Bradyrhizobium anhuiensis ASHBWH-23-9 with functions of promoting growth and inhibiting aspergillus flavus and application of bradyrhizobium anhuiensis ASHBWH-23-9

Abstract

The invention discloses bradyrhizobium yuanmingense ASHBWH-23-9 with functions of promoting growth and inhibiting aspergillus flavus and application of the bradyrhizobium yuanmingense ASHBWH-23-9, and the preservation number of the bradyrhizobium yuanmingense ASHBWH-23-9 is CCTCC (China Center for Type Culture Collection) M 20253053. The innovation of the invention lies in that the dual functions of generating cyclopentanone antibacterial volatile matters and efficiently symbiotically fixing nitrogen are integrated in the bradyrhizobium in the circular and clear garden for the first time. The molecular mechanism is that the generated volatile substance can accurately down-regulate the expression of a toxin synthesis key gene and a spore development core gene in aspergillus flavus. Compared with an existing microbial agent with a single function, nodulation and nitrogen fixation promotion and aspergillus flavus inhibition at the source can be synchronously realized at the rhizosphere of the peanuts through one-time inoculation, and particularly, aiming at the characteristics of overground flowering and underground fruiting of the peanuts, the microbial agent continuously plays a role in bacteriostasis and antipoison in soil in the pod development period.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Aspergillus flavus ASHBWH-23-9 that has both highly efficient growth-promoting and Aspergillus-inhibiting functions and its applications. Background Technology

[0002] As an important oilseed and cash crop, peanut sustainable production faces two core challenges: sustained yield increases and aflatoxin contamination control. Currently, the industry mainly relies on two parallel technological approaches:

[0003] Growth-promoting and yield-increasing technologies mainly involve the application of general plant rhizosphere growth-promoting bacteria (PGPR, such as Bacillus and Pseudomonas) to achieve non-specific growth promotion. However, these bacteria cannot effectively activate the root nodule symbiotic nitrogen-fixing system unique to leguminous crops, resulting in a biological bottleneck in yield-increasing effects.

[0004] Aflatoxin control technologies mainly include agricultural management (passive effect), physicochemical methods (high cost and risk of residue) and biological control (mainly relying on non-toxin-producing Aspergillus flavus, Bacillus and other antagonistic bacteria).

[0005] However, existing technologies have the following systemic defects: (1) Functional fragmentation and lack of synergy: Existing growth-promoting agents (including traditional rhizobia) generally lack the ability to effectively inhibit Aspergillus flavus, while biocontrol agents do not directly contribute to the symbiotic nitrogen fixation process. Simple combination of the two is prone to competitive antagonism, and the field effect is unstable. (2) Insufficient adaptation to the biological characteristics of peanuts: The unique habit of peanuts "flowering above ground and fruiting underground" means that the pods during the key development period are buried deep in the soil, making it difficult for traditional foliar application and seed treatment to form effective protection at the source of pollution - the soil fruiting layer. (3) Gap in technology source control: In particular, there is a lack of integrated biological solutions that can achieve "efficient symbiotic nitrogen fixation" and "targeted inhibition of Aspergillus flavus" on a single microbial carrier from the same ecological niche (rhizosphere).

[0006] To address the aforementioned industry bottlenecks and technological gaps, this invention provides an innovative *Aspergillus ashBWH-23-9*, a slow-growing rhizobium strain from the Yuanmingyuan plant. This strain is the first in its species to integrate highly efficient symbiotic nitrogen fixation capabilities with the dual function of directly inhibiting *Aspergillus flavus* through the production of specific antibacterial volatiles (cyclopentanones). Its innovative significance lies in the fact that a single inoculation can simultaneously achieve nitrogen nutrient supply and disease source control in the peanut rhizosphere, fundamentally overcoming the inherent shortcomings of existing technologies such as single-function operation, cumbersome procedures, and difficulty in effectively protecting underground pods during the fruiting period. This provides a novel, integrated microbial technology solution for green, high-yield, and safe peanut production. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a *Aspergillus flavus* strain ASHBWH-23-9 from the Yuanmingyuan Garden that combines growth promotion and aspergillus inhibition functions, along with its applications.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] The primary objective of this invention is to provide a *Bradyrhizobium yuanmingense* strain ASHBWH-23-9, which possesses both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Bradyrhizobium yuanmingense*, accession number is CCTCC M20253053, 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.

[0010] A second objective of this invention is to provide a microbial agent or preparation comprising at least one of *Syntrophus ashbWH-23-9*, its fermentation product, or a culture thereof; the fermentation product comprises fermentation broth, sterile supernatant, or an active substance extracted from the fermentation broth or sterile supernatant; the culture comprises a solid culture, a liquid culture, or a dried preparation thereof.

[0011] The third objective of this invention is to provide the application of *Syntrophus ashBWH-23-9*, or a microbial agent or preparation containing *Syntrophus ashBWH-23-9*, in promoting peanut growth and nodulation nitrogen fixation.

[0012] The fourth objective of this invention is to provide the application of *Aspergillus oryzae* ASHBWH-23-9, or a microbial agent or preparation containing *Aspergillus oryzae* ASHBWH-23-9, in inhibiting the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus*.

[0013] Preferably, the application specifically achieves the inhibitory function through the volatile organic compounds released by the *Syntrophus ashBWH-23-9*.

[0014] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Aspergillus ashBWH-23-9, or a microbial agent or preparation containing Aspergillus ashBWH-23-9, to the peanut growing environment or peanut plants.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1) Significant effect on promoting growth and nitrogen fixation: The Yuanmingyuan slow-growing rhizobium ASHBWH-23-9 can comprehensively promote peanut plant growth and nodule formation and nitrogen fixation. After inoculation, peanut plant height and biomass increased by 34.9% and 31.8%, respectively, and the number of root nodules and fresh weight of root nodules increased significantly by 114.7% and 29.0%, respectively. Transcriptomic analysis further revealed that this strain can specifically activate key genes in the symbiotic signaling pathways (NSP1, NSP2) and auxin signaling pathways (SAUR, AUXIN) in peanut roots, confirming the intrinsic basis of its highly efficient growth-promoting nitrogen fixation at the molecular mechanism level.

[0017] (2) Highly effective inhibition of Aspergillus flavus activity: The volatile organic compounds (VOCs) produced by the strain exhibit a strong and comprehensive inhibitory effect on Aspergillus flavus. Experiments show that its inhibition rates on Aspergillus flavus mycelial growth, spore reproduction, and aflatoxin B1 synthesis are as high as 76.3%, 97.4%, and 96.4%, respectively. Activated carbon adsorption experiments confirm that its antibacterial effect is mainly mediated by volatile substances.

[0018] (3) Key antibacterial components are clearly identified and the mechanism is well understood: Cyclopentanone was identified as the key volatile active substance responsible for its antibacterial effect. This compound exhibits a strong antibacterial effect even at low concentrations, with an inhibition rate of 97.9% after undiluted treatment, and shows a clear dose-response effect. Molecular mechanism studies have shown that the VOCs produced by ASHBWH-23-9 can precisely and significantly downregulate the expression of key regulatory genes (aflR, aflS) for aflatoxin synthesis and core genes (abaA, wetA) for spore development, thereby blocking the toxin production and reproduction of Aspergillus flavus at the transcriptional level.

[0019] (4) Significant advantages of integrated innovation and application: This invention is the first to successfully integrate the dual functions of "highly efficient symbiotic nitrogen fixation" and "cyclopentanone-mediated potent biocontrol" in the same strain of *Staphylococcus aureus* from Yuanmingyuan. With a single application, the dual goals of promoting nodulation and nitrogen fixation and inhibiting *Aspergillus flavus* can be achieved simultaneously in the peanut rhizosphere niche. This technology is particularly effective for the biological characteristics of peanuts, which "flower above ground and bear fruit underground," and plays a continuous protective role in the key soil microenvironment for pod development, providing an integrated and source-oriented green biotechnology solution to the industrial problem of increasing peanut yield and controlling toxins.

[0020] This invention provides a *Bradyrhizobium yuanmingense* strain ASHBWH-23-9, which possesses both growth-promoting and aflatoxin-inhibiting functions. This strain was obtained by the inventors through screening. The accession number for *Bradyrhizobium yuanmingense* is CCTCC M20253053, 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

[0021] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of Bradyrhizobium in peanut rhizosphere soil. The independent samples t-test was used for statistical analysis in the figure, and the data are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p < 0.001).

[0022] Figure 2 This invention constructs an ASHBWH-23-9 phylogenetic tree based on multiple genes (16S, atpD, recA, dnaK, glnII, 23S).

[0023] Figure 3 This invention relates to the phenotypic effects of inoculation with the slow-growing rhizobium bacillus ASHBWH-23-9 on peanut seedling growth and root nodule formation; wherein, Figure 3 A shows a comparison of the in-situ growth status of the plants in the flowerpots; Figure 3 B shows a close-up comparison of plant roots; in the figure, CK represents the blank control group and T represents the ASHBWH-23-9 treatment group.

[0024] Figure 4 This invention relates to the effects of inoculation with the slow-growing rhizobium bacillus ASHBWH-23-9 on key phenotypic indicators of peanut seedling growth and symbiotic nodulation; among which, Figure 4 A represents the effect on plant height; Figure 4 B represents the effect on the fresh weight of the plant; Figure 4 C represents the effect on the number of root nodules; Figure 4 D represents the effect on the fresh weight of root nodules; in the figure, CK represents the blank control group, and T represents the ASHBWH-23-9 treatment group; the column height represents the mean of each index, and the error bar represents the standard deviation (n ≥ 3); indicates that the difference between groups is extremely significant (independent samples t-test). p < 0.001).

[0025] Figure 5This is a schematic diagram illustrating the upregulation of key genes related to peanut root symbiosis and growth induced by the treatment of *Syntropha curcas* ASHBWH-23-9 in this invention; wherein, Figure 5 A represents the expression level of NSP1, a core transcription factor gene in the nodule signaling pathway; Figure 5 B represents the expression level of NSP2, a co-regulatory factor gene of the nodule signaling pathway; Figure 5 C represents the expression level of the auxin early response factor gene SAUR; Figure 5 D represents the expression level of the auxin early response factor gene AUXIN. In the figure, CK represents the blank control group, and T represents the APFJPT-23-4 treatment group; the column height represents the gene expression level (based on standardized counts); the error bar represents the standard deviation (n=3). , and The values ​​represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0026] Figure 6 This invention evaluates the inhibitory effect of *Aspergillus oryzae* ASHBWH-23-9 on the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus* using a double-plate method; wherein... Figure 6 A represents the diameter of Aspergillus flavus colonies; Figure 6 B represents conidia production; Figure 6 C represents aflatoxin production. In the figure, CK represents the blank control group, and T represents the ASHBWH-23-9 treatment group; quantitative data are expressed as mean ± standard deviation (n ≥ 3); indicates highly significant differences between groups (independent samples t-test). ).

[0027] Figure 7 This is a bar chart showing the quantitative statistical results of Aspergillus colony diameter in the blank control group (CK), blank control group + activated carbon (CK + Car), ASHBWH-23-9 treatment group (T), and ASHBWH-23-9 treatment group + activated carbon (T + Car); The figure shows... The differences between groups were statistically significant (p < 0.001).

[0028] Figure 8 This invention relates to the effect of treatment with the *Syntrophus ashBWH-23-9* on the expression of core regulatory genes for aflatoxin synthesis and spore development; wherein, Figure 8 A represents the expression level of aflatoxin synthesis core transcription factor gene aflR; Figure 8 B represents the expression level of the aflatoxin synthesis coactivator gene aflS; Figure 8 C represents the expression level of abaA, a key regulatory gene for conidial development; Figure 8D represents the expression level of wetA, a key regulatory gene for conidial maturation; in the figure, boxes represent quartile ranges, the midline represents the median, the whisker lines represent ranges, and dots represent individual cells. , and The values ​​represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0029] Figure 9 This invention uses headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) to detect and compare the relative abundance of cyclopentanone, a volatile component, in the blank control group (CK) and the ASHBWH-23-9 treatment group (T); the box plot in the figure shows the distribution of this compound in the two groups; This indicates that the difference between the two groups is extremely significant (independent samples t-test, p < 0.001). Detailed Implementation

[0030] The preferred embodiments of the present invention will be described below with reference to the figures. 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.

[0031] Example 1: Isolation, identification and preservation of the multifunctional slow-growing rhizobium ASHBWH-23-9.

[0032] The *Aspergillus oryzae* ASHBWH-23-9 of this invention was isolated from peanut rhizosphere soil treated with ARC microbial agent (a compound agent mainly composed of *Bacillus amyloliquefaciens*, *Brevibacillus laterosporus*, *Bacillus mucilaginosus*, and *Enterobacter ludwigii*). The effective viable count of *Bacillus amyloliquefaciens* in the microbial agent was ≥ 2 × 10⁻⁶. 9 CFU / g, effective viable count of Bacillus laterosporus brevis ≥ 2×10⁻⁶ 9 CFU / g, Bacillus subtilis ≥ 1×10 10 CFU / g, effective viable count of Ludwig's Enterobacter ≥ 1×10⁻⁶ 10 CFU / g, effective viable count of Flavobacterium breve ≥ 2×10⁻⁶ 9 CFU / gram.

[0033] Scientific basis: Microbial community analysis was performed on amplicon sequencing data of the 16S rRNA gene V3-V4 region from peanut rhizosphere soil samples, with a focus on the genus *Bradyrhizobium* spp., which is closely related to peanut symbiotic nitrogen fixation. For example... Figure 1 As shown, the average relative abundance of *Syntrophus* spp. in the CK group was 0.45%, while the average relative abundance in the ARC-treated group (T group) increased to 0.55%, a significant increase of 24.3% (p < 0.001). Statistical analysis indicated that this enrichment effect was extremely significant (p < 0.001). This result provides direct ecological evidence for the subsequent targeted isolation and screening of the functionally enhanced *Syntrophus ASHBWH-23-9* from the rhizosphere soil of the ARC-treated group, demonstrating that the ARC inoculant can reshape the rhizosphere microbiota and specifically promote the colonization and enrichment of beneficial symbiotic bacteria—*Syntrophus* spp.

[0034] Sample preparation: Take 1.0 g of fresh peanut rhizosphere soil sample from the ARC treatment group above, add 9.0 mL of sterile 0.85% NaCl solution, vortex for 5 minutes to prepare a soil mother suspension. Serial dilution: Take 1.0 mL of the mother suspension and perform seven consecutive 10-fold serial dilutions (10... -1 Up to 10 -7 ).

[0035] Spread culture: from 10 -5 10 -6 10 -7 100 μL of each of the three dilutions was spread onto YMA containing 0.0025% (w / v) Congo red (yeast extract 10.0 g / L, mannitol 10.0 g / L, KH2PO4 0.5 g / L). (NaCl 0.1 g / L, agar 15.0 g / L, pH 6.8-7.0) on a solid plate.

[0036] Colony selection and purification: After incubation in the dark at 28 ± 1℃ for 5-7 days, select typical slow-growing rhizobium single colonies that do not absorb Congo red, are milky white, raised, viscous, and have neat edges. Purify the culture using three consecutive streak plating tests to obtain a pure culture, designated ASHBWH-23-9.

[0037] Liquid culture: A single colony of *Syntrophus ashbWH-23-9* was inoculated into 5 mL of YMB liquid medium and cultured at 28°C with shaking at 180 rpm for 72 hours until the late logarithmic growth phase (OD200). 600(Values ​​are between 0.8 and 1.0). Short-term storage: Streak the above bacterial suspension on fresh YMA plates and store at 4°C, subculturing monthly. Long-term storage: Take the logarithmic growth phase bacterial suspension (OD value between 0.8 and 1.0). 600 ≈ 0.8) is mixed with an equal volume of sterile 60% (v / v) glycerol protectant to make a final glycerol concentration of 30%, dispensed into cryovials, and stored in an ultra-low temperature freezer at -80°C.

[0038] Genomic DNA extraction: Genomic DNA was extracted from the ASHBWH-23-9 pure culture using the TIANamp Bacteria DNA Kit according to the instructions. Its concentration and purity were measured using a spectrophotometer (A260 / A280 ratio between 1.8 and 2.0).

[0039] Gene sequencing and analysis: 16S rRNA gene: PCR amplification was performed using universal primers Fd1 and Rd1. The purified PCR product was sent for sequencing, yielding a sequence of approximately 1454 bp (sequence shown in SEQ ID NO: 3).

[0040] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0041] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3'.

[0042] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that ASHBWH-23-9 had a 16S rRNA gene similarity of >99.5% with type strains such as Bradyrhizobium yuanmingense CB1024.

[0043] Housekeeping gene identification: To accurately identify its species classification, partial sequences of the atpD, recA, dnaK, glnII, and 23S housekeeping genes were further amplified and determined.

[0044] atpD gene: Amplified using primers TSatpDf and TSatpDr, yielding a sequence of approximately 467 bp (the atpD sequence of ASHBWH-23-9 is shown in SEQ ID NO: 6).

[0045] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0046] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0047] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 481 bp (as shown in SEQ ID NO: 9).

[0048] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0049] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0050] dnaK gene: Amplified using primers BRdnaKf and BRdnaKr, yielding a sequence of approximately 674 bp (as shown in SEQ ID NO: 12).

[0051] The primer BRdnaKf sequence is shown in SEQ ID NO: 10: 5'- TTCGACATCGACGCSAACGG-3';

[0052] The primer BRdnaKr sequence is shown in SEQ ID NO: 11: 5'-GCCTGCTGCKTGTACATGGC-3'.

[0053] glnll gene: Amplified using primers TSglnIIf and TSglnIIr, a sequence of approximately 644 bp was obtained (sequence shown in SEQ ID NO: 15).

[0054] The primer TSglnIIf sequence is shown in SEQ ID NO: 13: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0055] The primer TSglnIIr sequence is shown in SEQ ID NO: 14: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0056] 23S gene: Amplified using primers FGPS1490 and FGPS130, yielding a sequence of approximately 596 bp (as shown in SEQ ID NO: 18).

[0057] The primer FGPS1490 sequence is shown in SEQ ID NO: 16: 5'-TGCGGCTGGATCACCTCCTT-3';

[0058] The primer FGPS130 sequence is shown in SEQ ID NO: 17: 5'-CCGGGTTTCCCCATTCGG-3'.

[0059] Multigene phylogenetic analysis: The gene sequences of ASHBWH-23-9 were aligned with the corresponding sequences of related slow-growing rhizobium type strains in GenBank. A phylogenetic tree was constructed using MEGA 11.0 software using the neighbor-joining method (bootstrap = 1000). The results are clearly shown (see...). Figure 2 ASHBWH-23-9 clustered with the type strain Bradyrhizobium yuanmingense CB1024, confirming its taxonomic position as Bradyrhizobium yuanmingense.

[0060] Example 2: Verification of the function of *Syntrophus ASHBWH-23-9* in promoting peanut growth and nitrogen fixation through nodulation.

[0061] Test materials: The tested peanut variety was Zhonghua 28. The soil was sandy loam, sterilized twice by autoclaving at 121℃ for 0.5 hours each time to eliminate the influence of native rhizobia. Inoculum preparation: *Syntrophus ashbWH-23-9* was cultured in YMB until mid-log (OD2). 600 (≈ 0.6), collect bacterial cells by centrifugation at 5000 rpm for 5 minutes, resuspend in sterile 0.85% NaCl solution and adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL (calibrated by plate count) was used as the inoculum suspension.

[0062] Experimental treatments: Treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned ASHBWH-23-9 bacterial suspension at a ratio of 10% (v / w) of seed weight. After slightly drying, the seeds were sown in pots. Blank control group (CK): Seeds were mixed with an equal volume of sterile 0.85% NaCl solution, and the remaining operations were the same as the treatment group. Experimental setup: A completely randomized block design was adopted, with 12 replicates (i.e., 12 pots) in each group. 8 seeds were sown in each pot, and 3 strong seedlings were retained after thinning. Culture conditions: Cultured in an artificial climate chamber with a photoperiod of 16 hours light / 8 hours dark, a day / night temperature of 28℃ / 22℃, and a relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0063] First, growth index measurement (results are shown in...) Figure 4 Table 1): Plants were harvested 30 days after sowing (seedling stage). Plant height was measured using a ruler (accuracy: 1 mm); the total fresh weight of the aboveground and underground parts of the plant was weighed using an analytical balance (accuracy: 0.01 g).

[0064] Plant height: The average plant height of the ASHBWH-23-9 treatment group (T) was 36.27 ± 2.02 cm, which was significantly increased by 34.9% compared with the blank control group (CK, 26.89 ± 2.51 cm) (p = 3.71 × 10⁻⁶). -6 ).

[0065] Fresh weight: The average fresh weight of group T was 80.425 ± 2.65 g, which was significantly higher than that of group CK (61.01 ± 3.33 g) by 31.8% (p = 2.24 × 10⁻⁶). -8 ).

[0066] Nodule count: The average number of root nodules in group T was 173.17 ± 17.85 / plant, which was significantly increased by 114.7% compared with group CK (80.67 ± 7.63 / plant) (p = 3.38 × 10⁻⁶). -9 ).

[0067] Nodule weight: The average fresh weight of root nodules in the T group was 0.196 ± 0.021 g / plant, which was significantly increased by 29.0% compared with the CK group (0.152 ± 0.024 g / plant) (p = 0.00035).

[0068] Conclusion: Inoculation with ASHBWH-23-9 significantly and comprehensively promoted the growth (plant height, fresh weight) and symbiotic nodulation ability (number of root nodules, root nodule weight) of peanut plants, indicating that this strain is a highly efficient peanut growth-promoting and symbiotic nitrogen-fixing strain.

[0069] Table 1: The promoting effect of ASHBWH-23-9 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0070]

[0071] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0072] Inoculation with the rhizobium ASHBWH-23-9 significantly promoted the growth and symbiotic nodulation of peanut seedlings, as evidenced by the obvious phenotypic differences. Figure 3As shown. In terms of overall morphology, the plants in the ASHBWH-23-9 treatment group (T) exhibited significant growth advantages: taller plants, more abundant stems and leaves, and more vigorous growth (as shown). Figure 3 A). Most importantly, direct observation of the root system ( Figure 3 B) It is clearly shown that the T group plants have a more developed root system, and the number and size of effective root nodules on their roots are far greater than those of the CK group. These striking visual evidences are highly consistent with the aforementioned measured precise quantitative data (Table 1) showing a highly significant increase in plant height (34.9%), a highly significant increase in plant fresh weight (31.8%), and highly significant increases in the number and fresh weight of root nodules (114.7% and 29.0%, respectively). Together, they constitute a complete chain of evidence that ASHBWH-23-9 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0073] Second, transcriptomics validation (molecular mechanism analysis).

[0074] Sample collection and processing: 30 days after sowing, root samples were randomly collected from 3 biological replicates of the ASHBWH-23-9 treatment group (T) and the blank control group (CK) (3 plants per replicate). The samples were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0075] RNA sequencing and analysis: Total RNA was extracted using the TRIzol® Reagent method. After quality control, paired-end 150 bp sequencing was performed using the Illumina NovaSeq 6000 platform. After quality control, the raw data were aligned to the peanut reference genome using HISAT2 software, and differentially expressed genes were analyzed using the DESeq2 R package (screening threshold: p < 0.05).

[0076] Results: To elucidate the molecular basis of ASHBWH-23-9's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that ASHBWH-23-9 treatment specifically activated a network of key genes in peanut roots related to symbiotic signal transduction and plant hormone signaling. Evidence for key gene expression is as follows (see results). Figure 5 Table 2):

[0077] Symbiotic nodulation pathway: The expression levels of NSP1 and NSP2, the core transcription factors of the nodulation signaling pathway, were significantly upregulated in group T, by 2.3-fold and 3.15-fold, respectively. These genes are the core regulatory switches that initiate the root nodulation process.

[0078] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 12.97-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 20.41-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0079] Conclusion: The above molecular evidence indicates that ASHBWH-23-9 inoculation systematically induces the co-regulation of key genes in the symbiotic signaling (NSP1, NSP2) and auxin signaling (SAUR, AUXIN) pathways in peanut roots. This reveals the intrinsic mechanism by which ASHBWH-23-9 promotes nodulation and growth at the transcriptional level: by mimicking or enhancing natural symbiotic dialogue signals, it initiates and accelerates the host's symbiotic developmental program and growth response.

[0080] Table 2: Effects of ASHBWH-23-9 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0081]

[0082] Example 3: Verification of the function of *Aspergillus flavus* ASHBWH-23-9 in inhibiting the growth and toxin production of *Aspergillus flavus*.

[0083] First, in vitro antagonism experiment (double-plate inverted method). To evaluate the antagonistic effect of ASHBWH-23-9 on Aspergillus flavus, an in vitro antagonism experiment was conducted using the double-plate inverted method.

[0084] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0085] Method: A double-layer plate interlocking method was used. The upper and lower plates were interlocked, the edges were sealed with Parafilm, and the plates were incubated in the dark at 28°C for 5 days.

[0086] Lower plate (90 mm in diameter): Treatment group (T) uniformly coated with 100 µL of 1×10⁻⁶ solution. 8 A CFU / mL fresh ASHBWH-23-9 bacterial suspension was prepared; the control group (CK) was spread onto an equal volume of sterile YMB medium. After the bacterial suspension was absorbed, the upper plate was inverted.

[0087] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0088] Measurement indicators:

[0089] Colony diameter: The diameter of Aspergillus flavus colonies was measured using the cross-hatching method, and the growth inhibition rate was calculated. Inhibition rate (%) = [(Coronary diameter of control group – Colony diameter of treatment group) / Colony diameter of control group] × 100%.

[0090] Sporulation: After culture, add 10 mL of sterile water containing 0.05% (v / v) Tween-80 to each dish, gently scrape the spores with a spreader, and count them using a hemocytometer.

[0091] Toxin determination: Mycelia were collected, lyophilized and ground, extracted with 70% methanol, purified by an immunoaffinity column, and the AFB1 content was detected by high performance liquid chromatography-fluorescence detector (HPLC-FLD) (column: C18 column; mobile phase: methanol:acetonitrile:water = 1:1:2; fluorescence detection wavelength: Ex 360 nm, Em 440 nm).

[0092] The results show (see) Figure 6 (Table 3) The ASHBWH-23-9 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by ASHBWH-23-9 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus simultaneously, achieving highly efficient source control.

[0093] Inhibition of mycelial growth: The colony diameter in the ASHBWH-23-9 treatment group (T) was 1.7 ± 0.07 cm, significantly lower than that in the blank control group (CK, 7.18 ± 0.08 cm), with an inhibition rate of 76.3% (p = 4.56 × 10⁻⁶). -14 ).

[0094] Inhibition of spore reproduction: The spore production in the ASHBWH-23-9 treatment group was 1.78 ± 0.13 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the blank control group (CK, 69 ± 10.79 × 10⁻⁶). 6 The inhibition rate reached 97.4% (p = 6.85 × 10⁻⁶). -7 ).

[0095] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the ASHBWH-23-9 treatment group was 21.44 ± 8.56 ng / plate, which was significantly lower than that in the blank control group (CK, 601 ± 150.49 ng / plate), with an inhibition rate of 96.4% (p = 3.58 × 10⁻⁶). -9 ).

[0096] Table 3: Inhibitory effect of ASHBWH-23-9 treatment on key life activities of Aspergillus flavus.

[0097]

[0098] Inhibition rate (%) = [(mean of CK group - mean of T group) / mean of CK group] × 100%. All data are expressed as mean ± standard deviation.

[0099] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0100] Based on the incubation apparatus, four treatment groups were set up. In all treatment groups, the upper plates were inoculated with a suspension of Aspergillus flavus spores (2 µL, 1×10⁻⁶) at the center point. 5 (spores / mL). Invert the two plates together, seal with Parafilm, and incubate in the dark at 28°C for 5 days.

[0101] CK group (blank control group): The lower plate was not inoculated with ASHBWH-23-9 and no activated carbon was used.

[0102] CK + Car group (activated carbon control): The lower plate was not inoculated with ASHBWH-23-9, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0103] Group T (with ASHBWH-23-9, without activated carbon): The lower plate was evenly coated with ASHBWH-23-9 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0104] T + Car group (with ASHBWH-23-9 and activated carbon): Half of the lower plate was coated with an equal amount of ASHBWH-23-9 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0105] Results and Analysis (See Results) Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of ASHBWH-23-9. This experiment proves that the inhibitory effect of ASHBWH-23-9 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0106] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.18 ± 0.08 cm) and the CK + Car group (7.14 ± 0.06 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0107] ASHBWH-23-9 releases potent antibacterial VOCs: The colony diameter in group T (1.7 ± 0.07 cm) was significantly smaller than that in group CK, with an antibacterial rate of 76.3%.

[0108] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (3.68 ± 0.08 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 48.8%, but was still significantly lower than that of the CK group.

[0109] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0110]

[0111] The key role of volatile organic compounds (VOCs) in inhibiting Aspergillus flavus mycelial growth was evaluated using activated carbon adsorption blocking experiments. Data are expressed as mean ± standard deviation. Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0112] Third, transcriptomics verification (molecular mechanism).

[0113] To elucidate the mechanism by which ASHBWH-23-9 inhibits toxin production and sporulation in Aspergillus flavus at the molecular level, transcriptome sequencing analysis was performed on the treated Aspergillus flavus hyphae. Differential expression analysis showed that ASHBWH-23-9 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0114] Evidence for key gene expression is as follows (see results) Figure 8 Table 5):

[0115] The expression of key genes regulating aflatoxin synthesis was suppressed: the expression levels of the core transcription factor gene aflR and its co-activator gene aflS in the ASHBWH-23-9 treatment group (T) decreased to 23.6% and 16.3% of the control group, respectively, with both downregulation reaching a significant level (p < 0.05). The aflR / aflS complex is the master switch for activating the entire aflatoxin synthesis gene cluster, and its significant inhibition directly leads to the shutdown of the aflatoxin synthesis pathway.

[0116] The expression of key genes regulating conidial development was strongly suppressed: the expression of abaA and wetA, core transcription factors regulating conidial development, was extremely suppressed, with expression levels in the treatment group only 1.08% and 8.5% of those in the control group, respectively, both downregulated to a highly significant level (p < 0.001). These two genes are essential for normal conidial formation and maturation, and their sharp decrease in expression directly explains the observed significant reduction in conidial yield at the molecular level.

[0117] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by ASHBWH-23-9 can precisely interfere with the core life activity programs of Aspergillus flavus, namely, by strongly inhibiting the core regulatory hubs of toxin production (aflR / aflS) and sporulation (abaA / wetA), thereby achieving the dual effects of antibacterial and toxicity reduction.

[0118] Table 5: Inhibitory effect of ASHBWH-23-9 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0119]

[0120] Example 4: Identification and verification of antibacterial active substances of *Syntrophus ashBWH-23-9*.

[0121] To identify the characteristic volatile compounds produced by *Syntrophus yunnanensis* ASHBWH-23-9, headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) was used to perform untargeted metabolomics analysis on the volatile components of the strain culture. By comparing the ASHBWH-23-9 treatment group (T) with the blank control group (CK), a compound with extremely significant differences was identified.

[0122] The compound was clearly identified by having a retention time of 10.549 min and a retention index of 1177.7 in the chromatogram, a similarity of 909 with cyclopentanone in the NIST standard library, and a molecular ion peak of m / z 55.

[0123] Quantitative and statistical analyses further confirmed the criticality of this compound (results see...). Figure 9 Table 6):

[0124] 1) Extremely significant enrichment: The relative abundance of this compound in the ASHBWH-23-9 treatment group was 64,000 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 7.63 × 10⁻⁶). -4 ).

[0125] 2) High model importance: In the orthogonal partial least squares discriminant analysis (OPLS-DA) model, its variable importance projection (VIP) value is as high as 1.35, indicating that it is one of the core volatile markers that distinguishes ASHBWH-23-9 from the blank control.

[0126] Conclusion: Comprehensive chromatographic-mass spectrometry identification and omics statistical analysis conclusively demonstrate that cyclopentanone is a characteristic volatile compound specifically and abundantly produced by ASHBWH-23-9. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0127] Table 6: Identification and omics analysis results of cyclopentanone, a characteristic volatile substance in ASHBWH-23-9.

[0128]

[0129] The retention index and high matching degree provide double verification for qualitative identification. The VIP value is much greater than 1.0, indicating that this compound is the most critical marker to distinguish the two groups of samples.

[0130] Second, purity verification experiment.

[0131] To directly verify the antibacterial activity of the key volatile substance cyclopentanone, the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus was determined using the fumigation method. The results (see Table 7) showed that cyclopentanone had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0132] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.9%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 81.9%.

[0133] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 55.4%) to a 40-fold dilution (inhibition rate 20.9%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0134] Determining the activity threshold: When the dilution factor reaches 40 times or more, there is no substantial difference in the colony diameter between each treatment group and the control group (inhibition rate < 10%), indicating that cyclopentanone has basically lost its observable antibacterial activity at this concentration.

[0135] Conclusion: This pure product verification experiment conclusively demonstrates that cyclopentanone itself is a potent inhibitor of Aspergillus flavus mycelial growth. It exerts a significant antibacterial effect even at low concentrations, a characteristic consistent with the highly specific and significant enrichment (64,000-fold) of this compound in the volatile substances of ASHBWH-23-9, strongly confirming that cyclopentanone is the key effector molecule mediating the volatile antibacterial effect of ASHBWH-23-9.

[0136] Table 7: Effects of different dosages of cyclopentanone on the growth of Aspergillus flavus mycelia.

[0137]

[0138] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0139] 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 *Aspergillus flavus* strain ASHBWH-23-9 from the Yuanmingyuan Garden, which possesses both growth-promoting and aspergillus-inhibiting functions, characterized in that... Its classification name is Bradyrhizobium yuanmingense, accession number is CCTCC M 20253053, accession date is December 30, 2025, and it is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent or preparation, characterized in that, It comprises at least one of the following: the *Syntrophus ashBWH-23-9* as described in claim 1, its fermentation product, or its culture; the fermentation product includes fermentation broth, sterile supernatant, or an active substance extracted from the fermentation broth or sterile supernatant; the culture includes a solid culture, a liquid culture, or a dried preparation thereof.

3. The application of the *Syntrophus ASHBWH-23-9* as described in claim 1 or the microbial agent or preparation as described in claim 2 in promoting peanut growth and nodulation nitrogen fixation.

4. The application of the *Aspergillus oryzae* ASHBWH-23-9 as described in claim 1 or the microbial agent or preparation as described in claim 2 in inhibiting the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus*.

5. The application according to claim 4, characterized in that, The specific application involves achieving the inhibitory function through the volatile organic compounds released by the *Syntrophus ashBWH-23-9*.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the *Syntrophus ashBWH-23-9* as described in claim 1 or the microbial agent or preparation as described in claim 2 to the peanut growing environment or peanut plants.

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