Saline-alkali-tolerant growth-promoting bacillus AH83 and application thereof

Abstract

The invention discloses a bacillus AH83 capable of resisting saline and alkaline and promoting growth and application thereof, and belongs to the technical field of microbiology and biology, the bacillus AH83 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC No: M 20241495, and the bacillus AH83 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC No: M 20241495. The AH83 provided by the invention is a new species of bacillus, can inhibit the growth of pathogenic bacteria of cotton fusarium wilt, and has the growth promoting functions of dissolving potassium, generating indoleacetic acid, cellulase, protease, siderophore and the like; meanwhile, the AH83 can survive in a highly saline-alkaline environment, can promote agronomic characters such as plant height, biomass, leaf area and root development in a corn seedling stage when applied in saline-alkaline soil, can also increase soil nutrients and enzyme activity content, and has a promoting effect on lavender seed germination. The strain is of great significance in improving crop yield and stress resistance and developing novel agricultural microbial agents.

Description

Technical Field

[0001] The present invention relates to the fields of microbiology and biotechnology, and specifically relates to a salt-tolerant and growth-promoting Bacillus AH83 and its application. Background Art

[0002] Saline-alkali stress seriously harms crop growth and poses a threat to global agricultural production, food security and sustainable development. Salinization damages the soil structure, leading to soil degradation, and approximately 10 million hectares of irrigated land are abandoned every year. Microorganisms are the most important components in the soil. Under salt stress, plants can attract functional microorganisms through root exudates to promote growth. Bacteria that are beneficial to plant growth are called plant growth-promoting bacteria, which can promote plant growth and increase crop yields by preventing pathogenic bacteria, improving the absorption and utilization efficiency of mineral nutrients by plants, and producing metabolites beneficial to plant growth. They can also improve the ability of plants to resist biotic and abiotic stresses. Plant endophytes are also one of the environments for the isolation of growth-promoting bacteria, with the advantages of being unaffected by the external environment and having a relatively stable effect. The PGPRs that have been discovered mainly include Azospirillum, Azotobacter, Gluconacetobacter, Pseudomonas, Bacillus, Burkholderia, etc. (Tabassum et al., 2021). Among them, Bacillus has been widely used because it can survive in extreme environments, is ubiquitous in nature, harmless to humans and animals, and non-pathogenic to plants, and has become one of the most deeply studied and widely used plant growth-promoting bacteria in the world.

[0003] There are many types of Bacillus, but the main types used in agriculture and forestry are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium, etc. Research by Ji Yan et al. showed that Bacillus subtilis enhanced the growth of maize by improving its root morphology and stress-resistant enzyme activity, and promoted the dry matter accumulation of maize under saline-alkali conditions; research by Lü Zhengyang et al. showed that Bacillus amyloliquefaciens PT6-1 increased the plant height, total fresh weight, total photosynthetic pigment content, peroxidase activity, and catalase of Pennisetum giganteum under saline-alkali conditions; research by Zhang Yanrui et al. showed that Bacillus pumilus JT8 promoted the germination of alfalfa seeds and the growth of seedlings when the concentration of saline-alkali solution was 100 mmol / L; research by Li Qingqing et al. showed that Bacillus megaterium had strong salt tolerance and phosphorus-solubilizing effects, and could better promote the seed germination of soybeans. At the same time, with the progress of Bacillus research, more and more new Bacillus strains with good biocontrol and growth-promoting effects have been discovered, such as Bacillus vallismortis. In recent years, there have been research reports that Bacillus vallismortis promotes plant growth, but mostly indirectly promotes plant growth through its antagonistic effect on pathogenic bacteria. For example, Xu Lingna et al. reported the antibacterial effect of the volatile substances produced by Bacillus vallismortis 12a on peach brown rot; Liu Zhihui et al. reported the antibacterial effect of Bacillus vallismortis 265ZY1 on dry rot of Solanum tuberosum caused by Fusarium solani; Zhang Guoyi et al. reported the antibacterial effect of Bacillus vallismortis HJ-5 on Verticillium wilt of cotton; Zhang Meng et al. reported the antibacterial effect of Bacillus vallismortis wm005 on various pathogenic bacteria such as Fusarium oxysporum f. sp. niveum, Sclerotinia sclerotiorum, and Rhizoctonia solani of cucumber.

[0004] However, there is a lack of exploration of new Bacillus species resources in saline-alkali environments. Through the screening of functions such as salt-alkali tolerance, nutrient activation, growth promotion, and enzyme production, it is expected to explore new strain resources with strong adaptability to high saline-alkali environments, which can improve saline-alkali soils, regulate the resistance of plants under saline-alkali soil conditions, and promote plant growth. Summary of the Invention

[0005] The purpose of the present invention is to provide a salt-tolerant and growth-promoting Bacillus to solve the problems raised in the above background technology.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A salt-tolerant and growth-promoting Bacillus sp. AH83 was deposited in the China Center for Type Culture Collection with the deposit number CCTCC No: M 20241495, the deposit address being Wuhan University, Wuhan, China, and the deposit date being July 8, 2024.

[0008] Another object of the present invention is to provide a bacterial agent containing the above-mentioned Bacillus AH83.

[0009] Another object of the present invention is to provide an application of the above-mentioned Bacillus AH83 or the above-mentioned bacterial agent in inhibiting the pathogen of cotton wilt disease.

[0010] Preferably, the pathogen of cotton wilt disease includes Fusarium oxysporum.

[0011] Another object of the present invention is to provide an application of the above-mentioned Bacillus AH83 or the above-mentioned bacterial agent in promoting plant growth.

[0012] Preferably, the plants include lavender and corn, and the plant growth promotion is for plants in ordinary soil or saline-alkali soil.

[0013] Another object of the present invention is to provide an application of the above-mentioned Bacillus AH83 or the above-mentioned bacterial agent in improving the soil structure of saline-alkali land or preparing a soil conditioner.

[0014] Another object of the present invention is to provide an application of the above-mentioned Bacillus AH83 or the above-mentioned bacterial agent in producing growth-promoting substances, and the growth-promoting substances include IAA, siderophore, protease and cellulase.

[0015] A salt-tolerant and growth-promoting Bacillus provided by the present invention can inhibit the growth of pathogens of cotton wilt disease such as Fusarium oxysporum, and has growth-promoting functions such as potassium solubilization, production of indole acetic acid, cellulase, protease, siderophore, etc.; at the same time, the Bacillus AH83 can survive in a highly saline-alkali environment, can promote agronomic traits such as plant height, biomass, leaf area and root development of corn seedlings in saline-alkali soil, can also increase soil nutrient and enzyme activity content, and has a promoting effect on the germination of lavender seeds. Description of the Drawings

[0016] Figure 1 It is a comparison chart of the inhibition rates of some strains against Fusarium oxysporum in the examples of the present invention.

[0017] Figure 2 It is the colony morphology of strain AH83 and the antagonistic effect diagram against Fusarium oxysporum in the examples of the present invention; among them, the left figure is the colony morphology of AH83 on LB medium, and the right figure is the antagonistic effect diagram of AH83 against Fusarium oxysporum.

[0018] Figure 3This is the phylogenetic tree of the 16S rRNA gene of strain AH83 in the embodiments of the present invention.

[0019] Figure 4 This is the phylogenetic tree based on the whole genome of strain AH83 in the embodiments of the present invention.

[0020] Figure 5 This is the graph showing the results of the salt and alkali tolerance ability of strain AH83 in the embodiments of the present invention.

[0021] Figure 6 This is the growth situation of strain AH83 in the medium with pH 10.0 and different salt concentrations in the embodiments of the present invention.

[0022] Figure 7 This is the graph showing the test results of the abilities of strain AH83 in the embodiments of the present invention to release potassium, produce siderophores, protease and cellulase; wherein, Figure a shows the growth situation of AH83 on the potassium release detection medium, Figures b and c respectively show the situations of AH83 producing protease and cellulase, and Figure d shows the transparent circle of siderophores produced by AH83.

[0023] Figure 8 This is the graph showing the growth promotion effect of strain AH83 on the germination and seedling growth of lavender seeds in the embodiments of the present invention; wherein, a is the photo of the germination of lavender seeds in the treatment group soaked with the AH83 bacterial suspension and the control group, b is the results of the seed germination rate and germination potential, c is the results of the stem length and root length of the seedlings, and d is the results of the fresh weight and dry weight of the seedlings (the data in the figure are the averages of 10 seedlings). Different letters indicate that each treatment has significant differences at the P<0.05 level.

[0024] Figure 9 This is the graph showing the growth promotion effect of strain AH83 on corn in the saline-alkali soil (original saline-alkali soil) of Xinjiang in the embodiments of the present invention; wherein, a is the photo of the potted plants in the treatment group with the AH83 fermentation broth (AH83-0) and the control group (CK-0), b is the photo of the roots of the plants in the AH83 treatment group and the control group, and c is the scanned image of the root morphology of the plants in the AH83 treatment group and the control group.

[0025] Figure 10 This is the graph showing the growth promotion effect of strain AH83 on corn in the non-saline-alkali soil (artificial saline-alkali soil) of Xinjiang with artificially added salts in the embodiments of the present invention; wherein, a is the photo of the plants in the treatment group with the AH83 fermentation broth (AH83-2) and the control group (CK-2), b is the photo of the roots of the plants in the AH83 treatment group and the control group, and c is the scanned image of the root morphology of the plants in the AH83 treatment group and the control group. Detailed implementation manners

[0026] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0027] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0028] The following embodiments are implementation cases of the technical solutions of the present invention in actual applications, but are not limited thereto. The reagents and experimental equipment involved are all commercially available products.

[0029] Example 1: This example provides a method for isolating, screening, and identifying Bacillus vallismortis with salt and alkali tolerance and growth promotion, which is as follows:

[0030] S1. Isolation and screening of strain AH83: In 2023, cotton plants were collected from Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Take 10 g of cotton stem samples, wash their surfaces with distilled water, soak them in 75% ethanol disinfectant for 5 min, soak them in 3% sodium hypochlorite for 5 min, and soak them in 75% ethanol for 5 min. Finally, rinse with sterile water 3 times, and spread the sterile water of the last rinse on an LB plate (5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, 15 g / L agar) as a blank control for the surface disinfection effect. Place the disinfected stem samples in a sterile mortar, add an appropriate amount of sterile normal saline, and grind them into a homogeneous slurry. Take 1 mL of the slurry and dilute it serially at 10 -1 、10 -2 、10 -3 、10 -4 , and spread them on plates, and incubate them at 28 °C for 2 d. If no colonies appear on the isolation medium plates of the control treatment, it indicates that the surface of the plant material is thoroughly disinfected and the isolated bacteria are endophytic bacteria. Use a sterile toothpick to pick different-shaped bacterial single colonies, streak and purify them, and then store them for later use. A total of 115 strains of bacteria were isolated.

[0031] The biocontrol effect of the isolated endophytic bacteria was detected by the confrontation culture method. First, activate Fusarium oxysporum on a PDA plate, use a 5 mm punch to punch out pathogen discs, inoculate them in the center of the PDA plate, and inoculate the 115 isolated endophytic bacteria 2.5 cm away from the discs respectively. Incubate at 25 °C for 4 - 7 days, measure the colony diameters of Fusarium oxysporum with and without inoculation of endophytic bacteria respectively, and calculate the inhibition rate according to the following formula:

[0032]

[0033] Among 115 strains, 49 strains with inhibitory effects on Fusarium oxysporum were initially screened out. Then, after re-screening, 5 strains with inhibition rates greater than 40% were selected, numbered AH83, AH21, AH74, BH32, and AH15 respectively (as Figure 1 shown). Among them, strain AH83 had the highest inhibition rate against Fusarium oxysporum (as Figure 1 and Figure 2 shown). Therefore, AH83 was selected for subsequent experiments. At the same time, this strain AH83 was deposited in the China Center for Type Culture Collection, with the deposit number CCTCC No: M20241495, the deposit address being Wuhan University, China, and the deposit time being July 8, 2024; Figure 2 Figure Figure 2 10 shows the colony morphology of strain AH83 and the antagonistic effect diagram against Fusarium oxysporum. In

[0034] Figure

[0034] 11, the left figure shows the colony morphology of AH83 on LB medium, and the right figure shows the antagonistic effect diagram of AH83 against Fusarium oxysporum.

[0034] S2. Identification of strain AH83: Based on the molecular biology detection results such as the morphological characteristics, physiological and biochemical properties, 16S rRNA gene, and genomic characteristics of strain AH83, it was identified as a new species of the genus Bacillus. Specifically as follows:

[0035] (1) Identification of morphological characteristics and physiological and biochemical characteristics: The morphological analysis results showed that the colony of strain AH83 was round, with a rough and wrinkled surface, positive for Gram staining, rod-shaped cells, spore-forming, negative for oxidase, and positive for catalase, indicating that strain AH83 belongs to the genus Bacillus.

[0036] (2) Phylogenetic analysis of 16S rRNA gene: The genomic DNA of strain AH83 was extracted using the DNA kit of Beijing Polymer Beauty Biotechnology Co., Ltd. The 16S rRNA gene was amplified using the existing well-known universal primers 27F (5’-AGAG TTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). The PCR reaction system and reaction program are shown in Table 1 and Table 2 respectively. After the PCR products were verified by agarose gel electrophoresis, they were sequenced and verified by Beijing Tsingke Biotechnology Co., Ltd., and the sequencing results were spliced using DNAMAN software. By comparing with the GenBank database using the NCBI BLAST program, it was found that the 16S rRNA sequence of strain AH83 had the highest similarity with the strains of the genus Bacillus. The phylogenetic tree constructed using MEGA 11.0 software also showed that strain AH83 belongs to the genus Bacillus (specifically as Figure 3 shown).

[0037] Table 1 PCR reaction system for 16S rRNA gene

[0038]

[0039] Table 2 PCR reaction conditions for 16S rRNA gene

[0040]

[0041] (3) Genome characteristics and phylogenetic analysis of strain AH83: After the total genomic DNA of strain AH83 extracted was qualified by purity detection, it was entrusted to Shanghai Sangon Biotech Co., Ltd. for genomic sequencing analysis, and the sequencing was completed by combining the PacBio RSII and Illumina Solexa sequencing platforms. The sequencing results showed that the genome size of strain AH83 was 4.04 Mb, the G+C content was 44.1 mol%, a total of 3,977 coding genes were predicted, and there were 10 rRNAs and 78 tRNAs. The identification results of the TYGS platform (https: / / tygs.dsmz.de / ) showed that AH83 was a potential new species. The phylogenetic tree constructed based on the whole genome by TYGS showed that strain AH83 clustered with Bacillus vallismortis DV1-F-3 T ), Bacillus inaquosorum KCTC 13429 T ), Bacillus spizizenii TU-B-10 T ), Bacillus rugosus SPB7 T ) in a branch and had a relatively close genetic relationship (as shown in Figure 3 ).

[0042] (4) Genome similarity analysis of strain AH83: The average nucleotide identity (ANI) between strain AH83 and related strains was calculated using JSpeciesWS (https: / / jspecies.ribohost.com / jspeciesws), and the DNA-DNA hybridization value (dDDH) was calculated using TYGS. The results showed that the genome similarity values (ANI and dDDH) between strain AH83 and related bacteria were both lower than the species threshold, indicating that AH83 was a new species of the genus Bacillus (Table 3). Among them, the type strain with the highest genome similarity to strain AH83 was Bacillus inaquosorum KCTC 13429 T , Bacillus spizizenii TU-B-10 T , Bacillus rugosus SPB7T and Bacillus vallismortis DV1-F-3 T , with ANI values of 92.58%-93.16% and dDDH values of 50.7%-52.7% (Table 3).

[0043] (5) Functional genomic analysis of strain AH83: Using antiSMASH (https: / / antismash.secondarymetabolites.org / #! / start), three secondary metabolite gene clusters were predicted in the genome of strain AH83, namely pulcherriminic acid, subtilosin, and bacilysin, which are related to functions such as stress resistance, nutrient utilization, and antibacterial activity.

[0044] Table 3 Sequence information used for genomic analysis of strain AH83 and ANI and dDDH values with related bacteria

[0045]

[0046] Example 2: This example verified the salt-alkali tolerance ability of strain AH83 as follows:

[0047] First, activate strain AH83 on LB liquid medium, and inoculate the activated strain into LB liquid medium containing 5%, 9%, 13%, 15%, 17% NaCl (w / v) and pH = 10.0 at an inoculation amount of 1%, and culture it at 28 °C and 150 rpm for 48 h, and measure its OD 600 value. At the same time, use non-inoculated LB medium as a control (CK), with 3 replicates for each treatment. The results are as Figure 5 and Figure 6 shown. The results show that the growth of strain AH83 was not significantly inhibited in the medium with pH = 10.0 and salt concentration below 15%, and significant inhibition occurred only in the 17% NaCl solution at pH = 10.0( Figure 5 ), indicating that strain AH83 has a high salt-alkali tolerance ability( Figure 6 ).

[0048] Example 3: This example verified the potassium-solubilizing function of strain AH83 as follows:

[0049] Prepare a potassium-solubilizing bacteria detection medium (5.0 g of glucose, (NH 4 )) 2 SO 4 0.5 g, 0.5 g of yeast powder, 0.3 g of MgSO 4 0.3 g, Na 2 HPO4 2.0 g, FeSO 4 0.03 g, MnSO 4 0.03 g, K 2 O·Al 2 O 3 ·6SiO 2 2.0 g, agar 15.0 g, distilled water 1000 mL, pH = 7.2, autoclaved at 121 °C for 15 min). After activating the strain AH83 on the LB medium, inoculate it onto the potassium-solubilizing bacteria detection medium and observe whether colonies appear on the potassium-solubilizing bacteria detection medium. The presence of colonies proves the ability to solubilize potassium. After detection, the strain AH83 can grow on the potassium-solubilizing bacteria detection medium, indicating its ability to solubilize potassium (as shown in Figure 7 a of

[0050] Example 4: This example verified the ability of strain AH83 to produce protease and cellulase, as follows:

[0051] First, activate the strain AH83 on the LB medium. Inoculate the activated strain onto the protease detection medium (tryptone 5.00 g, yeast extract 3.00 g, glucose 1.00 g, agar 15.00 g, distilled water 1000 mL, pH = 7.0, autoclaved at 121 °C for 30 min. When the sterilized detection medium is cooled to about 50 °C, add skim milk to the medium at a ratio of 10% and mix well, then pour it into a petri dish and wait for it to cool before use), culture at 28 °C for 48 h, repeat 3 times, and observe whether a clear zone appears. The results show that AH83 has a good ability to dissolve proteins, produces high levels of protease, the diameter (D) of its clear zone reaches 9.59 ± 0.63 mm, the diameter (d) of the colony is 5.61 ± 0.79 mm, and the enzyme production (D / d) is 2.92 ± 0.56 (specifically as shown in Figure 7 b of

[0052] First, activate the strain AH83 on the LB medium. Inoculate the activated strain onto the cellulose detection medium (MgSO 4 ·7H 2 O 0.25 g, K 2 HPO 4 0.50 g, (NH 4 ) 2 SO 40.5 g, sodium carboxymethyl cellulose 1.88 g, agar 15.0 g, distilled water 1000 mL, pH = 7.0, autoclaved at 121 °C for 30 min). Incubate at 28 °C for 5 days. After 5 days, add 5 mL of 0.2 mg / mL congo red staining solution to each plate, stain for 1 h, discard the congo red staining solution, add 1 M NaCl to wash for 1 h, discard the washing solution, observe the formation of hydrolysis zone around the colonies. The appearance of hydrolysis zone indicates the production of cellulase, and repeat 3 times. The results show that AH83 has good ability to dissolve cellulose, the diameter of its dissolution zone (D) reaches 2.08 ± 0.20 mm, the diameter of the colony (d) is 0.39 ± 0.04 mm, and the enzyme production (D / d) is 5.33 ± 0.26 (specifically as shown in Figure 7 as shown in c of

[0053] Example 5: This example verified the ability of strain AH83 to produce siderophores, specifically as follows:

[0054] Prepare the medium for detecting siderophores (glucose 100 g, peptone 20 g, MgSO 4 ·7H 2 O 0.5 g, CaCl 2 0.5 g, agar powder 20 g, distilled water 800 mL, pH = 7.0, autoclaved at 115 °C for 20 min, cooled to 60 °C, slowly add 100 mL of 10× buffer preheated to 60 °C and 0.06 g of CAS, FeCl 3 ·6H 2 O 0.0027 g, HDTMA 0.073 g, mix evenly without generating bubbles). After activating strain AH83 on LB medium, inoculate it onto the medium for detecting siderophores, repeat 3 times. After incubating at 28 °C for 5 days, observe whether a transparent zone is generated around the colonies. The results show that the inoculated strain AH83 has good ability to produce siderophores, the diameter of its transparent zone (D) reaches 7.88 ± 0.64 mm, the diameter of the colony (d) is 4.98 ± 0.55 mm, and the enzyme production (D / d) is 2.50 ± 0.47 (specifically as shown in Figure 7 as shown in d of

[0055] Example 6: This example verified the ability of strain AH83 to produce IAA, specifically as follows:

[0056] Prepare DF+Try culture medium for IAA detection (5.0 g of peptone, 1.5 g of yeast extract, 1.5 g of beef extract, 5.0 g of NaCl, 0.5 g of tryptophan, 1000 mL of distilled water, pH = 7.0, autoclave at 121 °C for 30 min). After activating the strain AH83 on LB medium, inoculate it into DF+Try culture medium at an inoculation amount of 1%, shake culture at 28 °C and 150 rpm for 7 days, take the bacterial liquid, centrifuge at 12000 rpm for 5 min, and determine the IAA content in the bacterial liquid by Salkowkin colorimetry, with 3 replicates. The results show that the yield of IAA synthesized by the strain AH83 is 3.26 ± 0.21 mg / L.

[0057] Example 7: This example verified the growth-promoting effect of the strain AH83 on the germination of lavender seeds and the growth of seedlings, as follows:

[0058] Pick a single colony of the strain AH83 and inoculate it into LB culture medium, culture at 28 °C and 150 rpm for 24 h, centrifuge to collect the bacterial cells, discard the culture supernatant, wash the bacterial cells 3 times with sterile water and then resuspend them, and dilute the bacterial liquid OD 600 values to three concentrations of 0.8 (high concentration), 0.4 (medium concentration), and 0.1 (low concentration) respectively. Select the main cultivated "French Blue" lavender variety in Yili area of Xinjiang, pick plump seeds of the same size, rinse the surface with sterile water, soak them in 75% alcohol for 2 min, rinse them three times with sterile water, and perform seed surface disinfection treatment. Divide the seeds into 5 groups and place them in equal amounts of sterile water (CK), 300 mg / L gibberellin solution (GA), low-concentration bacterial liquid (OD 600 = 0.1, AH83-L), medium-concentration bacterial liquid (OD 600 = 0.4, AH83-M), and high-concentration bacterial liquid (OD 600 = 0.8, AH83-H) for 4 h of seed soaking.

[0059] Sow the soaked seeds in a petri dish containing 3 layers of filter paper, add 7 mL of distilled water, germinate and culture them in a constant-temperature plant incubator at 25 °C for 20 days. Sow 30 seeds in each dish, and repeat each treatment in 5 dishes. Observe the germination of lavender seeds every 24 h until germination is completed. Seed germination is defined as the hypocotyl reaching 1 / 2 of the seed size as the germination standard, and add sterile water in a timely manner (add sterile water to the total weight of each dish to 22.5 mL) to keep the filter paper moist. Calculate the seed germination rate and germination potential of each treatment group according to the following formula:

[0060] Germination rate (%) = (number of germinated seeds / total number of seeds) × 100%

[0061] Germination potential (%) = (number of germinated seeds at the germination peak / number of test seeds) × 100%

[0062] After 20 days of cultivation, the seedlings were carefully removed from above the filter paper, avoiding damage to the root tissue, and the stem length, root length, and biomass were measured respectively.

[0063] The results are shown in Figure 8 , soaking seeds with 300 mg / L gibberellin and low, medium, and high concentrations of AH83 bacterial suspension all showed significant promoting effects on the germination of lavender seeds and the growth of seedlings. Among them, when soaking seeds with medium-concentration AH83 bacterial suspension (AH83-M), the growth-promoting effect was significantly better than that of low-concentration (AH83-L) and high-concentration (AH83-H) bacterial suspensions, and was also better than the gibberellin treatment (GA)( Figure 8 a); compared with CK, the germination rate and germination potential of lavender seeds in the AH83-M treatment group increased by 54% and 85% respectively( Figure 8 b), and the stem length, root length, fresh weight, and dry weight of the seedlings increased by 70%, 142%, 41%, and 48% respectively( Figure 8 c and d). The growth-promoting effects of the AH83-L and AH83-H treatment groups were slightly lower than that of AH83-M. The growth-promoting effects of the high-concentration bacterial liquid on the stems and roots of lavender seedlings decreased, possibly due to the relatively high content of IAA synthesized by the high-concentration bacterial suspension.

[0064] Example 8: This example verified the growth-promoting effect of strain AH83 on corn in saline-alkali soil and the soil improvement effect, as follows:

[0065] Soak the plump "Zhengdan 958" corn seeds in sterile water for 1 h, wash them, soak them in 3% sodium hypochlorite for 30 min, gently rub off the seed coating by hand, wash them, soak them in 70% ethanol for 10 min, and then wash them 3 times with sterile water, and soak them in sterile water at room temperature for 10 - 12 h. Lay 2 layers of sterile filter paper in a large petri dish, arrange the seeds on it, moisten the filter paper with 20 mL of sterilized distilled water, and cover it with a layer of sterile gauze. After covering the whole petri dish with a layer of plastic wrap, make several small holes on its surface, and culture at 28 °C until the seeds show white. Inoculate strain AH83 into LB liquid medium, and shake culture at 28 °C and 150 rpm until the OD of the bacterial liquid 600 reaches 1.0 as the seed liquid. Transfer the seed liquid to the fermentation medium (5.0 g of yeast powder, 10.0 g of peptone, 12.0 g of glucose, 5.0 g of NaCl, K 2 HPO 4 ·3H 2 O 0.1 g, 20.0 g of corn starch, 5.0 g of soybean meal powder, KH 2 PO 4 0.7 g, MgSO 40.2 g, 1000 mL of distilled water, pH = 7.0, autoclaved at 121 °C for 30 min), cultured with shaking at 28 °C and 150 rpm for 5 days, the bacterial concentration was measured and adjusted to 1×10 8 cfu / mL. Take 30 mL of the prepared bacterial suspension into a beaker, put in corn seeds with the same emergence, soak the seeds for 5 h, and at the same time, soak the seeds with the fermentation medium without inoculating the bacterial suspension as a blank control.

[0066] Set up corn pot experiments with two types of saline-alkali soils. One group is Xinjiang saline-alkali soil (original saline-alkali soil), and the other group is Xinjiang non-saline-alkali soil with 2 g / kg NaCl added externally (artificial saline-alkali soil). The basic physical and chemical properties of the two groups of soils are shown in Table 4. Each pot is filled with 1.5 kg of soil, and the amount of bacterial suspension added is 100 mL. At the same time, in order to maintain soil moisture, 100 mL of sterile water is added to the bacterial suspension. Spread the soil sample on kraft paper, evenly spray the bacterial suspension and sterile water, gently mix it after evenly sprinkling the bacterial suspension on each layer, and try to avoid soil disturbance until all the bacterial suspension is added. At the same time, use the fermentation medium without inoculating the bacterial suspension as a control. After spraying, weigh 1.5 kg of the soil and put it into a plastic flower pot with a diameter of 18 cm. Sow 3 corn seeds with the same germination in each pot, and the sowing depth is about 1 cm. Water 100 mL every two days after sowing. Thin out the seedlings 3 days after emergence, and keep two evenly sized corn plants in each pot. After thinning out the seedlings for 10 days, supplement and water with MS nutrient solution once.

[0067] Table 4 Basic physical and chemical properties of two types of saline-alkali soils

[0068]

[0069] On the 40th day after sowing, samples were collected to analyze the growth-promoting ability of the AH83 fermentation broth on maize plants. The maximum leaf was measured three times from top to bottom using a handheld chlorophyll meter (SPAD-502PIU), and then the average SPAD value was used as the SPAD value of the maize plants. The plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, maximum leaf area, root length, root surface area, root volume, and average root diameter of the maize plants were measured respectively. Further, the changes in soil physical and chemical properties and soil enzyme activities after applying the AH83 fermentation broth were detected. The determination of soil physical and chemical indexes was carried out according to the method of Bao Shidan. The soil conductivity and pH were measured using a conductivity meter and a pH meter respectively; the soil organic matter content was determined by the dichromate oxidation-external heating method; the soil nitrogen content was determined by the Kjeldahl method; the soil available nitrogen content was determined by the alkaline hydrolysis diffusion method. The total phosphorus content in the soil was determined by the NaOH alkali dissolution-molybdenum antimony anti-spectrophotometry method, and the available phosphorus content in the soil was determined by the sodium bicarbonate extraction-molybdenum antimony anti-colorimetry method; the total potassium content in the soil was determined by the NaOH alkali fusion-flame photometry method, and the available potassium content in the soil was determined by the ammonium acetate solution extraction-flame photometry method. The activities of soil catalase, sucrase, urease, and alkaline protease were determined using soil catalase, sucrase, urease, and alkaline protease kits respectively.

[0070] The results showed that the strain AH83 (AH83-0) had a significant growth-promoting effect on the aboveground and underground parts of maize in the original saline-alkali soil in Xinjiang (as Figure 9 shown). Compared with the non-inoculated control (CK-0), applying the strain AH83 increased the plant height of maize by 9.5%, the stem diameter by 29%, the aboveground fresh weight by 78.4%, the underground fresh weight by 38.8%, the aboveground dry weight by 31.6%, the leaf area by 37.3%, and the chlorophyll by 0.5%. It also significantly promoted the root development of maize, increasing the root length, root surface area, average root diameter, and root tip number of maize by 72.1%, 102.3%, 20.5%, 138.0%, and 127.9% respectively (as shown in Table 5). In the artificial saline-alkali soil, the strain AH83 (AH83-2) also showed a significant growth-promoting effect (as Figure 10 shown), increasing the plant height of maize seedlings by 5.8%, the stem diameter by 4.5%, the aboveground fresh weight by 9.9%, the underground fresh weight by 34.8%, the aboveground dry weight by 86.1%, the leaf area by 26.9%, and the chlorophyll by 5.9%. It increased the root length of maize by 12.3%, the root surface area by 33.0%, the average root diameter by 17.2%, the root volume by 57.7%, and the root tip number by 13.8% (as shown in Table 5).

[0071] In addition, applying strain AH83 to both groups of saline-alkali soils significantly improved the physical and chemical properties of the soils, and enhanced various indices of soil nutrients and enzyme activities (as shown in Table 6). Compared with the control group (CK-0), after inoculating AH83 in the original saline-alkali soil in Xinjiang (AH83-0), the soil pH value decreased by about 3%, while the contents of available phosphorus, available nitrogen, available potassium, total phosphorus and total nitrogen increased by 8.07%, 7.88%, 22.3%, 4.04% and 9.89% respectively, indicating that the application of the microbial agent could improve the soil acid-base balance and soil nutrient status. Meanwhile, the activities of soil catalase, sucrase and urease increased by 14.14%, 66.15% and 25.91% respectively (as shown in Table 6).

[0072] In the artificial saline-alkali soil, compared with the control group (CK-2), inoculating strain AH83 (AH83-2) increased the contents of available phosphorus, available nitrogen, available potassium, total phosphorus and total nitrogen in the soil by 5.01%, 15.06%, 26.41%, 12.9% and 16.01% respectively, the catalase by 6.25%, the sucrase by 136.21%, the urease content by 57.11%, and the alkaline protease content by 4.78% (as shown in Table 6).

[0073] In summary, the novel Bacillus species AH83 provided in the embodiments of the present invention can inhibit the growth of pathogenic bacteria such as Fusarium oxysporum causing cotton wilt, has growth-promoting functions such as potassium solubilization, indole acetic acid production, cellulase, protease, and siderophore production, and can promote the germination and seedling growth of lavender seeds. Meanwhile, strain AH83 can tolerate a highly saline-alkali environment, and applying it in the original and artificial saline-alkali soils can promote agronomic traits such as plant height, biomass, leaf area and root development of maize at the seedling stage, promote the root development of maize, improve the physical and chemical properties of the soil, increase soil nutrients and enzyme activities, and can be applied to the cultivation of crops such as maize and lavender in saline-alkali environments.

[0074] Table 5 Growth-promoting effects of strain AH83 on maize seedlings in two types of saline-alkali soils

[0075]

[0076] Note: Different letters indicate significant differences between the inoculation treatment group and the control group at the P<0.05 level.

[0077] Table 6 Effects of strain AH83 on the physical and chemical properties and enzyme activities of maize soil in two types of saline-alkali soils

[0078]

[0079] Note: Different letters indicate significant differences between the inoculation treatment group and the control group at the P<0.05 level.

[0080] Inspired by the above-described ideal embodiments of the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification.

Claims

1. A salt-alkali tolerant growth-promoting Bacillus sp. AH83, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC No: M 20241495.

2. A bacterial agent containing the Bacillus AH83 according to claim 1.

3. Use of the Bacillus AH83 according to claim 1 or the bacterial agent according to claim 2 in inhibiting pathogenic bacteria of cotton wilt.

4. The use according to claim 3, characterized in that: The cotton wilt pathogens include Fusarium oxysporum.

5. Use of the Bacillus AH83 according to claim 1 or the bacterial agent according to claim 2 in promoting plant growth.

6. The use according to claim 5, characterized in that: The plants include lavender and corn, and the plant growth promotion is normal soil or saline-alkali soil plant growth promotion.

7. Use of the Bacillus AH83 according to claim 1 or the bacterial agent according to claim 2 in improving the soil structure of saline-alkali land or preparing a soil conditioner.

8. Use of the Bacillus AH83 according to claim 1 or the bacterial agent according to claim 2 in producing growth-promoting substances, characterized in that: The growth-promoting substances include IAA, siderophore, protease and cellulase.

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