Bacillus subtilis for producing siderophore and application thereof

Abstract

The invention belongs to the technical field of rhizosphere microorganisms, and particularly relates to bacillus subtilis capable of producing siderophore and application of the bacillus subtilis. The preservation number of the bacillus subtilis M13 is CGMCC (China General Microbiological Culture Collection Center) No.26455, and the bacillus subtilis M13 is preserved on January 12, 2023. In the application, the bacillus subtilis strain with high siderophore yield is obtained by screening and separating from Chinese yam. Taking the cultivation of the hylocereus undatus as an example, after the fermentation liquor of the strain is directly irrigated to rhizosphere soil of the hylocereus undatus, the preliminary experiment and related detection results show that the polysaccharide content of the hylocereus undatus is obviously increased. The result proves that the bacillus subtilis strain with high siderophore yield, provided by the invention, has a relatively good application prospect in agricultural cultivation, and has relatively good practical value and popularization and application significance due to a relatively simple and convenient application mode.

Description

Technical Field

[0001] The present application belongs to the field of rhizosphere microorganism technology, and specifically relates to a siderophore-producing Bacillus subtilis and an application thereof. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) are a type of beneficial bacteria that live in the rhizosphere or soil of plants and can promote plant growth, increase plant yields, and even prevent and treat pests and diseases. Studies on plant growth environments have shown that PGPR can interact with plants and play an important role in plant stress resistance and promoting plant survival and growth. Therefore, in the research of plant growth regulation and crop yield improvement, the study of PGPR has received more and more attention.

[0003] Iron is one of the essential chemical elements for the growth and development of various plants, but iron usually only exists in the soil in the form of various soluble or insoluble compounds and cannot be directly absorbed and utilized by plants. Therefore, the absorption and utilization of iron by plants must rely on the absorption of plant roots and the ability to dissolve and transform various iron-containing compounds. In this process, the microenvironment formed by various microorganisms in the growth environment of plant roots has an important influence on the dissolution and transformation of iron-containing compounds.

[0004] Studies have shown that some microorganisms can synthesize a class of low-molecular-weight, low-molecular-weight organic substances that specifically chelate Fe3+ under low-iron environmental conditions, called siderophores. After being synthesized in microbial cells, siderophores are secreted to the surface of microbial cells or the external environment to bind to iron ions, and then transported into the cells for use by microorganisms. Since siderophores have a very strong and specific ability to bind to iron ions, they can take away iron from various water-soluble and water-insoluble compounds. Due to the relative lack of iron in the natural environment, under conditions of iron stress, in order to obtain the iron required for growth, many microorganisms contain corresponding siderophore genes, which are then used to produce siderophores and evolve different siderophore utilization mechanisms.

[0005] In the prior art, some researchers have isolated some siderophore-producing microbial strains from soil or other environments, and conducted application experiments in the growth of some plants and crops. The relevant experimental results show that the application of these strains can effectively improve or even enhance the growth of crops, showing good application prospects. However, due to the large number of rhizosphere microorganisms, the great differences in plant and crop types and soil environmental conditions, screening new siderophore-producing microbial strains is still the basis for current research work. Summary of the invention

[0006] The purpose of this application is to provide a strain of Bacillus subtilis that can produce siderophore Bacillus subtilis ) strains, thereby laying a certain technical foundation for improving the production effects of related crops and developing related bacterial fertilizer products.

[0007] The technical solution adopted in this application is briefly described as follows.

[0008] A siderophore-producing Bacillus subtilis, wherein the Bacillus subtilis ( Bacillus subtilis )M13, whose accession number is CGMCC No.26455, was submitted for preservation at the China General Microbiology Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on January 12, 2023, and its survival was confirmed by testing on the same day.

[0009] The biological agent prepared by using the siderophore-producing Bacillus subtilis is prepared by first inoculating the strain Bacillus subtilis M13 into a seed culture medium, culturing at 25°C and 120 r / min for 2 days to prepare a seed culture solution, then inoculating the seed culture solution into a sterilized fermentation medium at a 3% inoculation amount (volume ratio), and fermenting at 25-35°C for 48-96h (preferably 30°C for 72h); The seed culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 6.8±0.2; The fermentation medium comprises the following components: sucrose 20.0 g / L, (NH4)2SO4 3.5 g / L, glutamic acid 0.03 g / L, KH2PO4 1.0 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, and pH 6.8±0.2.

[0010] The siderophore-producing Bacillus subtilis or the bacterial agent prepared using the siderophore-producing Bacillus subtilis is used in the preparation of a microbial fertilizer preparation to increase the iron content in the soil environment around the plant root system.

[0011] The siderophore-producing Bacillus subtilis or the bacterial agent prepared therefrom is used in crop cultivation, wherein the crop is chrysanthemum or yam; a specific example of the chrysanthemum is goldenrod, and a specific example of the yam is Dioscorea opposita.

[0012] In the present application, the inventors screened and isolated a high-yield siderophore Bacillus subtilis strain from Chinese yam. Taking the cultivation of golden chrysanthemum as an example, after the fermentation liquid of the strain was directly irrigated into the rhizosphere soil of golden chrysanthemum, the experimental and related test results showed that the polysaccharide content of golden chrysanthemum was significantly improved. This result proves that the high-yield siderophore Bacillus subtilis strain provided by the present application has a good application prospect in agricultural cultivation, and because its application method is relatively simple, the fermentation liquid can be directly used to irrigate the roots of crops, and it can also be further used alone or in combination with other microorganisms as a bacterial fertilizer. Therefore, it has good practical value and promotion and application significance in improving the quality of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the growth morphology on the culture medium of the bacteria; Figure 2 Gram staining oil microscopic observation results of bacterial species; Figure 3 The results of the relevant physiological and biochemical identification experiments of the strains; CK in the figure is the blank control; Figure 4 This is a phylogenetic tree diagram constructed based on the comparison of bacterial species gene sequences; Figure 5 It is the curve diagram of the growth amount and relative content of siderophore of the strain at different time; Figure 6 To investigate the polysaccharide yield of Chrysanthemum chrysantha using different treatment methods; Figure 7 The results of soil urease activity detection in different treatment groups; Figure 8 The results of soil sucrase activity test in different treatment groups; Fig. 9 The results of soil neutral protease activity detection in different treatment groups are shown in Figure 2. Fig.10 Heat maps of the genus-level microbial communities of bacteria (left) and fungi (right) in the rhizosphere soil of annual chrysanthemum; Fig.11 Heat maps of bacterial (left) and fungal (right) microbial communities at the genus level in rhizosphere soil of continuous chrysanthemum. Fig.12 The phylum level composition and relative abundance of bacteria (left) and fungi (right) in the rhizosphere soil of annual chrysanthemum; Fig.13 The phylum-level composition and relative abundance of bacteria (left) and fungi (right) in the rhizosphere soil of continuously cropped chrysanthemum. DETAILED DESCRIPTION

[0014] The present application is further explained below with reference to the embodiments.

[0015] Example 1 This example is about the siderophore-producing Bacillus subtilis ( Bacillus subtilis ) The screening and acquisition process of M13 is briefly introduced as follows.

[0016] 1. Source of screening samples: Since the quality of yam is closely related to soil rhizosphere microorganisms, the inventors speculated that several beneficial strains could be isolated and screened from yam or yam rhizosphere soil. Therefore, the inventors collected several yam samples after planting and harvesting in Wuzhi County, Henan Province and other places in 2022 to isolate and screen the species (strains).

[0017] (II) Separation and screening process: The plate streak method was used to separate, purify and culture the bacterial species (strains) collected from the processed yam samples (cut into small sections after surface disinfection): first culture at a constant temperature of 25°C, then pick a single colony and inoculate it onto a nutrient agar slant and culture at a constant temperature of 25°C, and further screen and identify according to the purpose.

[0018] Considering that iron has an important influence on plant growth and crop quality, the inventors focused on screening related bacterial species (strains) that can produce siderophores based on CAS culture medium.

[0019] The iron salt and other components in the siderophore CAS culture medium plate are: 50 ml of chrome azuro blue S solution (60.5 mg of chrome azuro blue S dissolved in 50 mL of deionized water), 10 mL of trivalent iron solution (1 mmoL / L FeCl3.6H2O, 10 mmoL / L hydrochloric acid as solvent), 40 mL of CTAB solution (72.9 mg of CTAB dissolved in 40 mL of deionized water), after mixing evenly, the volume is fixed to 100 ml; after adjusting the pH to 7.0, sterilize at 121°C for 20 min; The screening criteria are: observe the surrounding conditions of the colonies after 3-5 days of culture, and those with an orange halo around the colonies are the strains that can produce siderophores. Finally, the purified strain with the largest halo (the largest halo also means the strongest ability to produce siderophores) is selected for further identification.

[0020] 3. Identification of bacterial species: Based on the above separation and screening results, the siderophore-producing Bacillus subtilis ( Bacillus subtilis ) The identification process and results of M13 are briefly introduced as follows.

[0021] Physiological and biochemical identification: When observing the growth morphology, the M13 strain to be identified was inoculated onto the seed solid culture medium with an inoculation loop and cultured by streaking at 30°C for 24 hours, and then its colony morphology was observed. Figure 1 As shown in the figure, it can be seen that the bacterial body is short rod-shaped, arranged singly or in pairs, and occasionally in chains; it has no motility.

[0022] The strain was further stained and identified using a Gram staining kit, and the morphology and color were observed under an oil microscope. Figure 2 As shown, it can be seen that the M13 strain appears purple, indicating that the strain is a Gram-positive bacterium.

[0023] Refer to the instructions in the Manual of Common Bacteria Identification to conduct carbon source decomposition, enzyme contact reaction, methyl red, VP experiment, starch hydrolysis, gelatin liquefaction reaction and other experimental tests. The relevant experimental results are as follows: Figure 3 As shown in Table 1 below.

[0024] Table 1. Results of some physiological and biochemical experiments Molecular Biology Identification: Combined with the existing conventional genome extraction and sequencing methods, the relevant gene sequences of the M13 strain were compared with the sequences in the existing database, and the phylogenetic tree was further constructed using the neighbor-joining method for highly homologous DNA sequences (such as Figure 4 as shown).

[0025] Based on the above physical and chemical identification and molecular biological analysis results, it can be clearly seen that the M13 strain to be protected in this application is Bacillus subtilis ( Bacillus subtilis ). Further, the inventor deposited it at the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on January 12, 2023, and its deposit number is: CGMCC No.26455.

[0026] Example 2 With respect to the strain screened in Example 1, the inventors conducted further experimental tests on the secretion of siderophore by the strain in combination with specific fermentation experiments. The relevant details are briefly described as follows.

[0027] 1. Fermentation culture First, the M13 strain was inoculated into the seed culture medium and fermented at 120 r / min and 30°C until OD600 was about 0.8, which was used as the seed culture medium for later use; The seed culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 6.8±0.2.

[0028] Subsequently, the seed culture solution was inoculated into the basic fermentation medium at a 3% inoculation rate (volume ratio) and fermented for 48 hours.

[0029] The basic fermentation medium comprises the following components: sucrose 10.0 g / L, (NH4)2SO4 3.5 g / L, L-aspartic acid 1.5 g / L, L-methionine 0.02 g / L, L-histidine 0.01 g / L, KH2PO4 1.0 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, pH 6.8±0.2.

[0030] On the basis of the above basic fermentation medium, after further combining experience and single factor optimization (the specific optimization process is omitted), the composition of the optimized fermentation medium was determined to be: sucrose 20.0 g / L, (NH4)2SO4 3.5 g / L, glutamate 0.02 g / L, KH2PO4 1.0 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, pH 6.8±0.2.

[0031] On the basis of this optimized medium, the fermentation culture was carried out for 48 h. At the same time, the siderophore production was used as the evaluation index. It was considered to add Fe 3+ .

[0032] When the above-mentioned optimized fermentation medium is used for fermentation culture, the concentration of glutamate (A), Fe 3+ Concentration (B) and temperature (C) were response factors, and the siderophore content (SU) was the response value to determine a three-factor three-level experiment.

[0033] The specific influencing factors and level design are shown in Table 2.

[0034] Table 2. Factor and level design .

[0035] Based on the above Box-Behnken design, experimental tests were carried out and the results of different combinations of factors and levels are shown in Table 3 below.

[0036] Table 3. Experimental results of different factors and level combinations .

[0037] Furthermore, the above test results were subjected to variance analysis and significance test, and the results are shown in Table 4.

[0038] Table 4. ANOVA of regression model Based on the results in Table 4 above, taking the siderophore content as the investigation index (Y), and the three response factors and their interactions as independent variables, the Design expert design software was used to perform regression fitting analysis on the experimental data in Table 3 to obtain the polynomial regression equation: Y=0.4257+0.0304A-0.1800B-0.0092C-0.0176AB-0.0140AC+0.0245BC+0.0643A²+0.0686B²-0.0338C².

[0039] The analysis shows that the F value of the model is 288.81. p <0.0001, indicating that the model is extremely significant and the lack of fit p The value is 0.2605, which is greater than 0.05, indicating that it is not significant, indicating that the experimental error is small and the predicted value R 2 Pred =0.9939 and the correction value R 2 Adj = 0.9727 is close, indicating that the experimental operation and theoretical prediction are close to being credible, and the regression equation can be used to replace the actual experimental point to calculate the experimental results.

[0040] In addition, A, B, A 2 , B 2 of p The value is less than 0.0001, indicating that these model items have a very significant effect on the response value, AB, AC, BC, C 2 of p The value is less than 0.05, indicating that these model terms have a significant effect on the response value. 3 + Concentration and temperature have a significant effect on the content of siderophore. According to the above results, the main effect order of siderophore content is: B>A>C, that is, Fe 3+ >Glutamic acid>temperature.

[0041] Based on the above analysis and regression equation model, the optimal process parameters are obtained: 3+ The inventors further conducted three parallel experiments when the concentration of glutamic acid was 0µg / mL, the concentration of glutamic acid was 0.03g / L, and the temperature was 30°C. The siderophore contents were 78.79%, 80.89%, and 79.16%, respectively, and the yields were 1.64%, 1.67%, and 1.66%, respectively, with an RSD value of 1.41%. This result also further verified the correctness of the relevant optimized process parameters.

[0042] It should be explained that during the experiment, when extracting and testing the siderophore in the fermentation culture medium, the following operation can be referred to: Take 50 mL of the fermentation product after fermentation as a sample, centrifuge at 7000 r / min for 10 min, discard the bacterial precipitate, collect the supernatant and transfer it to a separatory funnel, mix it evenly with an equal volume of ethyl acetate (shake it vigorously repeatedly to mix it evenly), and let it stand to separate; The organic phase was extracted and transferred to a rotary evaporator, and distilled under reduced pressure at 40°C to concentrate the sample; After final evaporation, dissolve it in 10 mL of methanol, centrifuge it at 12000×g for 10 min, filter it through a 0.22 μm filter membrane, discard the insoluble matter, and the resulting crude siderophore is obtained.

[0043] It should be explained that when determining the relative yield of siderophore, the specific method is as follows: After sampling the culture medium, centrifuge at 7000 r / min for 10 min, mix the supernatant with CAS detection solution (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.) at a volume ratio of 1:1 and let stand at room temperature for 1 h; OD was measured on a spectrophotometer 630 The absorbance value was calculated and the relative content of siderophore (Siderophore Units, SU%) was calculated according to the following formula: The formula of relative content of siderophore is SU(%)=(Ar-As) / Ar×100%; Wherein: Ar is the absorbance value of the mixture of blank culture medium and CAS detection solution; As is the absorbance value of the mixture of culture supernatant and CAS detection solution.

[0044] 2. Relative yield of siderophore under different fermentation times (SU) Based on the above optimized process parameters, the inventors further evaluated and determined the influence of fermentation time. The experimental process is as follows: The seed culture solution prepared above (OD600=0.8) was inoculated into the optimized fermentation medium at a 3% inoculum amount (volume ratio), and cultured at 30°C and 120r / min with shaking. The biomass (OD600) and relative siderophore production SU (%) of the strain were measured at different time conditions.

[0045] The results are as follows Figure 5 As shown in the analysis, it can be seen that the growth of the strain tends to be slow at 40h and the growth reaches the highest OD at 66h. 600 The relative content of siderophore was 1.88, and the siderophore content was not synchronized with the growth of the strain. The relative content of siderophore reached the highest level of 96.31% at 102h, and the yield was 0.94% / h.

[0046] Example 3 Taking the cultivation of Dendranthe mamorifolium (Ramat.) Tzvel. as an example, the inventors conducted further application experiments on the strain of the present application, and the specific details are briefly described as follows.

[0047] In the applicant's planting base in Kaifeng, goldenrod was conventionally planted in sandy land (before planting, soil sample testing showed that the effective iron content was 3.8 mg / kg, which was iron-deficient soil). In June 2023, the bacterial culture solution was irrigated in the rhizosphere of chrysanthemum (refer to the operation of Example 2, after the strain seed solution was inoculated into the optimized fermentation medium, it was shaken and cultured at 30°C and 120rmp for 102h). The experiment was designed into 2 groups, namely: In group A, each chrysanthemum plant was watered with 30 mL of fermentation liquid and the blank control plant was replaced with 30 mL of sterile water. The chrysanthemum plants in the continuous cropping soil of group B were watered with 30 mL of fermentation liquid, and the blank control plants were replaced with 30 mL of sterile water. After watering, routine management was carried out.

[0048] Five months after application, in November 2023, the goldenrod of each experimental group was collected to detect the chrysanthemum polysaccharide content. At the same time, soil samples were collected from each treatment group to detect soil enzyme activity and microbial diversity.

[0049] (1) Chrysanthemum polysaccharide content Chrysanthemum polysaccharide content detection Figure 6 As shown in the figure, the polysaccharide content of the golden chrysanthemum in group A was 248.84 mg / g after the application of bacterial culture solution, while the content of the blank control group was 96.64 mg / g; the polysaccharide content of the golden chrysanthemum in group B was 199.03 mg / g, while the content of the blank control group was 114.51 mg / g. This result shows that the bacterial fermentation liquid can significantly increase the polysaccharide content of chrysanthemum, whether it is planted in the same year or continuously planted.

[0050] (2) Soil urease Urease is widely present in biological systems such as animals, plants, microorganisms and soil, and has important biological and ecological significance. Urease can decompose urea to provide nitrogen to plants. Its activity is positively correlated with the content of organic matter in the soil, the number of soil microorganisms, and the content of total nitrogen and available nitrogen. Soil urease activity reflects the nitrogen status of the soil to a certain extent. For this reason, the soil urease test results of different treatment groups are as follows: Figure 7 shown.

[0051] When testing the urease content in the soil: define 1 μg NH3-N produced per gram of soil sample per day as one unit of enzyme activity; use the indigo blue colorimetric method (measured using the BC0120 kit of Beijing Solebow Technology Co., Ltd., refer to its instructions for operation) to measure the NH3-N produced by urease hydrolyzing urea.

[0052] The results show that: Group A is the annual chrysanthemum treated with bacterial solution, and the urease activity result is 944.43U / g, while the control group of group A is the annual chrysanthemum control group, and the result is 546.07U / g; Group B was the group of continuously planted chrysanthemums treated with bacterial liquid, and the result was 532.90U / g. Group B control group was the continuously planted chrysanthemum control group, and the result was 374.88U / g.

[0053] The above results show that the urease in the rhizosphere soil of each group of chrysanthemums is significantly increased after bacterial liquid treatment, among which the increase in urease for annual chrysanthemums is larger, indicating that it has a better effect on annual chrysanthemums, and the urease for continuous cropping chrysanthemums is significantly increased, but the increase is not large. In short, the increase in urease indicates that the bacterial agent prepared by the strain of the present application can increase the content of organic matter and the number of microorganisms in the soil, increase nitrogen elements, improve the utilization rate of nitrogen elements by chrysanthemums, and improve soil utilization.

[0054] (3) Soil sucrase Soil sucrase is an enzyme protein present in the soil, also known as soil glucosidase. It is one of the important enzymes for biomass decomposition and organic matter circulation in the soil. It can decompose sucrose in the soil into glucose and fructose, so that these carbon sources can be used and decomposed by soil microorganisms. The products of its enzymatic action are closely related to the organic matter, nitrogen and phosphorus content in the soil, the number of microorganisms and the respiration intensity of the soil. For this reason, the results of soil sucrase activity in different treatment groups are as follows: Figure 8 shown.

[0055] When testing the sucrase (S-SC) content in soil: define 1 mg of reducing sugar produced in each gram of soil sample at 37°C per day as one activity unit; use the BC0245 kit of Beijing Solebow Technology Co., Ltd. for determination (refer to the kit manual for operation). The determination principle is: S-SC catalyzes the degradation of sucrose to produce reducing sugar, which further reacts with 3,5-dinitrosalicylic acid to generate a brown-red amino compound with characteristic light absorption at 510nm. Within a certain range, the rate of increase of light absorption at 510nm is proportional to the activity of S-SC.

[0056] The results show that: Group A is the annual chrysanthemum treated with bacterial solution, and the result of sucrase activity is 20.16U / g, while the control group of group A is the annual chrysanthemum control group, and the result is 30.44U / g; Group B was the group of continuously planted chrysanthemums treated with bacterial solution, and the result was 12.64U / g. Group B control was the control group of continuously planted chrysanthemums, and the result was 18.06U / g.

[0057] The above results show that the use of the bacterial solution of the strain of the present application has a significant effect on reducing the soil sucrase of each group of chrysanthemum. Among them, the sucrase of the blank group of annual and continuously planted chrysanthemums also has a significant difference, indicating that the rhizosphere soil of continuously planted chrysanthemums has lower fertility than the rhizosphere soil of annual chrysanthemums, and the organic matter content decreases. In short, these results show that after treatment with the bacterial solution prepared by the strain claimed in the present application, the activity of soil sucrase and the respiration intensity of the soil can be reduced to varying degrees.

[0058] (4) Soil neutral protease Soil neutral protease is a type of enzyme protein present in the soil. It is mainly involved in the conversion of soil amino acids, proteins and other organic compounds containing protein nitrogen. Its hydrolysis product is one of the nitrogen sources for higher plants and can provide nutrients for soil microorganisms and plants. Due to the different types of soil proteases, their activities and mechanisms of action are also different. Generally, soil protease activity is closely related to environmental factors such as soil texture, temperature, humidity, soil pH, and organic matter content. These factors can affect the growth and metabolism of soil microorganisms, thereby affecting the activity of soil proteases. For this reason, the test results of different treatment groups are as follows: Fig. 9 shown.

[0059] When determining the content of soil neutral protease (S-NPT): define 1μmoL tyrosine produced in each gram of soil sample per day as one activity unit; use the BC0270 kit of Beijing Solebow Technology Co., Ltd. for determination (refer to its instruction manual for operation); the determination principle is: under neutral conditions, S-NPT can hydrolyze casein to produce tyrosine; under alkaline conditions, tyrosine reduces phosphomolybdic acid compounds to produce tungsten blue; there is a characteristic absorption peak at 680nm.

[0060] The results show that: Group A is the annual chrysanthemum treated with bacterial solution, and the protease activity result is 0.27U / g; Group A control group is the annual chrysanthemum control group, and the result is 0.25U / g; Group B was the group of continuously planted chrysanthemums treated with M13 bacterial solution, and the result was 0.23U / g. Group B control group was the continuously planted chrysanthemum control group, and the result was 0.14U / g.

[0061] The above results show that the treatment with the microbial agent has little effect on the activity of neutral proteases in the rhizosphere soil of annual chrysanthemum, but significantly improves the activity of neutral proteases in the rhizosphere soil of continuously cropped chrysanthemum. It is speculated that the reason may be that the content of organic matter in the continuously cropped soil increases, and the content of enzymes involved in the stage organic matter increases, which can produce more small molecules such as amino acids.

[0062] (5) Microbial diversity Since soil microorganisms are diverse and have various functions, they are important indicators for measuring soil health quality. Therefore, we further tested and analyzed the abundance and diversity of bacteria and fungi in the rhizosphere soil of annual chrysanthemum and continuous chrysanthemum. Fig.10 , Fig.11 Specifically: like Fig.10 As shown in the figure, the relative abundance of the bacterial community in the rhizosphere soil of annual chrysanthemum is significantly different, among which the bacterial genera such as Bacteroides, Sphingomonadaceae, Acidobacteria, Skermanella, Microcoleus and Micromonosporaceae are more different; The trends of fungal communities in fungal soils were somewhat similar to those of bacterial communities, with fungal genera such as Colletotrichum, Hannaella, Talaromyces, and Metarhizium showing greater differences in rhizosphere soils.

[0063] Fig.11 The results showed that the relative abundance of bacterial communities in the rhizosphere soil of continuous chrysanthemum was significantly different. Microcoleus ), Sphingomonas spp. ( Sphingomonadaceae ), Chlorotrichum spp. Chloronema ) and other bacterial genera vary greatly; The fungal community in the soil has some similarities with the bacterial community, among which the genus Anthozoa ( Ceratobasidium ), Microsporum ( Pyrenochaetopsis ), Myrothecium spp. Myrmecridium ), Gaiella spp. Guehomyces ), Pseudogycetes ( Pseudogymnoascus ) Acrophialophora ), Cladosporium ( Cladorrhinum ), Talaromyces ( Talaromyces ), Microenvironmentella ( Microdochium ), Penicillium and Aspergillus Aspergillus ) and so on showed great differences in the rhizosphere soil of fungi.

[0064] Different classification levels can specifically reflect the state of soil microbial community structure. The composition of bacteria and fungi in the rhizosphere soil of annual chrysanthemum was analyzed at the phylum classification level. Fig.11 , Fig.12 Specifically: like Fig.12 As shown in the figure, there are 12 main bacterial phyla, accounting for 96.03% and 96.52% in groups A and B, respectively, and there are 5 main fungal phyla, accounting for 99.92% and 99.99% in groups A and B, respectively. In general, the relative abundance of bacterial communities in the two groups of rhizosphere soils has changed to a certain extent. Among them, the proportion of Actinobacteriota in the treated soil increased, the proportion of Proteobacteria increased significantly, and the proportion of Acidobacteria and Cyanobacteria decreased significantly. The relative abundance of fungal communities is quite different. Among them, Ascomycota is the dominant phylum in both groups of soils, accounting for more than 60%. After the bacterial solution treatment, the proportion of Ascomycota and Zygomycota decreased significantly, and the proportion of Basidiomycota increased. The composition of bacterial and fungal communities in the rhizosphere soil of continuous chrysanthemum was analyzed at the phylum classification level.

[0065] like Fig.13 As shown in the figure, there are 11 main bacterial phyla, accounting for 95.29% and 95.72% in groups C and D, respectively. There are 5 main fungal phyla, accounting for 99.67% and 99.98% in the two groups, respectively. In general, the relative abundance of bacterial communities in the rhizosphere soil of the two groups has a large change. After the bacterial solution treatment, the proportion of Acidobacteriota decreased significantly, and the proportion of Cyanobacteria, Firmicutes and Myxococcota increased significantly. The relative abundance of fungal communities changed greatly, among which Ascomycota was the dominant colony, but after the bacterial solution treatment, the proportion of Zygomycota and Basidiomycota increased significantly, while the proportion of Ascomycota, the dominant colony, decreased significantly.

[0066] Studies have shown that continuous bacterial cropping reduces the number of bacteria in the soil and increases the number of fungi. The main reason for continuous cropping is that the soil changes from bacterial type to fungal type. Basidiomycetes have a strong ability to decompose lignocellulose. Most of the Ascomycetes and Zygomycetes are saprophytes, which play an important role in decomposing plant residues and degrading soil organic matter. The two phyla of Proteobacteria and Firmicutes participate in the early stage of cellulose degradation. Proteobacteria are more inclined to play a role in the late stage of cellulose degradation. Proteobacteria (Proteobacteria) is a beneficial bacteria of plant and animal symbiosis. It can survive for a long time under limited resources. At the same time, Proteobacteria and Firmicutes are the main participants in the carbon dioxide fixation pathway. Acidobacteria can degrade complex lignin and cellulose to provide nutrients for the soil.

[0067] After treatment with bacterial solution, the proportion of Proteobacteria in the rhizosphere soil microorganisms of annual chrysanthemum increased, the proportion of Basidiomycota increased, and the proportion of Ascomycota and Zygomycota decreased significantly. The proportion of bacteria that decompose organic matter in the soil decreased, which may have a reducing effect on the nutrition in the soil. After treatment, the proportion of Firmicutes, Myxomycota, and Basidiomycota in the rhizosphere soil microorganisms of continuous chrysanthemum increased significantly. From the above results, it can be clearly seen that after applying the specific M13 bacterial solution of the present application, the bacterial phylum that decomposes organic matter in the soil of Goldenrod can be increased, the activity of various soil enzymes can be increased, and then the soil nutrients can be improved, and finally the polysaccharide content of Goldenrod can be increased, thereby improving its quality.

[0068] Example 4 Since the strain of the present application was screened and obtained from Dioscorea opposita Thunb.., the inventor further conducted application experiments on the strain of the present application by taking the cultivation of Dioscorea opposita Thunb.. as an example, and the specific details are briefly described as follows.

[0069] In the planting base along the Yellow River in Wuzhi County, Henan Province, to which the applicant belongs, Chinese yam is conventionally planted in sandy soil (before planting, soil sample testing showed that the effective iron content was 2.9 mg / kg, which was iron-deficient soil). In May 2024, the rhizosphere of Chinese yam was irrigated with bacterial culture solution (refer to Example 3, after the strain seed solution was inoculated into the culture medium, it was shaken and cultured at 30°C and 120 rpm for 72 hours). In that year, the soil (the first time Chinese yam was planted within 10 years) was irrigated with 60 mL of fermentation solution for each Chinese yam plant, and the blank control plants were replaced with 60 mL of sterile water for routine management.

[0070] Five months after application, in October 2024, the yam was collected to test its soluble sugar content and reducing sugar content (using the BC0035 kit of Beijing Solebaugh Technology Co., Ltd. for plant soluble polysaccharide determination and BC0235 for reducing sugar detection, refer to the instructions of the kit for operation). The results showed that: After applying the bacterial culture fluid, the soluble sugar content increased from 383.4 mg / g to 482.1 mg / g, and the reducing sugar content increased from 3.74 mg / g to 4.89 mg / g. This result shows that the bacterial fermentation fluid can significantly increase the soluble polysaccharide and reducing sugar content of Huaishan yam and improve its quality.

Claims

1. A siderophore-producing Bacillus subtilis, characterized in that: The Bacillus subtilis ( Bacillus subtilis )M13, whose accession number is CGMCC No.26455, was submitted for preservation at the China General Microbiology Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on January 12, 2023, and its survival was confirmed by testing on the same day.

2. The biological agent is prepared by using the siderophore-producing Bacillus subtilis.

3. The biological agent prepared by the siderophore-producing Bacillus subtilis according to claim 2, characterized in that: When preparing the biological agent, first inoculate Bacillus subtilis M13 into the seed culture medium, culture at 25°C and 120r / min for 2 days to prepare the seed culture solution, then inoculate the seed culture solution into the fermentation medium at a volume ratio of 3%, and ferment at 25-35°C for 48-96h; The seed culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 6.8±0.2; The fermentation medium comprises the following components: sucrose 20.0 g / L, (NH4)2SO4 3.5 g / L, glutamic acid 0.03 g / L, KH2PO4 1.0 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, pH 6.8±0.

2.

4. The use of the siderophore-producing Bacillus subtilis according to claim 1 in the preparation of a microbial fertilizer preparation, characterized in that: Used to increase the iron content in the soil environment around plant roots.

5. The use of the siderophore-producing Bacillus subtilis according to claim 1 in crop cultivation, characterized in that: Used to increase the iron content in the soil environment around plant roots.

6. The use of the siderophore-producing Bacillus subtilis in crop cultivation as claimed in claim 5, characterized in that: The crop is chrysanthemum or yam.

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