Plant growth-promoting rhizobacteria and application thereof

Abstract

The invention relates to a plant growth-promoting rhizobacteria. The preservation number of the plant growth-promoting rhizobacteria is GDMCC No: 65494. Through rhizosphere soil microorganism sampling of Gannan navel oranges, the strain G41 with the characteristics of producing indoleacetic acid, producing siderophores and solubilizing phosphorus is successfully separated out. Furthermore, pot experiments verify that the strain group is inoculated, so that the plant height, stem diameter, biomass, chlorophyll content and antioxidant enzyme activity of navel orange seedlings are increased, and the strain has an excellent growth promoting effect. Through strain identification, the strain is formally named as the bacillus subtilis G41, and the bacillus subtilis G41 is named as the bacillus subtilis G41. In particular, it is worth mentioning that the strain has rich gene resources, 32 genes related to growth promoting characteristics are excavated from the gene level, and the genes are widely distributed in key processes such as indoleacetic acid biosynthesis, siderophore biosynthesis and transport and phosphorus circulation.

Description

Technical Field

[0001] The invention relates to a plant rhizosphere growth-promoting bacterium and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) are a type of beneficial microorganisms that can promote plant growth and maintain soil ecological balance. They are favored by researchers and practitioners because they are environmentally friendly, economical, practical and sustainable, and are beneficial to agriculture. PGPR live freely in the soil or are attached to plant roots, and promote plant growth by secreting specific substances, while inhibiting the growth of other harmful bacteria. It is reported that these microorganisms promote plant growth mainly through two mechanisms: one is direct growth-promoting effect, in which microorganisms can directly have a positive effect on plants by regulating nutrient utilization (dissolving phosphorus, potassium, and nitrogen fixation), secreting growth regulators (indoleacetic acid, ACC deaminase, gibberellins, abscisic acid), and producing siderophores; the other is indirect growth-promoting effect, in which microorganisms can indirectly act on plants by recruiting beneficial microorganisms, inhibiting the reproduction of pathogens, improving soil, and inhibiting abiotic stresses, thereby producing a growth-promoting effect. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a plant rhizosphere growth-promoting bacterium.

[0004] The present invention proposes a technical solution to solve the above technical problems: a plant rhizosphere growth-promoting bacterium, named Bacillus subtilis G41, which was deposited in Guangdong Microbiological Culture Collection Center, Guangdong, China on November 18, 2014, and its abbreviation is GDMCC, and the preservation number is No: 65494.

[0005] The above-mentioned method for cultivating plant rhizosphere growth-promoting bacteria comprises the following specific steps:

[0006] The rhizosphere soil samples were collected from a navel orange tree in a navel orange orchard in Ganzhou City, Jiangxi Province, at a depth of 20-30 cm near the drip line. The soil samples were mixed evenly, and 5 g of the sample was taken from the mixed soil to prepare a soil suspension. The soil suspension was placed in a constant temperature shaking water bath at 80 ° C and 180 r / min for 15 min; then the soil suspension dilution was prepared by gradient dilution method, and the dilution multiple was 10 -1 ~10 -6 Then, 100 μL of the dilution was spread on LB solid culture medium respectively. Finally, single colonies with unique colony structure were picked and purified three times on LB solid culture medium. The purified strains were inoculated on LB slant culture medium and stored at low temperature.

[0007] The soil samples were collected from a navel orange orchard in Ganzhou City, Jiangxi Province (25.48° N, 115.09° E). ​​The orchard was divided into six different areas, and five high-quality and high-yield navel orange trees were selected in each area as sampling objects. The rhizosphere soil was collected near the drip line of the selected navel orange tree, with a digging depth of 20-30 cm. The collected rhizosphere soil samples were then placed in sterile sealed bags and immediately brought back to the laboratory for storage in a 4°C refrigerator.

[0008] The purpose of the above-mentioned plant rhizosphere growth-promoting bacteria is to promote plant growth and maintain soil ecological balance.

[0009] The purpose of the plant rhizosphere growth-promoting bacteria is to promote the growth of navel orange seedlings.

[0010] The plant rhizosphere growth-promoting bacteria are used to produce indoleacetic acid, produce siderophores and biosynthesize siderophores.

[0011] The use of the above-mentioned plant rhizosphere growth-promoting bacteria is to promote phosphorus circulation between soil and plants (navel orange seedlings).

[0012] The purpose of the above-mentioned plant rhizosphere growth-promoting bacteria is to increase the chlorophyll content and enhance the activity of antioxidant enzymes.

[0013] The present invention has positive effects: the present invention successfully isolated the strain G41 which has the characteristics of producing indoleacetic acid, producing siderophores and dissolving phosphorus through the microbial resources of the rhizosphere soil of navel orange in southern Jiangxi. It was further verified by potted experiments that the inoculated strain group increased the plant height, stem diameter, biomass, chlorophyll content and antioxidant enzyme activity of navel orange seedlings, indicating that the strain has an excellent growth-promoting effect. Through strain identification, the strain was formally named Bacillus subtilis G41. It is particularly worth mentioning that the strain has rich gene resources. The present invention has excavated a total of 32 genes related to growth-promoting characteristics at the gene level. These genes are widely distributed in the key processes of indoleacetic acid biosynthesis, siderophore biosynthesis and transport, and phosphorus cycle. Therefore, Bacillus subtilis G41 can be used as a biofertilizer in the field of sustainable agriculture in the future, and the completion of its whole genome sequencing also provides a solid theoretical basis for in-depth exploration of the genetic mechanism of its growth-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the growth trend of navel orange seedlings and the control group;

[0015] Figure 2 is the growth trend of navel orange seedlings and the control group;

[0016] Figure 3 It was strain G41 that underwent whole genome sequencing;

[0017] Figure 4 It is the phylogenetic tree of the comparison between the housekeeping gene sequence of strain G41 and the known gene sequences in the database;

[0018] Figure 5 is the functional annotation of the genome of strain G41;

[0019] Figure 6 is the COG genome functional annotation classification statistics of strain G41;

[0020] Figure 7 is the GO genome functional annotation classification statistics of strain G41;

[0021] Figure 8 It is the KEGG genome functional annotation classification statistics of strain G41. DETAILED DESCRIPTION Example

[0022] 1. Isolation method of plant growth-promoting rhizosphere bacteria

[0023] The method for isolating plant rhizosphere growth-promoting bacteria in this embodiment comprises the following specific steps:

[0024] The rhizosphere soil samples were collected from the navel orange trees in a navel orange orchard in Ganzhou City, Jiangxi Province, 20-30 cm below the drip line. The soil samples were mixed evenly, and 5 g of the sample was taken from the mixed soil to make a soil suspension. The soil suspension was placed in a constant temperature shaking water bath at 80 ° C and 180 r / min for 15 min; then the soil suspension was diluted into a dilution solution by the gradient dilution method, and the dilution multiple was 10 -1 ~10 -6 Then, 100 μL of the dilution was spread on LB solid culture medium respectively. Finally, single colonies with unique colony structure were picked and purified three times on LB solid culture medium. The purified strains were inoculated on LB slant culture medium and stored at low temperature.

[0025] The soil samples of this example were collected from a navel orange orchard in Ganzhou City, Jiangxi Province (25.48° N, 115.09° E). ​​The orchard was divided into six different areas, and five high-quality and high-yield navel orange trees were selected in each area as sampling objects. The rhizosphere soil was collected near the drip line of the selected navel orange tree, and the digging depth was 20-30 cm. The collected rhizosphere soil samples were then placed in sterile sealed bags and immediately brought back to the laboratory and stored in a 4°C refrigerator for later use.

[0026] 2. Determination of growth-promoting properties of strains

[0027] The ability of strains to secrete indole-3-acetic acid (IAA) was determined by the Salkowski colorimetric method. The isolated and purified strain G41 was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured at 37°C and 150 r / min for 24 h. 10 mL of the bacterial suspension was centrifuged at 8000 r / min for 10 min. The supernatant was mixed with the Salkowski reagent at a volume ratio of 1:1. The mixture was allowed to react in the dark for 30 min. The absorbance of the mixture at 530 nm was measured, and the IAA content was calculated based on the standard curve.

[0028] The quantitative determination of the iron carrier production of the strain was based on Payne's method. The strain was inoculated into MSA medium〔23〕, cultured at 37℃ and 150 r / min for 48 h, and then 10 mL was taken and centrifuged at 8000 r / min for 10 min. 2 mL of the supernatant and 2 mL of the CAS detection solution were taken and mixed thoroughly. After 1 h of reaction, OD630 nm (As) was measured. In addition, 2 mL of CAS detection solution and 2 mL of supernatant of MSA medium without the inoculated strain were taken and mixed evenly. After 1 h of reaction, OD630 nm (Ar) was measured. The iron carrier production of the strain is expressed in iron carrier units. The specific formula is: .

[0029] Method for testing the phosphorus solubility characteristics of strains. Strain G41 was inoculated into a conical flask containing 50 ml of liquid inorganic phosphorus formula culture medium and cultured at 37 °C and 150 r / min for 7 days. After the experiment, the liquid culture medium was centrifuged at 8000 r / min for 10 minutes, and finally the effective phosphorus content of the supernatant was determined by the molybdenum antimony colorimetric method.

[0030] 3. Experimental Design of Potted Growth Promotion for Navel Orange Seedlings

[0031] The "Newhall" navel orange seedlings with consistent growth, strong growth, and no diseases and damage were selected as the research objects. The isolated strains preserved on the slant culture medium were picked up with an inoculation loop and placed in a conical flask containing 50 ml of liquid LB culture medium, and activated and cultured at 37 ℃ and 150 r / min to the logarithmic phase. Then the bacterial activation culture solution was inoculated into the liquid LB culture medium, and the culture was shaken at 37 ℃ and 150 r / min for 24 hours. The concentration of the bacterial solution was controlled to OD600=1 with sterile distilled water for watering the navel orange seedlings. The experimental design was divided into two treatments: a control group and a strain inoculation experimental group, each treatment was repeated 6 times, with a total of 12 pots. The experimental period was a total of nine weeks, and the bacteria were watered once a week.

[0032] Measurement of various indicators of navel orange seedlings

[0033] The specific methods for measuring the plant height, stem diameter, aboveground biomass, and underground biomass of navel orange seedlings are as follows: (1) Plant height: the physiological plant height of navel orange seedlings is measured from the grafting point to the highest point; (2) Stem diameter: the diameter 2 cm away from the grafting point is measured with a vernier caliper; (3) The soil at the root of the navel orange seedlings is washed away with clean water, and then the aboveground biomass and underground biomass are weighed, and both are measured in fresh weight. The chlorophyll content in the leaves of navel orange seedlings was determined by ethanol extraction. Superoxide dismutase (SOD) was determined using the kit of Nanjing Jiancheng Bioengineering Research Institute. Peroxidase (POD) and catalase (CAT) were both determined using the kit of Beijing Solebaugh Technology Co., Ltd.

[0034] Whole genome sequencing and gene mining of strain G41

[0035] The present invention adopts de novo sequencing technology for whole genome sequencing of isolated strains. This sequencing technology can sequence and analyze the bacterial genome without relying on any reference sequence, and assemble the genome sequence from scratch using bioinformatics methods. The scanning diagram uses the second-generation sequencing platform Illumina to construct and insert a fragment of about 400bp for DNA samples that have passed the quality inspection, and perform PE150 (pair-end) sequencing, i.e. double-end sequencing, and the single-end sequencing read length is 150bp. Each sample is required to provide a raw sequencing data volume (raw data) with a coverage depth of not less than 100 times the genome, and finally assemble to obtain multiple genome scaffolds. The predicted genes are compared and analyzed with the database to obtain annotation results, and the KEGG database can further obtain KEGG Name, KO ID, KO Description and other annotation information, which helps us understand the biological function of genes from a system level. The whole genome sequencing work was completed by Shanghai Meiji Biomedical Technology Co., Ltd.

[0036] According to the sequencing results, 31 housekeeping genes were selected as comparison sequences, and the comparison analysis was performed in the local database of the sequencing company using the software BlAST+ (Version 2.3.0). The 19 bacterial strains closest to the experimental strains at the species level were obtained. Finally, the NJ (Neighbor-Joining) method was selected using the software MEGA (Version 10.1.7) to construct a phylogenetic tree.

[0037] result.

[0038] The strain G41 obtained by the above isolation method showed multiple growth-promoting properties and was officially named Bacillus subtilis G41. It was deposited in Guangdong Microbiological Culture Collection Center on November 18, 2014, with the deposit number GDMCC No: 65494.

[0039] 1. Growth-promoting properties of strain G41.

[0040] The selected strain Bacillus licheniformis G41 showed a variety of growth-promoting properties. After one day of cultivation, the indoleacetic acid production of strain G41 was 23.22 ± 0.03 mg / L. The quantitative determination of siderophore was expressed in siderophore units, and the result was 62.17 ± 0.37%. The phosphate solubilization capacity of the strain was determined by the molybdenum antimony colorimetric method and was 100.18 ± 5.87 mg / L, as shown in Table 1.

[0041] Table 1 Table 1 Plant growth-promoting properties of strain G41

[0042] 2. Effect of strain G41 inoculation on the growth of navel orange seedlings

[0043] The navel orange seedlings inoculated with strain G41 showed a significantly better growth trend than the control group ( Figure 1 , 2), specifically in terms of plant height ( Figure 2 A) Main diameter ( Figure 2 B) Aboveground biomass ( Figure 2 C) and belowground biomass ( Figure 2 D) These indicators. The average plant height of the navel orange seedlings in the control group was 55.7 cm, the average stem diameter was 8.02 mm, and the aboveground biomass and underground biomass were weighed to be 49.76 g and 20.23 g, respectively. In contrast, the plant height of the navel orange seedlings inoculated with the strain increased to 65.8 cm, an increase of 18.13%; the stem diameter increased slightly to 8.2 mm, but this difference did not reach a statistically significant level; the aboveground biomass increased significantly to 60.14 g, an increase of 20.86%; the underground biomass also increased significantly to 23.83 g, an increase of 17.8%. In summary, the strain G41 significantly promoted the overall growth of navel orange seedlings after inoculation, especially in terms of biomass accumulation.

[0044] Effects of strain G41 inoculation on chlorophyll content in leaves of navel orange seedlings

[0045] analyze Figure 2According to the data shown in E, 2F, and 2G, the average chlorophyll a content of the control group reached 0.97 mg / g, and the chlorophyll b content was 0.34 mg / g. The sum of the two, that is, the average total chlorophyll content, was 1.31 mg / g. In contrast, the experimental group performed more significantly, with the average chlorophyll a content increasing to 1.33 mg / g, and the average chlorophyll b content increasing to 0.49 mg / g, which in turn caused the average total chlorophyll content to jump to 1.82 mg / g. Specifically, compared with the control group, the chlorophyll a content of the experimental group increased by 37.11%, the chlorophyll b content increased by 44.12%, and the total chlorophyll content increased by 38.93%. This significant growth trend shows that there are obvious differences between the experimental group and the control group in terms of chlorophyll a, b and total chlorophyll content.

[0046] Effects of strain G41 inoculation on antioxidant enzyme activities in leaves of navel orange seedlings

[0047] according to Figure 2 H, 2I, 2J show that the average activity levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the control group were 819.95 U / g, 7180.55 U / g, and 323.18 U / g, respectively, and the average activity levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the strain inoculation treatment group were 1054.77 U / g, 13552.7 U / g, and 759.36 U / g, respectively, which increased by 28.64%, 88.74%, and 134.97% compared with the control group. The above results show that the SOD activity, POD activity, and CAT activity of the inoculation treatment group were significantly different from those of the control group.

[0048] Strain G41 genome profile and phylogenetic analysis

[0049] In order to further reveal the growth-promoting properties of the strain at the genetic level, the present invention sequenced the whole genome of strain G41. The sequencing results showed that the genome size of the strain was 4610067bp, the total number of scaffolds was 21, the average GC content was 46.17%, the total number of genes was 4700, the number of tRNAs was 82, the number of rRNAs was 3, and the genome type was circular. The specific characteristics of the genome are as follows Figure 3In order to identify the taxonomic affiliation of strain G41, its housekeeping gene sequence was compared with the known gene sequences in the database. The final results revealed that there was a sequence similarity of up to 99.1% between strain G41 and Bacilluslicheniformis (GCF_000011645.1), and it was determined to be a Bacillus sp. G41, and the phylogenetic tree is shown in the figure below. Figure 4 shown.

[0050] Functional annotation of strain G41 genome

[0051] like Figure 6 , 7 As shown in Figure 8, the predicted 4700 genes were compared with the three major databases of COG, GO, and KEGG to obtain the corresponding functional annotation information. Through the annotation of the COG database, the 3779 genes in strain G41 were classified into 23 specific types. Among them, there are 402 genes related to carbohydrate transport and metabolism, which is the most annotated functional type; followed by the transcription type, which contains 394 genes; the amino acid transport and metabolism type involves 367 genes; in contrast, the extracellular structure type has the least gene annotation, with only 5 genes. A total of 3117 genes were annotated in the GO database and given functional labels, including three major types of biological processes, cell components, and molecular functions, corresponding to the annotations of 1591, 1545, and 2471 genes, respectively. Among them, protein hydrolysis, membrane, and ATP binding dominate in their respective classifications. 3447 genes were annotated in the KEGG database, and these genes were divided into 6 primary classifications and 43 secondary classifications. In particular, 404 and 2616 genes were annotated in the environmental information processing and metabolism pathways, respectively. In the environmental information processing pathway, there were 212 and 189 genes related to signal transduction and membrane transport, respectively, accounting for 52.48% and 46.78% of the total number of genes in the pathway, respectively. In the metabolic pathway, the number of genes was mainly concentrated in the global and overview maps, sugar metabolism, and amino acid metabolism, with 1019, 381, and 283 related genes, respectively, accounting for 38.95%, 14.56%, and 10.82%, respectively, revealing the high activity of strain G41 in these basic metabolic processes.

[0052] Mining of Growth-Promoting Genes of Strain G41

[0053] The results of whole genome sequencing showed that strain G41 has many genes related to growth-promoting properties. As shown in Table 2, a total of 32 functional genes were mined, including 3 indoleacetic acid biosynthesis genes, 11 siderophore biosynthesis and transport genes, and 18 phosphorus cycle genes. Among these genes, aofH, amiE, and genes encoding aldehyde dehydrogenase are related to indoleacetic acid biosynthesis. Monoamine oxidase is responsible for converting tryptamine to indole-3-acetaldehyde in the tryptamine (TAM) pathway. Amidase is the last step in the indole-3-acetamide (IAM) pathway and can convert indole-3-acetamide to indole-3-acetic acid. Aldehyde dehydrogenase is a key enzyme in the indole-3-pyruvate (IPyA) pathway that can convert indole-3-acetaldehyde to indole-3-acetic acid. Genes entA, entB, entC, entE, dhbF, asbA, and menF are involved in the biosynthesis of non-ribosomal peptides of the iron carrier family; three genes related to the synthesis of iron carriers were found in the cell ABC transporter pathway, namely fecB, fecC, and fecD; hemH is involved in the synthesis of protoporphyrin / coproporphyrin ferrochelatase in the porphyrin metabolic pathway, which is related to the chelation and transport of iron. Among the phosphorus cycle functional genes, there are five genes related to phosphate mineralization, namely glpQ, glpA, glpK, phoD, and a gene encoding 3-phytase. There are a total of 7 inorganic phosphorus solubilization genes, all of which are related to the production of organic acids, including gdh, ackA, gltA, pyk, pyc, buk, and aroK. At the same time, the strain also has genes pstA, pstB, pstC, and pstS that encode proteins related to the high-affinity inorganic phosphate transport system. Phosphorus deficiency-induced response regulatory genes include phoR and phoP, as shown in Table 3.

[0054] Table 2

[0055] Table 3

[0056] 8. The present invention isolated a strain of Bacillus subtilis G41 with the ability to produce indoleacetic acid, produce siderophores, and dissolve phosphorus from a navel orange orchard in southern Jiangxi. The strain was then inoculated into the roots of navel orange seedlings for potted experiments. The results showed that various traits of the plants can be effectively promoted, further confirming the important role of microorganisms in promoting plant growth. Specifically, Shin et al. found that B. velezensis BS1 with the ability to produce hydrolases (cellulase and protease) and synthesize siderophores can promote the growth of pepper seedlings. Similarly, Sharma et al. isolated a strain of B. subtilis KU21 with the activity of dissolving phosphorus, fixing nitrogen, indole-3-acetic acid (IAA), siderophores, hydrogen cyanide (HCN), producing lyases, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase from rosemary roots. The results of subsequent tomato growth promotion experiments showed that the seed germination, plant growth parameters, nutrient acquisition, and soil quality parameters (NPK) of the experimental group were significantly higher than those of the control group. Devi et al. recovered two plant growth-promoting Bacillus strains (B. licheniformis MNNITSR2 and B. velezensis MNNITSR18) with multiple growth-promoting traits. Subsequent pot experiments showed that the rice plant height, root length, root number, tiller number, leaf number, dry weight and yield of the single inoculation and mixed inoculation treatment groups were significantly improved compared with the control group. A recent study has shown that inoculation with B. subtilis CG-6 not only significantly increased the plant height, root length, fresh weight and dry weight of alfalfa, but also significantly increased the content of antioxidant enzymes such as catalase, peroxidase, superoxide dismutase and lipoxygenase in alfalfa leaves, with an increase of 15.52% to 34.03%.

[0057] As the earliest discovered plant hormone, auxin represented by indole-3-acetic acid (IAA) is an indispensable key factor in regulating plant growth and development. It is worth noting that the existence of this biologically active substance is not limited to plants. Many microorganisms, including bacteria, yeast and fungi, have also been found to have the ability to synthesize IAA. In-depth exploration of the mechanism of microbial synthesis of IAA, existing literature has revealed five major L-tryptophan-dependent pathways: indole-3-acetamide (IAM), indole-3-acetonitrile (IAN), tryptophan side chain oxidase (TSO), tryptamine (TAM) and indole-3-pyruvate (IPyA). This conclusion has been confirmed in many research articles. For example, Ji et al. observed a complete indole-3-pyruvate (IPyA) pathway in the whole genome sequencing analysis of Bacillus amyloliquefaciens Ba13. In addition, Batista et al. clarified the existence of two synthesis pathways of IPyA and TAM in B. thuringiensisRZ2MS9. In the research field of Lysinibacillus, relevant researchers have not only discovered the genes amiE and aldH encoding enzymes related to the indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) pathways, but also key genes for the IPyA and TAM pathways. This study has discovered key genes in the three pathways of IAM, TAM, and IPyA.

[0058] Siderophores are low molecular weight compounds synthesized by bacteria, actinomycetes, fungi, some algae and plants under low iron stress. Their uniqueness lies in their ability to specifically chelate trivalent iron ions, thereby effectively alleviating iron deficiency stress. To date, the scientific community has confirmed that more than 500 compounds belong to the siderophore family. Based on the differences in the chemical groups that chelate with Fe3+, they are mainly divided into the following three categories: catechol siderophores, hydroxamate siderophores and carboxylate siderophores. In the process of exploring the synthesis mechanism of siderophores, Sheng et al. first used genome mining technology to analyze the siderophore synthesis genes of Brevibacillus brevis GZDF3, and constructed the phylogenetic tree of each synthesis gene. Finally, the strain GZDF3 was identified to have the ability to synthesize catechol siderophores by CAS liquid phase detection and Arnow method. At the same time, the whole genome sequencing results of B. subtilis TY-1 also contained gene clusters related to the synthesis of catechol siderophores bacillibactin. Chandwani et al. sequenced the whole genome of a strain of Bacillus subtilis (CWTS 5) that can produce siderophores and found many genes related to the biosynthesis and transport of siderophores. This paper systematically summarizes the genes related to the synthesis and transport of siderophores from three aspects: the biosynthesis of non-ribosomal peptides of the siderophore family, the cellular ABC transporter pathway, and the porphyrin metabolic pathway.

[0059] Phosphate solubilizing microorganisms can not only effectively promote the dissolution of inorganic phosphorus (Pi) by secreting protons, organic acids, inorganic acids and other substances, but also mineralize organic phosphorus (Po) by secreting a variety of phosphatases, thereby increasing the content of soluble phosphorus in the soil. This process enriches the soil phosphorus nutrient pool and accelerates the absorption and utilization efficiency of available phosphorus by plants. Organic acids can lower the pH value of the surrounding environment and promote the conversion of insoluble phosphates into soluble phosphates. A study on the solubilization of different inorganic phosphates by rhizobacteria of leguminous plants in acidic soils found that the pH value of the culture medium supernatant decreased during the entire reaction process and was between 5.23 and 3.29, which enhanced the dissolution of phosphates. Xu et al. analyzed the intrinsic mechanism of B. subtilis YB-04 in promoting the growth of cucumber seedlings based on genome sequencing and found that the genome of the strain contains genes pstA, pstB, pstC, and pstS encoding proteins related to the phosphate-specific transport system, response regulatory genes phoR and phoP under phosphate deficiency conditions, and alkaline phosphatase genes phoA and phoD. These genes together constitute the strain's response strategy to adapt to low-phosphorus environments. Another article not only screened out genes encoding a variety of phosphatases and proteins related to the phosphate-specific transport system, but also obtained multiple genes related to organic acid secretion. The PstABCS system has a high affinity and can be used under phosphate-limited environmental conditions. The phosphate deficiency response regulation of strain G41 is controlled by the two-component regulatory system phoR-phoP. PhoR responds to phosphate deficiency by phosphorylating phoP, and then the phosphorylated phoP can bind to specific sequences on DNA to accurately activate or inhibit gene transcription.

[0060] The present invention successfully isolated the strain G41 with the characteristics of producing indoleacetic acid, producing siderophores, and dissolving phosphorus through the microbial resources of the rhizosphere soil of navel orange in southern Jiangxi. It was further verified by potted experiments that the inoculated strain group increased the plant height, stem diameter, biomass, chlorophyll content and antioxidant enzyme activity of navel orange seedlings, indicating that the strain has an excellent growth-promoting effect. Subsequently, whole genome sequencing was performed on strain G41, and its genomic information was deeply revealed. Through strain identification, the strain was formally named Bacillus subtilis G41. It is particularly worth mentioning that the strain has rich gene resources, and a total of 32 genes related to growth-promoting characteristics have been excavated from the gene level. These genes are widely distributed in the key processes of indoleacetic acid biosynthesis, siderophore biosynthesis and transport, and phosphorus cycle.

[0061] Obviously, the above embodiments are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. However, these obvious changes or modifications derived from the spirit of the present invention are still within the scope of protection of the present invention.

Claims

1. A plant rhizosphere growth-promoting bacterium, characterized in that: The deposit number is GDMCC No: 65494.

2. Use of the plant rhizosphere growth-promoting bacteria as described in claim 1 in promoting plant growth.

3. Use of the plant rhizosphere growth-promoting bacteria as described in claim 1 in promoting the growth of navel orange seedlings.

4. Use of the plant growth-promoting rhizobacteria as described in claim 1 in the production of indoleacetic acid, siderophore production and siderophore biosynthesis.

5. Use of the plant rhizosphere growth-promoting bacteria as claimed in claim 1 in promoting phosphorus circulation between soil and plants.

6. The plant rhizosphere growth-promoting bacteria as claimed in claim 1 increase the chlorophyll content of navel orange seedlings.

7. Use of the plant rhizosphere growth-promoting bacteria as claimed in claim 1 in improving the antioxidant enzyme activity of navel orange seedlings.

8. A fertilizer, characterized in that: Contains the plant rhizosphere growth-promoting bacteria as described in claim 1.

9. A soil conditioner, characterized in that: Contains the plant rhizosphere growth-promoting bacteria as described in claim 1.

10. A method for screening plant rhizosphere growth-promoting bacteria, characterized in that: The following steps are involved: Soil samples were collected from 20-30 cm below the drip line of navel orange trees in a navel orange orchard in Ganzhou City, Jiangxi Province. The soil samples were mixed evenly, and 5 g of the sample was taken from the mixed soil to make a soil suspension. The soil suspension was placed in a constant temperature shaking water bath at 80°C and 180 r / min for 15 min; then the soil suspension was diluted into a dilution solution by a gradient dilution method, and the dilution multiple was 10 -1 ~10 -6 Then, 100 μL of the dilution was spread on LB solid culture medium respectively. Finally, single colonies with unique colony structure were picked and purified three times on LB solid culture medium. The purified strains were inoculated on LB slant culture medium and stored at low temperature.

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