening plate and observe whether the strain can produce an iron-loving zone on the CAS plate.

[0060] Figure 5 This is a qualitative test to determine the ability of the cold-resistant short bacillus TRM86011 to secrete iron. Based on the iron-loving rings produced by the strain on CAS medium plates, it can be determined that the bacterium has the growth-promoting characteristic of secreting iron.

[0061] Example 6 This embodiment describes the effect of the cold-resistant short bacillus TRM86011 on wheat growth under salt stress.

[0062] Preparation of sterile wheat seedlings: Wheat seeds were soaked in 5% sodium hypochlorite for 3 minutes, rinsed 3 times with sterile water, then soaked in 70% ethanol for 5 minutes, rinsed 5 times with sterile water, germinated in water at 25℃ for 1 day, and then transferred to a soil containing nutrient soil:vermiculite in a volume ratio of 2:1 (total volume 180cm³). 3 It sprouted in the flowerpot.

[0063] Preparation of bacterial suspension: Same as in Example 5.

[0064] Inoculation: The prepared bacterial suspension was inoculated into the roots of aseptically cultured wheat plants with leaves 4-5 cm in length, 5 mL per pot. The plants were then allowed to grow at 25℃ for 30 days. During this period, a compound saline solution (NaCl, Na2SO4, NaHCO3 and Na2CO3 in a molar ratio of 1:9:9:1) was added to each pot until the concentration of the compound saline solution in the pot reached 200 mmol / L or 400 mmol / L, pH=8.0-8.5. The growth of wheat was observed. After 30 days of growth, the plant height, root length, fresh weight of stems and leaves, and fresh weight of roots were measured to analyze whether the cold-resistant short bacillus TRM86011 promoted the growth of wheat under NaCl stress. Among them, TRM86011 represents plants treated with a suspension of cold-resistant brevicorba (negative control); CK represents plants treated with sterile water (blank control); TRM86011-200 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 200 mmol / L compound saline-alkali solution (experimental group); CK-200 represents plants treated with sterile water and 200 mmol / L compound saline-alkali solution (positive control); TRM86011-400 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 400 mmol / L compound saline-alkali solution (experimental group); CK-400 represents plants treated with sterile water and 400 mmol / L compound saline-alkali solution (positive control).

[0065] Figure 6 The data shows the growth of wheat seedlings in each group, and it can be seen that there are differences in the growth of wheat plants between the experimental group and the control group. Figure 7 The plant height and root length of wheat seedlings in each group ( Figure 7 A) Fresh weight of stems and leaves and fresh weight of roots ( Figure 7 The bar chart in section B). Figure 7It can be seen that, through the analysis of wheat plant biomass, the height, fresh weight of stems and leaves, and fresh weight of roots of the experimental group were all significantly higher than those of the control group. p (≤0.05), the root fresh weight of the treatment groups was higher than that of the control group.

[0066] Example 7 This embodiment describes the effect of the cold-resistant short bacillus TRM86011 on the growth promotion of maize under salt stress conditions.

[0067] Preparation of sterile corn seedlings: Soak corn seeds in 2% sodium hypochlorite for 3 minutes, rinse 3 times with sterile water, then soak in 70% ethanol for 5 minutes, rinse 5 times with sterile water, germinate in water at 25℃ for 3 days, and then transfer to a soil containing nutrient soil:vermiculite in a 2:1 ratio (total volume 180cm³). 3 It sprouted in the flowerpot.

[0068] Preparation of bacterial suspension: Same as in Example 5.

[0069] Inoculation: The prepared bacterial suspension was inoculated into the roots of aseptically cultured maize plants at the two-leaf-one-heart stage (plant height 4-5cm), with 3mL inoculated per pot. The plants were then allowed to continue growing at 25℃ for 30 days. During this period, a compound saline-alkali solution (molar ratio of NaCl, Na2SO4, NaHCO3, and Na2CO3 of 1:9:9:1) was added to each pot until the concentration of the compound saline-alkali solution in the pot reached 200mmol / L or 400mmol / L, pH=8.0-8.5. The growth status of the maize was observed. After 30 days of growth, the plant height, root length, fresh weight of stems and leaves, and fresh weight of roots were measured to analyze whether the cold-resistant short bacillus TRM86011 had a promoting effect on the growth of maize under NaCl stress. Among them, TRM86011 represents plants treated with a suspension of cold-resistant brevicorba (negative control); CK represents plants treated with sterile water (blank control); TRM86011-200 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 200 mmol / L compound saline-alkali solution (experimental group); CK-200 represents plants treated with sterile water and 200 mmol / L compound saline-alkali solution (positive control); TRM86011-400 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 400 mmol / L compound saline-alkali solution (experimental group); CK-400 represents plants treated with sterile water and 400 mmol / L compound saline-alkali solution (positive control).

[0070] Figure 8 This shows the growth status of the corn seedlings in each group. Figure 9 The plant height and root length of wheat seedlings in each group ( Figure 9 A) Fresh weight of stems and leaves and fresh weight of roots ( Figure 9 The bar chart in section B). Figure 9It can be seen that there are differences in the growth of maize plants between the experimental group and the control group. Through the analysis of maize plant biomass, the plant height and fresh weight of stems and leaves of maize plants in the experimental group are significantly higher than those in the control group. p (≤0.05), the root fresh weight of the treatment groups was higher than that of the control group.

[0071] Example 8 This example describes the effect of the cold-resistant short bacillus TRM86011 on cotton growth under salt stress.

[0072] Preparation of sterile corn seedlings: Cotton seeds were soaked in 2% sodium hypochlorite for 3 minutes, rinsed 3 times with sterile water, then soaked in 70% ethanol for 5 minutes, rinsed 5 times with sterile water, and germinated in water at 25℃ for 3 days. The seeds were then transferred to a soil containing nutrient soil and vermiculite in a 2:1 ratio (total volume 180 cm³). 3 It sprouted in the flowerpot.

[0073] Preparation of bacterial suspension: Same as in Example 5.

[0074] Inoculation: The prepared bacterial suspension was inoculated into the roots of aseptically cultured cotton plants at the 4-leaf stage (plant height 4-5 cm), with 3 mL inoculated per pot. The plants were then allowed to continue growing at 25℃ for 30 days. During this period, a compound saline-alkali solution (molar ratio of NaCl, Na2SO4, NaHCO3, and Na2CO3 of 1:9:9:1) was added to each pot until the concentration of the compound saline-alkali solution in the pot reached 200 mmol / L or 400 mmol / L, pH=8.0-8.5. The growth status of the cotton plants was observed. After 30 days of growth, the plant height, root length, fresh weight of stems and leaves, and fresh weight of roots of the cotton plants were measured to analyze whether the cold-resistant short bacillus TRM86011 had a promoting effect on the growth of cotton under NaCl stress. Among them, TRM86011 represents plants treated with a suspension of cold-resistant short bacilli (negative control); CK represents plants treated with sterile water (blank control); TRM86011-200 represents plants treated with a suspension of cold-resistant short bacilli TRM86011 and 200 mmol / L compound saline-alkali solution (experimental group); and CK-200 represents plants treated with sterile water and 200 mmol / L compound saline-alkali solution (positive control).

[0075] Figure 10 This shows the growth status of cotton seedlings in each group. Figure 11 The plant height and root length of wheat seedlings in each group ( Figure 11 A) Fresh weight of stems and leaves and fresh weight of roots ( Figure 11 The bar chart in section B). Figure 11 It can be seen that there are differences in the growth of cotton plants between the experimental group and the control group. Through the analysis of cotton plant biomass, the plant height, root length and fresh weight of stems and leaves of cotton plants in the experimental group are significantly higher than those in the control group. p (≤0.05), the root fresh weight of the treatment groups was higher than that of the control group.

[0076] Example 9 This embodiment describes the effect of the cold-resistant short bacillus TRM86011 on the growth promotion of tomatoes under salt stress.

[0077] Preparation of sterile corn seedlings: Tomato seeds were soaked in 2% sodium hypochlorite for 3 minutes, rinsed 3 times with sterile water, then soaked in 70% ethanol for 5 minutes, rinsed 5 times with sterile water, and germinated in water at 25℃ for 3 days. The seeds were then transferred to a soil containing nutrient soil and vermiculite in a 2:1 ratio (total volume 180 cm³). 3 It sprouted in the flowerpot.

[0078] Preparation of bacterial suspension: Same as in Example 5.

[0079] Inoculation: The prepared bacterial suspension was inoculated into the roots of aseptically cultured 4-leaf stage (plant height 4-5cm) tomatoes at 3mL per pot. The plants were then allowed to grow at 25℃ for 30 days. During this period, a compound saline-alkali solution (molar ratio of NaCl, Na2SO4, NaHCO3 and Na2CO3 of 1:9:9:1) was added to each pot until the concentration of the compound saline-alkali solution in the pot reached 200mmol / L or 400mmol / L, pH=8.0-8.5. The growth status of the corn was observed. After 30 days of growth, the plant height, root length, fresh weight of stems and leaves, and fresh weight of roots of the tomatoes were measured to analyze whether the cold-resistant short bacillus TRM86011 had a promoting effect on the growth of tomatoes under NaCl stress. Among them, TRM86011 represents plants treated with a suspension of cold-resistant brevicorba (negative control); CK represents plants treated with sterile water (blank control); TRM86011-200 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 200 mmol / L compound saline-alkali solution (experimental group); CK-200 represents plants treated with sterile water and 200 mmol / L compound saline-alkali solution (positive control); TRM86011-400 represents plants treated with a suspension of cold-resistant brevicorba TRM86011 and 400 mmol / L compound saline-alkali solution (experimental group); CK-400 represents plants treated with sterile water and 400 mmol / L compound saline-alkali solution (positive control).

[0080] Figure 12 This shows the growth status of each group of tomato seedlings. Figure 13 The plant height and root length of wheat seedlings in each group ( Figure 12 A) Fresh weight of stems and leaves and fresh weight of roots ( Figure 12 The bar chart in section B). Figure 13 It can be seen that there are differences in the growth of tomato plants between the experimental group and the control group. Through the analysis of tomato plant biomass, the plant height, root length, stem and leaf fresh weight, and root fresh weight of the tomato plants in the experimental group are all significantly higher than those in the control group. p (≤0.05), the root fresh weight of the treatment groups was higher than that of the control group.

[0081] In summary, this invention isolated a cold-resistant short bacillus strain, TRM86011, from meadow soil samples in the Pamir Plateau region of Kashgar, Xinjiang Uygur Autonomous Region. This strain possesses the ability to grow in saline-alkali environments and secretes IAA, siderophores, and extracellular polysaccharides. Verification showed that the cold-resistant short bacillus TRM86011 can promote the growth of wheat, corn, cotton, and tomato under salt stress, helping plants resist salt stress. The cold-resistant short bacillus TRM86011 shows promising application prospects in the preparation of microbial fertilizers and microbial agents suitable for saline-alkali areas.

[0082] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A cold-resistant short bacillus, characterized in that, The psychrogenic short bacillus is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2023038.

2. A microbial inoculant, characterized in that, The microbial agent is the supernatant, fermentation product, filtrate, or extract of the psychrogenic short bacillus and / or its culture as described in claim 1.

3. The application of the cold-resistant short bacillus of claim 1 or the microbial agent of claim 2 in plant growth promotion.

4. The application according to claim 3, characterized in that, The plant growth promotion includes promoting plant growth in salt-stressed, alkali-stressed, or saline-alkali environments.

5. A microbial fertilizer, characterized in that, The active ingredients of the microbial fertilizer include the supernatant, fermentation product, filtrate, or extract of the psychrophilic short bacillus and / or its culture as described in claim 1.

6. A method for promoting plant growth under adverse conditions, characterized in that, include: This enables the cold-resistant short bacillus of claim 1 or the microbial fertilizer of claim 5 to act on plants and / or their planting sites; The adversity is a salt stress environment, an alkaline stress environment, or a saline-alkali environment.

7. The method for promoting plant growth under alkaline stress according to claim 6, characterized in that, The cold-resistant short bacillus has the ability to grow in the aforementioned adverse environment, and promotes plant growth in the adverse environment by secreting IAA, siderophores and extracellular polysaccharides, thus helping the plant resist the stress of the adverse environment.

8. A method for increasing the content of plant growth-promoting substances in soil, characterized in that, Apply the cold-resistant short bacillus of claim 1 or the microbial fertilizer of claim 5 to the soil.

9. The method for increasing the content of plant growth-promoting substances in soil according to claim 8, characterized in that, The plant growth-promoting substances include at least one of IAA, siderophores, and extracellular polysaccharides.

10. The method for increasing the content of plant growth-promoting substances in soil according to claim 8, characterized in that, The soil includes one of saline soil, alkaline soil, or saline-alkali soil.

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