Method for efficiently screening rhizobium with functions of promoting growth and inhibiting aspergillus flavus and application of rhizobium

Abstract

The invention discloses a method for efficiently screening rhizobium with functions of promoting growth and inhibiting aspergillus flavus and application of the method, the field ecological pre-enrichment effect of a microbial agent with an ARC function is introduced into a rhizobium screening process for the first time, and the abundance of a target flora is directionally increased from the source through the toxin control and bacterium enrichment effect of the rhizobium. The method comprises the following steps: synchronously collecting rhizosphere soil, root system and root nodule samples, and comprehensively separating rhizobium; a high-throughput primary screening system with parallel growth-promoting and antibacterial functions is constructed, and the nitrogen-fixing growth-promoting capability and the antibacterial effect of volatile substances of candidate strains are synchronously evaluated in an early stage through a pot experiment and a double-vessel buckling method. And further performing functional quantification, multi-gene phylogenetic identification and omics mechanism analysis on the positive strain. The method overcomes the defects that a traditional method is single in function and low in efficiency, and an effective way for rapidly, directionally and systematically screening multifunctional rhizobium resources from an optimized rhizosphere ecosystem is provided.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial resource screening and evaluation technology, specifically relating to a method for efficiently screening rhizobia that have both growth-promoting and aflatoxin-inhibiting functions, and its application. Background Technology

[0002] Leguminous crops such as peanuts and soybeans are important global sources of food and oil. Their sustainable production faces two core challenges: first, they rely on nitrogen fixation through symbiotic relationships with rhizobia, but under natural conditions, nodulation efficiency is low, limiting yield increases; second, their fruits are susceptible to aflatoxin contamination, seriously threatening food safety. Traditional solutions involve applying growth-promoting agents such as rhizobia and biocontrol agents separately, but these methods suffer from limitations such as single-function application, increased costs, and unstable effectiveness.

[0003] In recent years, Academician Li Peiwu's team at the Chinese Academy of Agricultural Sciences has achieved a breakthrough in ARC (Aflatoxin control, Rhizobium-nodulation induction, Coupling) functional microbial agent technology. This technology, through soil treatment with specific compound microbial agents, achieved a synergistic effect of reducing the abundance of Aspergillus flavus toxin-producing bacteria in the field by more than 60%, significantly increasing the accumulation and nodulation of peanut rhizobia, and increasing average yield by 19.67% without the application of exogenous rhizobia. This achievement demonstrates that by regulating the rhizosphere microbiota, it is entirely possible to simultaneously achieve the dual goals of "toxin control" and "nitrogen fixation."

[0004] ARC technology creates a favorable microenvironment in the field for the colonization and expression of multifunctional beneficial microorganisms, especially rhizobia that may possess both growth-promoting and antibacterial properties. However, a systematic approach is still lacking in how to efficiently and accurately isolate and identify rhizobium strains that perform core coupling functions from this optimized ecosystem and develop them into directly inoculated microbial products. Existing rhizobium screening methods mostly focus on single nitrogen-fixing and growth-promoting abilities, with isolation sources typically limited to root nodules. Furthermore, they fail to incorporate the important biocontrol trait of inhibiting Aspergillus flavus into the core screening process, resulting in single-function strains that cannot meet the urgent need for "multi-functional" microbial preparations in green production.

[0005] Therefore, developing an efficient screening method that can effectively utilize the ecological advantages created by ARC technology, systematically isolate from multi-dimensional rhizosphere habitats, and evaluate the dual functions of growth promotion and antibacterial activity in parallel is of great theoretical and applied value for exploring novel multifunctional rhizobium resources, deepening the mechanism of ARC technology, and developing a new generation of microbial inoculants. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for efficiently screening rhizobia that both promote growth and inhibit Aspergillus flavus, and its application.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] The first objective of this invention is to provide a method for efficiently screening rhizobia that both promote growth and inhibit Aspergillus flavus, comprising the following steps:

[0009] (1) Ecological pre-enrichment treatment of leguminous rhizosphere soil using ARC functional microbial agents;

[0010] (2) Collect three types of samples simultaneously from the crop treated in step (1): rhizosphere soil, roots and root nodules;

[0011] (3) The three types of samples were pretreated and serially diluted, spread on rhizobium selective medium, isolated and purified to obtain rhizobium single colonies, and a candidate strain library was established.

[0012] (4) Perform bifunctional parallel primary screening on the strains in the candidate strain library, and select strains that meet the standards of both primary screening results as primary screening positive strains.

[0013] (41) Initial screening of growth-promoting function: evaluate its promoting effect on host plant growth and nodulation through pot experiments;

[0014] (42) Initial screening of antibacterial function: The inhibitory effect of its volatile substances on the growth of Aspergillus flavus mycelium was evaluated by the double-plate inverted method;

[0015] (5) The positive strains in the initial screening are re-screened to quantify their growth-promoting and antibacterial effects, and the selected strains are identified and their functional mechanisms are analyzed.

[0016] Preferably, the ARC functional microbial agent in step (1) comprises at least three of Bacillus amyloliquefaciens, Bacillus lateralis, Bacillus mucilaginosus, and Enterobacter ludwig's bacillus; the effective viable count of the Bacillus amyloliquefaciens is ≥22 x 10⁻⁶. 9 cfu / g, the effective viable count of the *Bacillus laterosporus* ≥22 x 10⁻⁶ 9 cfu / g, the effective viable count of the Bacillus mucilaginosus is ≥1x10⁻⁶. 10 cfu / g, the effective viable count of *Enterobacter ludwig vannamei* ≥ 1 x 10⁻⁶ 10 cfu / gram.

[0017] Preferably, in step (41), the standard for the initial screening of growth-promoting function is: 30 days after inoculation with candidate strains, the growth rate of the host plant height relative to the blank control is ≥25%, and the number of root nodules per plant is not less than 150% of the blank control.

[0018] Preferably, in step (42), the standard for initial screening of antibacterial function is: the relative inhibition rate of candidate strains on the diameter of Aspergillus flavus colonies is ≥50%.

[0019] Preferably, the screening step in step (5) is as follows: the quantification standard for its promoting effect is to significantly increase the height of the host plant and the number of root nodules; the quantification standard for its antibacterial effect is to make the candidate strain inhibit the colony diameter of Aspergillus flavus by ≥60%, the inhibition rate of conidia production by ≥90%, and the inhibition rate of aflatoxin B1 production by ≥90%.

[0020] Preferably, the species identification in step (5) is a phylogenetic identification based on multiple housekeeping genes, including sequence analysis of the strain's 16S rRNA gene and at least two housekeeping genes selected from atpD, recA, dnaK, and glnII genes, and construction of a phylogenetic tree.

[0021] Preferably, the functional mechanism parsing in step (5) includes:

[0022] (51) The characteristic volatile antibacterial substances produced by the strain were identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry.

[0023] (52) The strain was analyzed to upregulate the expression of genes related to host plant symbiosis and / or downregulate the expression of genes related to aflatoxin synthesis and spore development by transcriptome sequencing technology.

[0024] Preferably, the characteristic volatile antibacterial substance in step (51) is dimethyl disulfide or 2-methylbutyric acid.

[0025] The second objective of this invention is to provide a rhizobium strain that combines growth-promoting and aflatoxin-inhibiting functions, obtained by screening using the method described above. This includes *Bradyrhizobium japonicum* (a slow-growing rhizobium of soybean) APFJPT-23-4 or *Bradyrhizobium guangzhouense* (a slow-growing rhizobium of Guangzhou). *Bradyrhizobium japonicum* APFJPT-23-4 is classified as *Bradyrhizobium japonicum* and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20253050. *Bradyrhizobium guangzhouense* (a slow-growing rhizobium of Guangzhou) is also deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20253048.

[0026] The third objective of this invention is to provide an application of a rhizobium with both growth-promoting and aflatoxin-inhibiting functions in the preparation of microbial agents, wherein the microbial agents are used to promote the growth of leguminous crops, nodulation and nitrogen fixation, and / or inhibit aflatoxin contamination.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] First, the screening efficiency is significantly improved: using ARC microbial agents for field pre-enrichment increases the proportion of target functional microbial groups from the source, greatly improving the "hit rate" of screening and overcoming the problem of low efficiency in blindly screening from natural soil.

[0029] Second, the screening coverage is more comprehensive: the three-in-one separation strategy of rhizosphere soil, roots and root nodules can capture various rhizobia colonizing in different rhizosphere microenvironments (soil, root surface and root nodules), especially those root surface colonizing bacteria that have not yet formed root nodules but have antibacterial potential, avoiding the limitations of a single source.

[0030] Third, the functional evaluation system is improved: For the first time, a high-throughput primary screening system with both growth-promoting and antibacterial functions has been established. This system provides dual checks in the early stages of screening to ensure that the selected strains possess both core functions, providing clear guidance and saving on later verification costs.

[0031] Fourth, the identification and mechanism of the strains are clear: by integrating multi-level analysis methods such as multi-gene phylogenetics, metabolomics and transcriptomics, it can not only accurately classify and identify the strains, but also deeply reveal the material basis and molecular mechanism of their growth-promoting and antibacterial functions, laying a solid scientific foundation for the protection of intellectual property rights and subsequent application development of the strains.

[0032] Fifth, its application value is clear: the method of this invention directly serves the research and development of novel multifunctional microbial inoculants. The strains screened by this method (such as the preserved APFJPT-23-4 and APHBXY-23-1) are themselves product candidate strains with great application potential. They can form a synergistic effect model of "ecological regulation + functional strain inoculation" with ARC inoculant technology, providing an innovative solution for green yield increase and toxin source control in legume crops.

[0033] Cell Preservation:

[0034] This invention provides a rhizobium with both growth-promoting and aflatoxin-inhibiting functions, including the slow-growing rhizobium of soybean APFJPT-23-4, classified as Bradyrhizobium japonicum. The slow-growing rhizobium of soybean APFJPT-23-4 was obtained by the inventors of this invention through screening. The accession number of the slow-growing rhizobium of soybean APFJPT-23-4 is CCTCCNO:M20253050, the accession date is December 30, 2025, and the depositary institution is the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University.

[0035] This invention provides a rhizobium with both growth-promoting and aflatoxin-inhibiting functions, including *Bradyrhizobium guangzhouense* APHBXY-23-1. This *Bradyrhizobium guangzhouense* was obtained by the inventors through screening. The accession number of *Bradyrhizobium guangzhouense* APHBXY-23-1 is CCTCC NO:M 20253048, the accession date is December 30, 2025, and it is deposited at the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. Attached Figure Description

[0036] Figure 1 This is a flowchart illustrating the overall process of the screening method described in this invention.

[0037] Figure 2 This is a schematic diagram of the multi-source sample (rhizosphere soil, root system, root nodules) collection and processing process in Embodiment 1 of the present invention.

[0038] Figure 3 This is a schematic diagram of the dual-function parallel primary screening system in Embodiment 1 of the present invention, showing the parallel operation of potted plant growth-promoting primary screening and double-plate inverted antibacterial primary screening.

[0039] Figure 4 This is a phylogenetic tree of rhizobium APFJPT-23-4 based on multiple gene sequences in Example 1 of the present invention. Detailed Implementation

[0040] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0041] Example 1: Screening of multifunctional rhizobium APFJPT-23-4 using the method of the present invention (see Figure 1 ).

[0042] First, ecological pre-enrichment and sample collection.

[0043] Pre-concentration treatment: In a selected peanut experimental field, ARC functional microbial inoculant was applied as a basal agent on the day of sowing. The ARC inoculant is a compound microbial preparation, whose main functional components include Bacillus amyloliquefaciens (22 x 10⁻⁶). 9 CFU / g, used to inhibit pathogens), *Brevibacillus laterosporus*, 22 x 10⁻⁶ CFU / g, used to inhibit pathogens; *Brevibacillus laterosporus*, 22 x 10⁻⁶ CFU / g. 9 CFU / g, produces antibacterial substances), Bacillus mucilaginosus (1x10⁻¹⁰). 10 CFU / g (activates soil nutrients) and Enterobacter ludwigii (1x10⁻¹) 10 (cfu / gram, producing plant hormones). Application method: Mix 2 kg of the inoculant per acre with an appropriate amount of dry, fine soil, spread it in the sowing furrow, cover with a thin layer of soil, and then sow. This step aims to reshape the rhizosphere microbiome, creating enrichment conditions for rhizobia with both growth-promoting and antibacterial functions.

[0044] Sample collection (see) Figure 2 During the peak flowering period of peanuts (approximately 60 days after sowing), healthy, disease-free plants were randomly selected from the treatment area, and the root ball was completely dug up with a shovel. After gently shaking off large clumps of soil, the following categories were collected: ① Rhizosphere soil: Collect soil tightly adhering to the roots, mix well, and weigh 1.0 gram; ② Roots: Cut off approximately 0.5 grams of secondary roots with abundant root hairs, and quickly rinse three times with sterile water to remove loose impurities; ③ Root nodules: Use tweezers to remove 5-10 bright red, plump, round, effective root nodules from the main root and lateral roots.

[0045] Second, candidate strains were isolated.

[0046] The three types of samples were placed in centrifuge tubes containing glass beads and 9.0 mL of sterile physiological saline (0.85% NaCl), respectively, and vortexed for 5 minutes to prepare soil mother suspension, root grinding solution, and root nodule grinding solution. 1.0 mL of each mother suspension was then serially diluted 10-fold to 10⁻⁶. -7 From 10 of each sample -5 10 -8 10 -7Three dilutions, 100 µL each, were spread onto yeast extract-mannitol agar (YMA) plates containing 0.0025% (w / v) Congo red indicator. All operations were performed in a laminar flow hood. Plates were incubated upside down at 28°C in the dark for 5–7 days. Picking criteria: Select typical slow-growing rhizobium single colonies that were milky white (do not absorb or weakly absorb Congo red), raised, with a smooth, viscous surface and regular edges. Purification was performed using three consecutive streak plating tests to obtain pure cultures. In this example, 10 µL of the yeast extract was obtained from a rhizosphere soil sample. -6 A target bacterium, designated APFJPT-23-4, was isolated from the dilution plate.

[0047] Third, dual-function parallel primary screening (see...) Figure 3 ).

[0048] Preliminary screening for growth-promoting function (micropot method): The tested peanut variety was Zhonghua 28. APFJPT-23-4 was inoculated into YMB liquid medium and cultured at 28°C with shaking at 180 rpm until mid-log phase (OD2). 600 (≈0.6), resuspended in sterile physiological saline and adjusted to a concentration of 1×10. 8 CFU / mL. Plump seeds were selected and treated with the above bacterial suspension at a ratio of 10% (v / w) of seed weight. After drying, the seeds were sown in pots (15 cm in diameter) filled with sterile sandy loam soil. A sterile physiological saline-treated seed control (CK) was used. Six seeds were sown per pot, and three seedlings were retained after thinning. Six replicates were set for each group. The plants were cultured in an artificial climate chamber (photoperiod 16h / 8h, day / night temperature 28°C / 22°C), under standard freshwater management, without nitrogen fertilizer. Measurements were taken 30 days after sowing. The initial screening threshold was set as follows: the average plant height of the treatment group ≥ 125% of CK, and the average number of root nodules per plant ≥ 150% of CK. The APFJPT-23-4 treatment group had a plant height approximately 132% of CK and a root nodule count approximately 180% of CK, thus passing the initial screening.

[0049] Preliminary screening for antibacterial function (simplified double-plate inverted method): The pathogen was the standard strain of Aspergillus flavus LNZW-1, which produces toxins. 20 µL of APFJPT-23-4 bacterial suspension was added to the lower YMA plate and spread evenly using a glass rod. 2 µL of Aspergillus flavus spore suspension (1×10⁻⁶) was inoculated at the center of the upper PDA plate. 5 (spores / mL). Quickly invert the upper and lower plates together, seal the edges with Parafilm, and incubate in the dark at 28°C for 4 days. The initial screening threshold was set as follows: the diameter of Aspergillus colonies in the treatment group was ≤50% of that in the control group (i.e., inhibition rate ≥50%). The colony diameter in the APFJPT-23-4 treatment group was approximately 45% of that in the control group, thus passing the initial screening.

[0050] This invention employs a parallel strategy of potted plant growth-promoting primary screening and double-plate inoculated antibacterial primary screening for high-throughput, targeted screening of candidate rhizobia. The left section illustrates the growth-promoting function screening process: after inoculation and pot culture for 30 days, plant height and root nodule count are measured, with a primary screening threshold of "plant height growth rate ≥ 25% and root nodule count ≥ 150% of control". The right section illustrates the antibacterial function screening process: using a double-plate inoculated device, Aspergillus flavus is fumigated with volatile substances produced by the candidate strain, and after culture, a primary screening threshold of "colony diameter inhibition rate ≥ 50%" is used. Only when the same strain reaches the threshold in both primary screening channels (i.e., simultaneously "positive for growth-promoting primary screening" and "positive for antibacterial primary screening") is it identified as a target strain with dual functional potential and proceeds to the next stage of rescreening. This parallel screening system is the core of this method for achieving efficient and accurate screening.

[0051] Fourth, efficient strain rescreening and accurate identification.

[0052] Plant growth promotion function rescreening: The experimental scale was expanded to 12 replicates per group. Harvest was conducted 30 days after sowing for precise measurement. The plant height in the APFJPT-23-4 treatment group was 38.85 ± 2.15 cm, which was highly significant (independent samples t-test). The root nodules were significantly higher than those in the control group (28.33 ± 3.10 cm), an increase of 37.1%; the number of root nodules per plant was 177.33 ± 26.14, which was significantly higher than those in the control group (83.88 ± 11.90), an increase of 111.4%.

[0053] Antibacterial function rescreening (standard double-plate inverted method): 100 µL of fresh APFJPT-23-4 bacterial suspension (1×10⁻⁶) was evenly spread on the lower plate. 8 The concentration of APFJPT-23-4 was measured. Aspergillus spores were inoculated at the center of the upper plate. After 5 days of sealed incubation, quantitative analysis was performed. APFJPT-23-4 showed an inhibition rate of 67.1% on Aspergillus colony diameter, 96.0% on conidia production, and 96.6% on aflatoxin B1 (AFB1) production, all meeting the optimal criteria (all >60%).

[0054] Precise species identification: Genomic DNA was extracted from APFJPT-23-4 using a bacterial genomic DNA extraction kit. The full-length 16S rRNA gene and partial sequences of the atpD, recA, and glnII housekeeping genes were amplified and sequenced by PCR. Commonly used primers in this field were employed. The obtained sequences were compared with those of relevant type strains in the GenBank database, and a multigene phylogenetic tree was constructed using MEGA software with a neighbor-joining method (see [link to MEGA software]). Figure 4The results showed that APFJPT-23-4 clustered with the type strain of Bradyrhizobium japonicum, with a bootstrap support of 99%, and was therefore classified and identified as Bradyrhizobium japonicum. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC M 20253050.

[0055] Functional Mechanism Analysis:

[0056] Identification of volatile compounds (see Table 1): Characteristic volatiles of APFJPT-23-4 were analyzed using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Dimethyl disulfide was identified as a specific, high-abundance volatile compound by comparison with a standard spectral library. The half-maximal inhibitory concentration (EC50) of this pure product against Aspergillus flavus mycelial growth was... 50 The concentration was 1.61 mg / plate.

[0057] Table 1: Core results of the identification of dimethyl disulfide, a volatile component of Rhizobium APFJPT-23-4.

[0058]

[0059] Transcriptome analysis: RNA-Seq sequencing (Illumina platform) was performed on peanut roots inoculated with APFJPT-23-4 and CK. Analysis showed that the expression level of NSP1, a core transcription factor gene of the nodulation signaling pathway, was significantly upregulated in the treatment groups. Simultaneously, RNA-Seq analysis of Aspergillus flavus treated with its volatile substances revealed that the expression levels of aflR and aflS, core regulatory genes for toxin synthesis, were significantly inhibited.

[0060] Example 2: Screening of multifunctional rhizobium APHBXY-23-1 using the method of the present invention.

[0061] This example aims to demonstrate that highly efficient bacterial strains can be screened from samples of different sources (root nodules) and to showcase the reproducibility of the method. The ecological pre-enrichment is the same as in Example 1, except that:

[0062] First, sample collection and strain isolation: the focus was on isolation from the collected root nodules. The root nodules were surface-sterilized with 75% ethanol for 30 seconds, rinsed three times with sterile water, ground, and then spread onto YMA-Congo red plates. One purified strain was selected and numbered APHBXY-23-1.

[0063] Second, dual-function parallel primary screening: the method in Example 1 was followed. APHBXY-23-1 performed well in both the growth-promoting primary screening (plant height growth rate of about 128%, root nodule number growth rate of about 165%) and the antibacterial primary screening (colon diameter inhibition rate of about 58%), and passed the primary screening.

[0064] Third, efficient strain rescreening and accurate identification.

[0065] Functional re-screening: In terms of growth promotion, APHBXY-23-1 increased peanut plant height by 31.9% and root nodule number by 110.2%; in terms of antibacterial properties, its inhibition rates on Aspergillus colony diameter, sporulation amount and AFB1 yield were 76.6%, 97.0% and 96.3%, respectively.

[0066] Species identification: Through multi-gene (16S rRNA, atpD, recA, dnaK) phylogenetic analysis, it was identified as *Bradyrhizobium guangzhouense*. This strain is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC M 20253048.

[0067] Mechanism analysis: GC-MS identified 2-methylbutanoic acid as the key volatile antibacterial substance. Transcriptome analysis showed that this strain upregulated the NSP1, NSP2 (symbiotic signaling) and SAUR (auxin response) genes in peanut roots, and downregulated the expression of aflR, aflS (toxin synthesis), abaA, and wetA (spore development) genes in Aspergillus flavus.

[0068] Example 3: Verification of the screening efficiency of the method of the present invention.

[0069] To verify the efficiency advantage of the method of the present invention over traditional methods in screening "dual-function" rhizobia, a comparative experiment was designed.

[0070] Group A (method group of this invention): From fields pretreated with ARC inoculant, screening was carried out according to the method of this invention (multi-source collection, dual-function initial screening). Approximately 300 rhizobium strains were initially isolated.

[0071] Group B (conventional method control group): Rhizobia were collected from conventional fields without ARC inoculant treatment, and only root nodules were isolated. Initial screening was performed solely based on growth-promoting ability (number of root nodules). Approximately 300 rhizobia strains were initially isolated. The strains that passed their respective initial screenings (15 strains in Group A, 20 strains in Group B) from both groups underwent a unified and rigorous bifunctional secondary screening (standards as in Example 1).

[0072] Results: Among the 15 strains in group A, 2 strains (APFJPT-23-4 and APHBXY-23-1) fully met all the preferred criteria for growth promotion and antibacterial activity, accounting for 13.3%. Among the 20 strains in group B, although 5 strains showed excellent growth promotion effects, none of them showed significant inhibitory ability against Aspergillus flavus (inhibition rate <30%).

[0073] Conclusion: The method of this invention (ARC pre-enrichment + multi-source isolation + dual-function primary screening) can selectively and efficiently screen for rhizobia that truly possess both highly efficient growth-promoting and potent antibacterial functions, while traditional methods struggle to obtain such multifunctional strains. This demonstrates the significant progress and practical value of this invention in solving specific technical problems.

[0074] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for efficiently screening rhizobia that possess both growth-promoting and aflatoxin-inhibiting functions, characterized in that, Includes the following steps: (1) Ecological pre-enrichment treatment of leguminous rhizosphere soil using ARC functional microbial agents; (2) Collect three types of samples simultaneously from the crop treated in step (1): rhizosphere soil, roots and root nodules; (3) The three types of samples were pretreated and serially diluted, then spread on rhizobium selective medium, and single colonies of rhizobium were isolated and purified to establish a candidate strain library. (4) Perform dual-function parallel primary screening on the strains in the candidate strain library, and select strains that meet the standards of both primary screening results as primary screening positive strains. (41) Initial screening of growth-promoting function: The effect of promoting growth and nodulation of host plants was evaluated through pot experiments; (42) Initial screening of antibacterial function: The inhibitory effect of its volatile substances on the growth of Aspergillus flavus mycelium was evaluated by the double-plate inverted method; (5) The positive strains in the initial screening are re-screened to quantify their growth-promoting and antibacterial effects, and the selected strains are identified and their functional mechanisms are analyzed.

2. The method according to claim 1, characterized in that, The ARC functional microbial agent in step (1) contains at least three of the following: Bacillus amyloliquefaciens, Bacillus lateralis, Bacillus mucilaginosus, and Enterobacter ludwig's bacillus; the effective viable count of the Bacillus amyloliquefaciens is ≥22 x 10⁻⁶. 9 cfu / g, the effective viable count of the *Bacillus laterosporus* ≥22 x 10⁻⁶ 9 cfu / g, the effective viable count of the Bacillus mucilaginosus is ≥1x10⁻⁶. 10 cfu / g, the effective viable count of *Enterobacter ludwig vannamei* ≥ 1 x 10⁻⁶ 10 cfu / gram.

3. The method according to claim 1, characterized in that, In step (41), the standard for the initial screening of growth-promoting function is: 30 days after inoculation with candidate strains, the growth rate of the host plant height relative to the blank control is ≥25%, and the number of root nodules per plant is not less than 150% of the blank control.

4. The method according to claim 1, characterized in that, In step (42), the standard for initial screening of antibacterial function is: the relative inhibition rate of candidate strains on the diameter of Aspergillus flavus colonies is ≥50%.

5. The method according to claim 1, characterized in that, The specific screening step in step (5) is as follows: its quantification standard for promoting efficacy is to significantly increase the height of the host plant and the number of root nodules; The quantitative standard for its antibacterial efficacy is that the candidate strain inhibits the colony diameter of Aspergillus flavus by ≥60%, the conidia production by ≥90%, and the aflatoxin B1 production by ≥90%.

6. The method according to claim 1, characterized in that, In step (5), species identification is based on phylogenetic identification of multiple housekeeping genes, including sequence analysis of the strain's 16S rRNA gene and at least two housekeeping genes selected from atpD, recA, dnaK, and glnII genes, and construction of a phylogenetic tree.

7. The method according to claim 1, characterized in that, The functional mechanism analysis in step (5) includes: (51) The characteristic volatile antibacterial substances produced by the strain were identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry. (52) The strain was analyzed to upregulate the expression of genes related to host plant symbiosis and / or downregulate the expression of genes related to aflatoxin synthesis and spore development by transcriptome sequencing technology.

8. The method according to claim 7, characterized in that, The characteristic volatile antibacterial substance in step (51) is dimethyl disulfide or 2-methylbutyric acid.

9. A rhizobium strain with both growth-promoting and aflatoxin-inhibiting functions, obtained by screening using the method described in any one of claims 1-8, characterized in that... This includes either *Bradyrhizobium japonicum* or *Bradyrhizobium guangzhouense*, specifically *Bradyrhizobium japonicum*, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20253050; or *Bradyrhizobium guangzhouense*, which is also deposited at the same collection with accession number CCTCC M 20253048.

10. An application of the rhizobium with both growth-promoting and aflatoxin-inhibiting functions as described in claim 9 in the preparation of microbial inoculants, characterized in that, The microbial agent is used to promote the growth of leguminous crops, nodulation and nitrogen fixation, and / or inhibit aflatoxin contamination.

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