Saline-alkali-tolerant growth-promoting bacillus velezensis AH216 and application thereof

Abstract

The invention discloses a saline-alkaline tolerant growth-promoting bacillus velezensis AH216 and application thereof, and belongs to the technical field of microbiology and biologica.Bacillus velezensis AH216 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No: M 20241496. The saline-alkaline tolerant growth-promoting bacillus velezensis AH216 has the advantages that the saline-alkaline tolerant growth-promoting bacillus velezensis AH216 can be used for preparing saline-alkaline tolerant growth-promoting bacillus velezensis AH216; the bacillus velezensis AH216 provided by the invention 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 bacillus velezensis AH216 can tolerate a highly saline-alkaline environment, can improve agronomic characters such as the plant height, the biomass and the leaf area in the corn seedling stage in common soil and saline-alkaline soil, and can promote the development of a corn root system.

Description

Technical Field

[0001] The present invention relates to the fields of microbiology and biotechnology, and specifically to a salt-tolerant and growth-promoting Bacillus velezensis AH216 and its applications. Background Art

[0002] Soil salinization, as a prominent problem of soil degradation, threatens the soil health and food security in China. Compared with other improvement measures, the use of microbial inoculants prepared from functional microorganisms to improve saline-alkali soil can achieve better improvement effects while having the advantages of ecological environmental protection, pollution-free, and long-lasting improvement effects. The beneficial functional microorganisms in microbial inoculants can not only activate nutrients such as phosphorus and potassium in saline-alkali soil, but also secrete growth-promoting substances such as indole acetic acid to promote crop growth and increase crop yield. At present, the application of functional microorganisms has become one of the important measures for saline-alkali soil improvement.

[0003] Bacillus is a type of Gram-positive bacteria that are aerobic or facultatively anaerobic and can produce spores. It can affect plant growth in two ways: direct action means that functional microorganisms directly promote plant growth and development through processes such as biological nitrogen fixation, production of auxin (IAA), and activation of phosphorus and potassium nutrients in the soil during their growth and metabolism. Indirect action means that functional microorganisms indirectly achieve growth-promoting effects by inhibiting the growth of pathogenic bacteria through antibiotic action or producing substances such as ACC-deaminase and siderophores to relieve plant damage and improve soil nutrient status. Therefore, Bacillus has been widely studied and applied as a model species and has become one of the most intensively studied and widely applied plant growth-promoting and biocontrol bacteria in the world.

[0004] Bacillus velezensis is an important member of the genus Bacillus. It was named after being initially isolated from the waters of the Velez River in Spain (Joshi et al., 2010). Bacillus velezensis is commonly found in environments such as plant tissues, rhizosphere soil, air, and fermented foods. It is now mainly isolated from plant tissues and soil environments. Bacillus velezensis has several obvious advantages, including specific colonization in certain parts of a given crop, formation of biofilms, production of antibacterial substances, and induction of systemic resistance in plants.

[0005] In the prior art, Han Xing et al. applied Bacillus velezensis LJ02 in the field to control Verticillium wilt of cotton, and the control effect reached 61.10%, and significantly promoted the growth of cotton. Zhang et al. confirmed that Bacillus velezensis E2 had an obvious inhibitory effect on the growth of Aspergillus westerdijkiae fc-1 and the OTA (Ochratoxin A) toxin produced by it, and believed that Bacillus velezensis E2 could be used to control fungal diseases caused by Aspergillus westerdijkiae in agricultural products. Yan Haohao et al. used Bacillus velezensis SDTB038 and fluxapyroxad to synergistically control potato late blight, and the control effect reached 79.45% in the field experiment. Laura et al. applied Bacillus velezensis XT1 to the roots of tomatoes to control gray mold, and the study found that through the mechanism of inducing systemic resistance of plants, it not only reduced the incidence and severity of diseases, but also increased the biomass of plants.

[0006] In summary, there have been many studies showing that Bacillus velezensis has the effect of antagonizing plant pathogens and is a biocontrol bacterium with development potential. However, there are few studies on the activation of nutrients, growth promotion, enzyme production, etc. of Bacillus velezensis, especially its adaptability to high salinity, and there are no reports on the regulation of plant salt tolerance, promotion of plant growth and improvement of saline-alkali soil under saline-alkali soil conditions. Summary of the Invention

[0007] The purpose of the present invention is to provide a salt-tolerant and growth-promoting Bacillus velezensis AH216 to solve the problems raised in the above background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] A salt-tolerant and growth-promoting Bacillus velezensis AH216, which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC No: M 20241496, the preservation address is Wuhan University, China, and the preservation time is July 8, 2024.

[0010] Another purpose of the present invention is to provide a bacterial agent containing the above-mentioned Bacillus velezensis AH216.

[0011] Another purpose of the present invention is to provide an application of the above-mentioned Bacillus velezensis AH216 or the above-mentioned bacterial agent in inhibiting the pathogen of cotton fusarium wilt.

[0012] Preferably, the pathogen of cotton fusarium wilt includes Fusarium oxysporum.

[0013] Another object of the present invention is to provide an application of the above-mentioned Bacillus velezensis AH216 or the above-mentioned bacterial agent in promoting plant growth.

[0014] Preferably, the plants include lavender and corn, and the plant growth promotion is for promoting the growth of plants in ordinary soil or saline-alkali soil.

[0015] Another object of the present invention is to provide an application of the above-mentioned Bacillus velezensis AH216 or the above-mentioned bacterial agent in improving the soil structure of saline-alkali land or preparing a soil conditioner.

[0016] Another object of the present invention is to provide an application of the above-mentioned Bacillus velezensis AH216 or the above-mentioned bacterial agent in producing growth-promoting substances, and the growth-promoting substances include IAA, siderophore, protease and cellulase.

[0017] A strain of salt-tolerant and growth-promoting Bacillus velezensis AH216 provided by the present invention can inhibit the growth of cotton wilt pathogens 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 velezensis AH216 can survive in a highly saline-alkali environment, and can improve the agronomic traits such as plant height, dry weight, and leaf area of corn seedlings in different saline-alkali soils, promote the root development of corn, and can also increase the soil nutrient and enzyme activity content and has a promoting effect on the germination of lavender seeds. Description of the Drawings

[0018] Figure 1 It is a comparison chart of the inhibition rates of some strains on Fusarium oxysporum in the examples of the present invention.

[0019] Figure 2 It is a graph of the colony morphology of strain AH216 and its inhibitory effect on Fusarium oxysporum in the examples of the present invention; among them, the left figure is a photo of the colony morphology of AH216 on LB medium, and the right figure is an inhibitory effect diagram of AH216 on Fusarium oxysporum.

[0020] Figure 3 It is a 16S rRNA gene phylogenetic tree of strain AH216 in the examples of the present invention.

[0021] Figure 4 It is a phylogenetic tree of strain AH216 based on the whole genome in the examples of the present invention.

[0022] Figure 5 It is a graph of the salt-tolerant ability results of strain AH216 in the examples of the present invention.

[0023] Figure 6 It is the growth situation of strain AH216 in a medium with pH = 10.0 and different salt concentrations in the examples of the present invention.

[0024] Figure 7 It is a graph showing the test results of the potassium-solubilizing, siderophore-producing, protease-producing, and cellulase-producing abilities of strain AH216 in the embodiments of the present invention. Among them, Figure a shows the growth of AH216 on the potassium-solubilizing detection medium, Figures b and c show the protease-producing and cellulase-producing situations of AH216 respectively, and Figure d shows the transparent circle of siderophore produced by AH216.

[0025] Figure 8 It is a graph showing the growth promotion effect of strain AH216 on the germination and seedling growth of lavender seeds in the embodiments of the present invention. Among them, a is a photo of the germination of lavender seeds in the AH216 bacterial suspension soaking treatment group and the control group, b is the result of the seed germination rate and germination potential, c is the result of the seedling stem length and root length, and d is the result 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 a significant difference at the P<0.05 level.

[0026] Figure 9 It is a graph showing the growth promotion effect of strain AH216 on corn in the saline-alkali soil (original saline-alkali soil) of Xinjiang in the embodiments of the present invention. Among them, a is a photo of the potted plants in the AH216 fermentation broth treatment group and the control group, b is a photo of the roots of the plants in the AH216 treatment group and the control group, and c is a scanned image of the root morphology of the plants in the AH216 treatment group and the control group.

[0027] Figure 10 It is a graph showing the growth promotion effect of strain AH216 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. Among them, a is a photo of the plants in the AH216 fermentation broth treatment group and the control group, b is a photo of the roots of the plants in the AH216 treatment group and the control group, and c is a scanned image of the root morphology of the plants in the AH216 treatment group and the control group. Detailed implementation manners

[0028] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0029] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0030] 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.

[0031] Example 1: This example provides a method for isolating, screening, and identifying a salt-tolerant and growth-promoting Bacillus velezensis AH216, which is as follows:

[0032] S1. Isolation and screening of strain AH216: In 2023, cotton plants were collected from Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. 10 g of cotton leaves were taken, and the surface was washed with distilled water and then soaked and disinfected in 75% ethanol, 3% sodium hypochlorite, and 75% ethanol solutions for 5 min in sequence. Finally, it was rinsed 3 times with sterile water, and the sterile water from the last rinse was spread on an LB plate (yeast extract 5 g / L, tryptone 10 g / L, NaCl 5 g / L, pH = 7.0 - 7.2, agar 15 g / L) as a blank control for the surface disinfection effect. The disinfected leaf samples were placed in a sterile mortar and ground with an appropriate amount of sterile normal saline until homogenized. 1 mL of the homogenate was serially diluted according to 10 -1 、10 -2 、10 -3 、10 -4 and spread on plates respectively, and cultured at a constant temperature of 28°C for 2 d. If no colonies appear on the isolation medium plate of the surface disinfection blank control, it indicates that the surface of the plant material is thoroughly disinfected and the isolated bacteria are endophytic bacteria. Bacterial single colonies with different morphologies were picked with a sterile toothpick, purified by streaking, and stored for later use. A total of 87 endophytic bacteria were isolated. The biocontrol effect of the isolated endophytic bacteria was detected by the confrontation culture method. First, Fusarium oxysporum was activated on a PDA plate, and a pathogen agar disc was punched with a 5 mm punch and inoculated in the center of the PDA plate. The 87 isolated endophytic bacteria were respectively inoculated 2.5 cm away from the agar disc and cultured at 25°C for 4 - 7 days. The colony diameters of Fusarium oxysporum with and without inoculation of endophytic bacteria were measured respectively, and the inhibition rate was calculated according to the following formula:

[0033]

[0034] Among the 87 strains, 27 strains were found to have an inhibitory effect on Fusarium oxysporum through primary screening. Then, through re-screening, 5 strains with an inhibition rate of more than 50% on the mycelial growth of Fusarium oxysporum were selected. The screening results are shown in Figure 1 ; among them, strain 1# (AH216) had the strongest inhibitory effect and was selected for subsequent experiments. At the same time, this strain AH216 was deposited in the China Center for Type Culture Collection, with the deposit number CCTCC No: M20241496, the deposit address being Wuhan University, Wuhan, China, and the deposit time being July 8, 2024; Figure 2 is the colony morphology of strain AH216 and the inhibitory effect on Fusarium oxysporum; Figure 2 In it, the left figure is the colony morphology of AH216 on the LB medium, and the right figure is the inhibitory effect diagram of AH216 on Fusarium oxysporum.

[0035] S2. Identification of strain AH216: Based on the morphological and molecular biological detection results of strain AH216, it was identified as Bacillus velezensis. The specific details are as follows:

[0036] (1) Phylogenetic analysis of 16S rRNA gene: The genomic DNA of strain AH216 was extracted using the bacterial DNA kit of Beijing Juhemei Biotechnology Co., Ltd. The 16S rRNA gene was amplified using the universal primers 27F (5’-AGAGTTTG ATCCTGGCTCAG-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 AH216 had the highest similarity with strains of the genus Bacillus. The phylogenetic tree constructed using MEGA 11.0 software also showed that strain AH216 belongs to the genus Bacillus (as shown in Figure 3 ).

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

[0038]

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

[0040]

[0041] (2) Genomic characteristics of strain AH216: The total genomic DNA of strain AH216 extracted was entrusted to Shanghai Sangon Biotech Co., Ltd. for genomic sequencing analysis after passing the DNA purity test. Sequencing was completed using a combination of PacBio RS II and Illumina Solexa sequencing platforms. The sequencing results showed that the genome size of strain AH216 was 3.47 Mb, the G+C content was 47.2 mol%, there were a total of 3,743 coding genes, the coding region accounted for 89.3% of the genome, and there were 10 rRNA genes and 80 tRNA genes. The results of species identification based on the whole genome using TYGS (http: / / ggdc.dsmz.de) showed that strain AH216 was Bacillus velezensis. At the same time, the constructed whole-genome phylogenetic tree also showed that strain AH216 and Bacillus velezensis were in the same branch (as shown in Figure 4as shown

[0042] S3. Genome similarity analysis of strain AH216: The average nucleotide identity (ANI) between strain AH216 and other Bacillus velezensis 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 strain AH216 had the highest similarity with Bacillus velezensis NRRL B-41580 T , with an ANI of 98.1% and a dDDH of 84.5%, both within the species threshold range, indicating that AH216 belongs to Bacillus velezensis, but there are certain differences in the genome compared with the type strain, which may be related to the differences in the adaptability and physiological functions of the strain under specific environmental conditions (Table 3).

[0043] Table 3 Sequence information used for genome analysis of strain AH216 and ANI and dDDH values with related bacteria

[0044]

[0045] S4. Secondary metabolic functional genome analysis of strain AH216: Using the antiSMASH analysis platform (https: / / antismash.secondarymetabolites.org / #! / start), 11 secondary metabolic gene clusters were predicted in the genome of strain AH216, encoding antibacterial substances difficidin, fengycin, bacillaene, macrolactin H, bacilysin, and surfactin, as well as the siderophore bacillibactin, demonstrating the potential of strain AH216 for biocontrol and growth promotion at the genetic level.

[0046] Example 2: This example verified the salt and alkali tolerance ability of strain AH216, as follows:

[0047] LB culture media containing 0, 5%, 9%, 13%, 15%, and 17% NaCl (w / v) with a pH of 10.0 were prepared respectively. Single colonies of strain AH216 were picked and inoculated into LB liquid medium, cultured at 28 °C and 150 rpm for 12 - 16 h as the seed solution. The seed solution was inoculated into the saline-alkali culture medium at an inoculation amount of 1%, and cultured at 28 °C and 150 rpm for 48 h, and the OD of the bacterial solution was measured 600Value, analyze the growth of the strain in the saline-alkali medium environment. At the same time, use the LB medium containing salt without inoculating the strain AH216 as the control (CK), with 3 replicates for each treatment. The results are as Figure 5 and Figure 6 shown. The results show that within the range of pH = 10.0 and salt content of 0 - 13%, the strain AH216 grows well, and the saline-alkali stress brings mild growth inhibition. The inhibition rate of 13% NaCl is 35%, while a large-scale growth inhibition only appears in the 15% NaCl solution.

[0048] Example 3: This example verified the potassium-solubilizing function of the strain AH216, specifically as follows:

[0049] Prepare the 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 HPO 4 2.0 g, 0.03 g of FeSO 4 0.03 g of MnSO 4 ·0.03 g, K 2 O·Al 2 O 3 ·6SiO 2 2.0 g, 15.0 g of agar, 1000 mL of distilled water, pH = 7.2, autoclave at 121 °C for 15 min). After activating the strain AH216 on the LB medium, inoculate it onto the potassium-solubilizing bacteria detection medium. After detection, the strain AH216 can grow on the detection medium, indicating its potassium-solubilizing ability (as shown in Figure 7 a of).

[0050] Example 4: This example verified the ability of the strain AH216 to produce protease and cellulase, specifically as follows:

[0051] Prepare the protease detection medium (5.0 g of tryptone, 3.0 g of yeast extract, 1.0 g of glucose, 15.0 g of agar, 1000 mL of distilled water, pH = 7.0, autoclave at 121 °C for 30 min. When the sterilized detection medium is cooled to about 50 °C, add 10% sterile skim milk, mix well and pour the plate). After activating the strain AH216 on the LB medium, inoculate it onto the protease detection medium, culture at 28 °C for 48 h, with 3 replicates, and observe whether there is a dissolution zone. The appearance of a dissolution zone indicates the production of protease.

[0052] Prepare the cellulase detection medium (MgSO 4 ·7H 2O 0.25 g, K 2 HPO 4 0.50 g, (NH 4 ) 2 SO 4 0.5 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). After activating the strain AH216 on the LB medium, inoculate it onto the medium for cellulose detection. Incubate at 28 °C for 5 days, with 3 replicates. After the incubation, add 5 mL of Congo red solution (0.2 mg / mL) to each plate, stain for 1 h, discard the Congo red solution, then add 1 M NaCl solution to wash for 1 h, discard the washing solution, and observe the formation of a hydrolysis zone around the colonies. The appearance of a hydrolysis zone indicates the production of cellulase. The results show that the strain AH216 has good abilities to dissolve proteins and cellulose, and is a high producer of protease and cellulase. The diameter (D) of the protease hydrolysis zone reaches 21.50 ± 0.72 mm, the colony diameter (d) is 10.85 ± 0.45 mm, and the enzyme production (D / d) is 3.94 ± 0.21 (as shown in Figure 7 b); the diameter (D) of the cellulase hydrolysis zone reaches 3.18 ± 0.12 mm, the colony diameter (d) is 0.50 ± 0.07 mm, and the enzyme production (D / d) is 6.38 ± 0.37 (as shown in Figure 7 c).

[0053] Example 5: This example verified the ability of strain AH216 to produce siderophores, as follows:

[0054] Prepare the medium for siderophore detection (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. Cool to 60 °C, and 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 well without generating bubbles). After activating the strain AH216 on the LB medium, inoculate it onto the siderophore detection medium, with 3 replicates. Incubate at 28 °C for 5 days, and then observe whether a clear zone appears around the colonies. The results show that a distinct clear zone appears after inoculating the strain AH216, indicating that AH216 has the ability to synthesize siderophores. The diameter (D) of the clear zone reaches 6.47 ± 0.45 mm, the colony diameter (d) is 5.15 ± 0.30 mm, and the enzyme production (D / d) is 1.29 ± 0.49 (as shown inFigure 7 as shown in d of

[0055] Example 6: This example verified the ability of strain AH216 to produce IAA, 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 strain AH216 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 measure the IAA content in the bacterial liquid by the Salkowkin colorimetric method, with 3 replicates. The results showed that strain AH216 had the ability to synthesize IAA, and the IAA yield was 4.04 ± 0.14 mg / L.

[0057] Example 7: This example verified the promoting effect of strain AH216 on the germination and seedling growth of lavender seeds, as follows:

[0058] Pick a single colony of AH216 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 OD 600 value of the bacterial liquid to three concentrations of 0.8 (high concentration), 0.4 (medium concentration) and 0.1 (low concentration) respectively. Select the seeds of the main cultivated lavender variety "French Blue" in Yili area, Xinjiang, pick plump seeds of the same size, wash 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, AH216-L), medium-concentration bacterial liquid (OD 600 = 0.4, AH216-M) and high-concentration bacterial liquid (OD 600 = 0.8, AH216-H) for seed soaking for 4 h.

[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 based on the hypocotyl reaching 1 / 2 of the seed size as the germination standard, and sterile water is added 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 peak germination stage / Number of seeds of the tested variety) × 100%

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

[0063] The results are shown in Figure 8 , gibberellin, and soaking treatments with AH216 bacterial suspensions at low, medium, and high concentrations all showed significant promoting effects on the germination of lavender seeds and the growth of seedlings. Among them, when soaking seeds with the medium-concentration AH216 bacterial suspension (AH216-M), the growth-promoting effect was significantly better than that of the low-concentration (AH216-L) and high-concentration (AH216-H) bacterial suspensions, and was better than the treatment with 300 mg / L gibberellin (GA)( Figure 8 a); compared with CK, the germination rate and germination potential of lavender seeds in the AH216-M treatment group increased by 61% and 83% respectively( Figure 8 b), and the stem length, root length, fresh weight, and dry weight of the seedlings increased by 81%, 57%, 40%, and 114% respectively( Figure 8 c and d). The stem length of the AH216-L and AH216-H treatment groups was slightly higher than that of GA, while the root length was slightly lower than that of GA. The germination rate, germination potential, and biomass were comparable to those of GA. The promoting effect of the high-concentration bacterial solution treatment on stem length and root length was slightly lower than that of the low-concentration bacterial solution, which may be due to the higher content of IAA synthesized by the high-concentration bacterial suspension.

[0064] Example 8: This example verified the growth-promoting effect of strain AH216 on corn in saline-alkali soil and the soil improvement effect, as follows:

[0065] Select "Zhengdan 958" corn seeds with uniform and plump grains, soak them in sterile water for 1 h, wash them, soak them in 3% sodium hypochlorite solution for 30 min, gently rub off the seed coating by hand, wash them, soak them in 70% ethanol for 10 min, then wash them 3 times with sterile water, and soak them 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 sterile water, and cover it with a layer of sterile gauze. After covering the entire 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 AH216 into LB liquid medium, and shake-culture at 28 °C and 150 rpm until the OD of the bacterial liquid 600 reaches 1.0 to obtain the seed liquid. Inoculate the seed liquid into the fermentation culture medium at an inoculation amount of 5% (5.0 g of yeast powder, 10.0 g of peptone, 12.0 g of glucose, 5.0 g of NaCl, K 2 HPO4 3H 2 O 0.1 g, corn starch 20.0 g, soybean meal powder 5.0 g, KH 2 PO 4 0.7 g, MgSO 4 0.2 g, distilled water 1000 mL, pH = 7.0, autoclaved at 121 °C for 30 min), cultured with shaking at 28 °C and 150 rpm for 5 days, diluted and spread on plates to determine the viable count of the bacterial solution, and diluted it to a bacterial suspension of 1.0×10 8 cfu / mL. Take 30 mL of the prepared bacterial suspension into a petri dish, put in corn seeds with consistent emergence, soak the seeds for 5 h, and at the same time soak the seeds with the fermentation medium without inoculating the bacterial solution in equal amount as a blank control.

[0066] Set up corn pot experiments with two types of saline-alkali soils. One group selects Xinjiang saline-alkali soil (original saline-alkali soil), and the other group selects Xinjiang non-saline-alkali soil with 2 g / kg of NaCl added exogenously (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 100 mL of the bacterial suspension is evenly applied. At the same time, in order to maintain soil humidity, 100 mL of sterile water is added to the bacterial solution. Spread the soil sample on kraft paper, evenly spray the bacterial solution and sterile water, gently mix evenly after evenly sprinkling the bacterial solution on each layer, and try to avoid soil disturbance during this period until all the bacterial solution is added. At the same time, use the blank fermentation medium as a control. After adding, 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 consistent 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 corn plants of uniform size in each pot. 10 days after thinning, 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] Samples were collected on the 40th day after sowing. The largest leaf was measured three times from top to bottom using a handheld chlorophyll meter (SPAD-502PIU), and then the average SPAD value was taken as the relative chlorophyll content value of the maize plants. The plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, largest leaf area, root length, root surface area, root volume, and average root diameter of maize seedlings in each treatment group were measured respectively. In addition, the effects of the AH216 fermentation broth on the physical and chemical properties of saline-alkali soil and soil enzyme activities were determined. The determination of soil physical and chemical indexes referred 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 potassium dichromate oxidation-external heating method. The soil nitrogen content was determined by the Kjeldahl method. The available nitrogen content in the soil was determined by the alkali hydrolysis diffusion method. The available phosphorus content in the soil was determined by the sodium bicarbonate extraction-molybdenum antimony anti-colorimetric method. The total phosphorus content in the soil was determined by the NaOH alkali dissolution-molybdenum antimony anti-spectrophotometry method. The total potassium content in the soil was determined by the NaOH alkali fusion-flame photometry method. 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 in the original saline-alkali soil environment in Xinjiang, the AH216 fermentation broth (AH216-0) had a significant growth-promoting effect on both the aboveground and underground parts of maize (as Figure 9 shown), increasing the maize plant height by 11.1%, the stem diameter by 23.7%, the aboveground and underground fresh weights by 108.9% and 46.3% respectively, the aboveground and underground dry weights by 108.0% and 53.0% respectively, the relative chlorophyll content by 4.7%, and significantly promoting the development of maize roots, increasing the maize root length by 120.1%, the root surface area by 130.4%, the average root diameter by 5.0%, the root volume by 140.3%, and the number of root tips by 128.7% (as shown in Table 5). In the artificial saline-alkali soil, soaking seeds with the AH216 bacterial solution (AH216-2) also had a significant growth-promoting effect on maize (as Figure 10 shown), increasing the maize seedling plant height by 27.5%, the stem diameter by 11.3%, the aboveground and underground fresh weights by 58.3% and 24.7% respectively, the aboveground and underground dry weights by 178.8% and 129.4% respectively, the relative chlorophyll content by 15.9%, and increasing the maize root length by 62.3%, the root surface area by 90.2%, the average root diameter by 16.6%, the root volume by 122.9%, and the number of root tips by 42.0% (as shown in Table 5).

[0071] In addition, inoculating the strain AH216 into the original saline-alkali soil in Xinjiang significantly improved the physical and chemical properties of the soil, and increased the soil nutrients and fertility. Compared with the CK, after inoculating the strain AH216, the soil pH value decreased by 2.1%, and at the same time, the contents of available phosphorus (15.4%), available nitrogen (3.9%), available potassium (30.3%), total phosphorus (8.1%) and total nitrogen (13.2%) in the soil increased significantly. Compared with the CK, the inoculation of the strain AH216 significantly increased the activities of soil catalase (5.0%), sucrase (44.2%) and urease (35.8%), while the content of alkaline protease did not change significantly (as shown in Table 6). In the artificial saline-alkali soil, the strain AH216 also significantly improved the soil quality. Compared with the control group, the contents of available phosphorus, available nitrogen, available potassium, total phosphorus and total nitrogen in the soil increased by 18.8%, 13.4%, 34.8%, 9.7% and 20.7% respectively, and the activities of soil sucrase, urease and alkaline protease increased by 60.4%, 59.4% and 15.3% respectively, while there was no significant difference in the content of catalase (as shown in Table 6).

[0072] In summary, the present invention provides Bacillus velezensis AH216, which can inhibit the growth of cotton wilt disease pathogens such as Fusarium oxysporum, 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. At the same time, the Bacillus velezensis AH216 can survive in a highly saline-alkali environment, and can improve the agronomic traits such as plant height, biomass, and leaf area of maize seedlings in both the original and artificial saline-alkali soils, promote the root development of maize, improve the physical and chemical properties of the soil, increase the soil nutrients and fertility, and can be applied to the planting of crops such as maize in saline-alkali environments.

[0073] Table 5 Growth-promoting effects of strain AH216 on maize in two types of saline-alkali soils

[0074]

[0075] Note: Different letters indicate significant differences between the inoculated treatment group and the control group at the P<0.05 level.

[0076] Table 6 Effects of strain AH216 on the physical and chemical properties and enzyme activities of maize soil in two types of saline-alkali soils

[0077]

[0078]

[0079] Note: Different letters indicate significant differences between the inoculated 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 this invention. The technical scope of this invention is not limited to the content in the specification.

Claims

1. A salt-alkali tolerant growth-promoting Bacillus velezensis AH216, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC No: M 20241496.

2. A bacterial agent containing the Bacillus Velezii AH216 according to claim 1.

3. Use of the Bacillus Velez AH216 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 Velez AH216 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 Velez AH216 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 Velez AH216 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.

Images