Bradyrhizobium anhuiensis APXJTC-23-3 with functions of promoting growth and inhibiting aspergillus flavus and application of bradyrhizobium anhuiensis APXJTC-23-3

Abstract

The invention discloses a bradyrhizobium anhuoshanense APXJTC-23-3 with functions of promoting peanut growth and inhibiting aspergillus flavus and application of the bradyrhizobium anhuoshanense APXJTC-23-3, the bradyrhizobium anhuoshanense APXJTC-23-3 is classified and named as Bradyrhizobium anhuoshanense, and the preservation number of the bradyrhizobium anhuoshanense APXJTC-23-3 is CCTCC M 20253052. The innovation of the invention lies in that the dual functions of efficient symbiotic nitrogen fixation and production of bacteriostatic volatile matters such as 3-methylbutyric acid are integrated in the bradyrhizobium in the circular and clear garden for the first time. Through one-time application, two core functions of promoting nodulation, nitrogen fixation and growth of peanuts can be synchronously realized in a rhizosphere ecological niche of the peanuts; and infection of aspergillus flavus and toxin synthesis are directly inhibited in soil in the key period of legume development. The invention provides a green biological solution for realizing integration of yield increase and poison prevention from a root source especially aiming at the traditional prevention and control problem caused by overground flowering and underground fruiting of peanuts.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a slow-growing rhizobium strain APXJTC-23-3 from Yuanmingyuan that has both growth-promoting and aflatoxin-inhibiting functions and its applications. Background Technology

[0002] Peanuts (Arachis hypogaea L.) are an important global oilseed and cash crop, and their safe production has long faced two interrelated challenges: sustained yield increases and aflatoxin contamination control. Currently, the industry mainly relies on two parallel but separate technological pathways:

[0003] I. Growth-promoting and yield-increasing technologies primarily rely on the application of general-purpose plant rhizosphere growth promoters (PGPRs), such as Bacillus spp. and Pseudomonas spp. These agents mainly promote growth through non-specific pathways such as dissolving phosphorus and potassium or secreting plant hormones. However, for legumes like peanuts, the most crucial and efficient yield-increasing mechanism depends on the formation of root nodules and biological nitrogen fixation with specific symbiotic nitrogen-fixing microorganisms—rhizobia. Existing general-purpose PGPRs cannot effectively replace or activate this specific symbiotic system, and their yield-increasing effect has a biological upper limit.

[0004] II. Aflatoxin contamination control technologies mainly include:

[0005] 1. Agricultural management measures (such as crop rotation and selection of resistant varieties) have passive and unstable effects.

[0006] 2. Physicochemical methods (such as irradiation, chemical fumigants (such as propionate), and adsorbents (such as montmorillonite)) are mostly used for post-production processing. They are costly and pose risks of affecting quality and chemical residues. Their application in food-grade raw materials is subject to strict regulations.

[0007] 3. Biological control methods are currently a hot research topic and direction, mainly utilizing microbial antagonists, such as non-toxin-producing Aspergillus flavus, Bacillus, Pseudomonas, and Trichoderma spp. Their mechanisms of action include competing for nutrients and space, secreting antimicrobial substances, or inducing systemic resistance in plants.

[0008] Systemic defects and bottlenecks in existing technologies:

[0009] 1. Limited Function and Lack of Synergistic Effect: Existing growth-promoting microbial agents (including conventional rhizobium inoculants) generally lack efficient and direct inhibition capabilities against Aspergillus flavus. Conversely, existing biocontrol agents typically do not directly contribute to promoting the core symbiotic nitrogen fixation process in leguminous crops. Simultaneous application of both types of products in the field not only increases costs and operational complexity but may also lead to unstable or even mutually negating effects due to interspecies competition or antagonism among microorganisms.

[0010] 2. Fragmented Key Technological Pathways: "Increased Yield" and "Virus Prevention" are treated as two separate technical issues. There is a lack of an integrated solution that combines the dual functions of "efficient symbiotic nitrogen fixation" and "targeted inhibition of Aspergillus flavus" on a single microbial carrier, starting from the same ecological niche (rhizosphere). Simple microbial agent formulations struggle to achieve stable and synergistic efficacy in complex field environments.

[0011] 3. Insufficient control over the unique biological characteristics and pollution sources of peanuts: Aspergillus flavus infection originates in the field soil. The unique growth habit of peanuts—flowering above ground and fruiting underground—means that the crucial pod development period is deeply buried in the soil. This leads to physical obstacles for traditional control methods during critical periods: seed treatments at the sowing stage are unlikely to maintain their effectiveness until the fruiting stage; foliar application during the fruiting stage cannot reach the underground pods; and applying chemical fungicides to the soil through irrigation systems disrupts the rhizosphere microecological balance and easily induces drug resistance in pathogens. Existing technologies mostly focus on post-harvest treatment, with a severe lack of proactive and efficient methods to block pollution sources in the field.

[0012] 4. Lack of Integrated Core Biological Functions: To date, no Bradyrhizobium spp. strain, particularly Bradyrhizobium yuanmingense, has been reported in publicly available literature and commercial products to possess the dual functions of efficiently promoting symbiotic nodulation and nitrogen fixation in peanuts while directly and effectively inhibiting the growth of Aspergillus flavus. This technological gap leaves a lack of effective biological tools for simultaneously addressing peanut yield increase and toxin control at their root causes.

[0013] Therefore, there is an urgent need in this field for an innovative biological solution: a novel multifunctional rhizobium strain that can function as both a highly efficient symbiotic nitrogen-fixing bacterium and an in-situ biocontrol bacterium. This strain should be able to simultaneously achieve the dual objectives of promoting nodulation and nitrogen fixation and inhibiting Aspergillus flavus at its source within the same ecological niche of the rhizosphere during the same peanut growth cycle, thereby overcoming the industry bottleneck of existing technologies that are fragmented and only address the symptoms without addressing the root cause. Summary of the Invention

[0014] To address the shortcomings of existing technologies, this invention provides a *Aspergillus flavus* strain APXJTC-23-3 from Yuanmingyuan, which combines growth-promoting and aspergillus-inhibiting functions, and its applications.

[0015] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0016] The first objective of this invention is to provide a *Bradyrhizobium yuanmingense* strain APXJTC-23-3, which has both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Bradyrhizobium yuanmingense*, accession number is CCTCC M20253052, 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.

[0017] The second objective of this invention is to provide a microbial agent or preparation comprising at least one of *Pseudomonas yuanmingyuanensis* APXJTC-23-3, its fermentation product, or its culture; the fermentation product includes fermentation broth, sterile supernatant, or active substances extracted from the fermentation broth or sterile supernatant; the culture includes solid culture, liquid culture, or a dried preparation thereof.

[0018] The third objective of this invention is to provide the application of *Phyllostachys yuanmingyuanensis* APXJTC-23-3, or a microbial agent or preparation containing *Phyllostachys yuanmingyuanensis* APXJTC-23-3, in promoting peanut growth and nodulation nitrogen fixation.

[0019] The fourth objective of this invention is to provide the application of *Aspergillus oryzae* APXJTC-23-3, or a microbial agent or preparation containing *Aspergillus oryzae* APXJTC-23-3, in inhibiting the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus*.

[0020] Preferably, the application specifically achieves the inhibitory function through the volatile organic compounds released by the Yuanmingyuan slow-growing rhizobium APXJTC-23-3.

[0021] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned *Aspergillus oryzae* APXJTC-23-3, or a microbial agent or preparation containing *Aspergillus oryzae* APXJTC-23-3, to the peanut growing environment or peanut plants.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) Excellent effect on promoting growth and nitrogen fixation: APXJTC-23-3, a slow-growing rhizobium from Yuanmingyuan, can effectively promote the growth and symbiotic nodulation of peanut plants. After 30 days of inoculation, the plant height and biomass increased significantly by 36.4% and 37.6% respectively compared with the control; at the same time, the number of root nodules and the fresh weight of root nodules increased significantly by 114.7% and 51.3% respectively. Transcriptome analysis further revealed that this strain can specifically upregulate the expression of key genes in the nodulation signaling (such as NSP1, NSP2) and auxin signaling (such as SAUR) pathways in peanut roots, confirming its synergistic mechanism of promoting symbiosis and growth at the molecular level.

[0024] (2) Comprehensive and outstanding inhibitory activity against Aspergillus flavus: The volatile organic compounds (VOCs) released by the strain have a strong and broad-spectrum inhibitory effect on Aspergillus flavus. In vitro fumigation experiments showed that the inhibition rates on Aspergillus flavus mycelial growth, spore production, and aflatoxin B1 synthesis were as high as 76.0%, 97.2%, and 96.3%, respectively. Activated carbon adsorption experiments confirmed that this antibacterial effect is mainly mediated by volatile substances.

[0025] (3) The antibacterial mechanism is clear: the key antibacterial component was identified as 3-methylbutyric acid, which showed strong dose-dependent antibacterial activity even at low concentrations, with an inhibition rate of 97.7% after treatment with the original solution. Molecular mechanism studies showed that the strain VOCs could specifically and significantly downregulate the expression of the core regulatory genes (aflR, aflS) for aflatoxin synthesis and the key genes (abaA, wetA) for spore development, thereby blocking toxin production and reproduction at the transcriptional level.

[0026] (4) Integrated Functions and Strong Field Applicability: This invention is the first to achieve the innovative integration of two major functions, "efficient symbiotic nitrogen fixation" and "strong antibacterial effect mediated by volatile substances," in a single strain of *Staphylococcus aureus* from Yuanmingyuan. With a single inoculation, it can simultaneously promote growth and increase yield while preventing viral infection at the source in the same ecological niche of peanut rhizosphere. In particular, it provides a green and efficient integrated biological solution to address the traditional chemical control obstacles caused by peanut's "flowering above ground and fruiting underground."

[0027] Cell Preservation:

[0028] This invention provides a *Bradyrhizobium yuanmingense* strain APXJTC-23-3, which possesses both growth-promoting and aflatoxin-inhibiting functions. This strain is classified as *Bradyrhizobium yuanmingense*. *Bradyrhizobium yuanmingense* APXJTC-23-3 was obtained by the inventors of this invention through screening. The accession number of *Bradyrhizobium yuanmingense* APXJTC-23-3 is CCTCC M20253052, the accession date is December 30, 2025, and it is deposited at 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. Attached Figure Description

[0029] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of Bradyrhizobium spp. in peanut rhizosphere soil. The independent samples t-test was used for statistical analysis, and the data are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p < 0.001).

[0030] Figure 2 This invention constructs the APXJTC-23-3 phylogenetic tree based on multiple genes (16S, atpD, recA, dnaK, glnII, 23S).

[0031] Figure 3 This invention demonstrates the effects of inoculation treatment with the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan on peanut seedling growth and root nodule formation. Figure 3 A shows a comparison of the in-situ growth status of the plants; Figure 3 B shows a comparison of the phenotypic characteristics of the entire plant after washing; Figure 3 C represents a comprehensive comparison of mature plants, showing that after inoculation with APXJTC-23-3, the plants were taller, had darker green leaves, more developed root systems, more pods, and more root nodules. In the figure, CK represents the blank control group, and T represents the APXJTC-23-3 inoculation treatment group.

[0032] Figure 4 This invention relates to the effects of inoculation with the Yuanmingyuan slow-growing rhizobium APXJTC-23-3 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 APXJTC-23-3 inoculation treatment group; the column height represents the average value of each indicator, and the error bar represents the standard deviation (n ≥ 3). The differences between groups were statistically significant (p < 0.001).

[0033] Figure 5 This is a schematic diagram illustrating the upregulation of key genes related to peanut root symbiosis and growth induced by treatment with the Yuanmingyuan slow-growing rhizobium APXJTC-23-3; wherein, Figure 4 A represents the expression level of NSP1, a core transcription factor gene in the nodule signaling pathway; Figure 4 B represents the expression level of NSP2, a co-regulatory factor gene of the nodule signaling pathway; Figure 4 C represents the expression level of the auxin early response factor gene SAUR; Figure 4 D represents the expression of the auxin signaling pathway gene AUXIN; in the figure, CK represents the blank control group, and T represents the APFJPT-23-4 inoculation 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 numbers indicate that the differences between groups were significant (p < 0.05) and highly significant (p < 0.01), respectively.

[0034] Figure 6 This invention evaluates the inhibitory effect of the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan 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 ASHBQJ-24-3 treatment group; quantitative data are expressed as mean ± standard deviation (n ≥ 3); indicates highly significant differences between groups (independent samples t-test). ).

[0035] Figure 7 This is a bar chart showing the quantitative statistical results of Aspergillus flavus colony diameter in the blank control group (CK), blank control group (CK) + activated carbon (Car), APXJTC-23-3 treatment group (T), and APXJTC-23-3 treatment group (T) + activated carbon (Car); In the figure, The differences between groups were statistically significant (p < 0.001).

[0036] Figure 8 This invention relates to the effect of treatment with the Yuanmingyuan slow-growing rhizobium APXJTC-23-3 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 8C represents the expression level of abaA, a key regulatory gene for conidial development; Figure 8 D represents the expression level of wetA, a key regulatory gene for conidial maturation; in the figure, CK represents the blank control group, T represents the APXJTC-23-3 treatment group; 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.

[0037] Figure 9 This invention employs headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) to detect and compare the relative abundance of the volatile component 3-methylbutyric acid in the blank control group (CK) and the APXJTC-23-3 treatment group (T); box plots show the distribution of this compound in the two groups; in the figure, This indicates that the difference between the two groups is extremely significant (independent samples t-test, p < 0.01). Detailed Implementation

[0038] 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.

[0039] Example 1: Isolation, identification and preservation of APXJTC-23-3, a slow-growing rhizobium from Yuanmingyuan.

[0040] The *Pseudomonas amyloliquefaciens* APXJTC-23-3 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 is ≥ 2 × 10⁻⁶. 9 CFU / g, effective viable count of Bacillus laterosporus brevis ≥ 2 × 10⁻⁶ 9 CFU / g, Bacillus mucilaginosus ≥ 1 × 10⁻⁶ 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.

[0041] 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 showed 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 pyrenoidosa* APXJTC-23-3 from the rhizosphere soil of the ARC-treated group, indicating that the ARC inoculant can reshape the rhizosphere microbiota and specifically promote the colonization and enrichment of beneficial symbiotic bacteria—*Syntrophus pyrenoidosa*.

[0042] 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 ).

[0043] Spread culture: from 10 -5 10 -6 10 -7 100 μL of each of the three dilutions was plated onto YMA (yeast extract 10.0 g / L, mannitol 10.0 g / L, KH2PO4 0.5 g / L) containing 0.0025% (w / v) Congo red. (NaCl 0.1 g / L, agar 15.0 g / L, pH 6.8-7.0) on a solid plate.

[0044] Colony selection and purification: After incubation in the dark at 28 ± 1°C 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 by three consecutive streak plating tests to obtain a pure culture, designated APXJTC-23-3.

[0045] Liquid culture: A single colony of *Pseudomonas yuanmingyuanensis* APXJTC-23-3 was inoculated into 5 mL of YMB liquid medium and cultured at 28℃ 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 bacterial suspension in the logarithmic growth phase (OD200). 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.

[0046] Genomic DNA extraction: Genomic DNA was extracted from the APXJTC-23-3 pure culture using the TIANamp Bacteria DNA Kit according to the instructions, and its concentration and purity (A260 / A280 ratio between 1.8 and 2.0) were detected using a NanoDrop™ 2000 spectrophotometer.

[0047] 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 1426 bp (as shown in SEQ ID NO: 3).

[0048] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0049] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3'.

[0050] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that APXJTC-23-3 had a 16S rRNA gene similarity of >99.5% with type strains such as Bradyrhizobium yuanmingense CB1024.

[0051] 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.

[0052] atpD gene: Amplified using primers TSatpDf and TSatpDr, yielding a sequence of approximately 473 bp (the atpD sequence of APXJTC-23-3 is shown in SEQ ID NO: 6).

[0053] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0054] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0055] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 474 bp (as shown in SEQ ID NO: 9).

[0056] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0057] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0058] dnaK gene: Amplified using primers BRdnaKf and BRdnaKr, yielding a sequence of approximately 628 bp (as shown in SEQ ID NO: 12).

[0059] The primer BRdnaKf sequence is shown in SEQ ID NO: 10: 5'- TTCGACATCGACGCSAACGG-3';

[0060] The primer BRdnaKr sequence is shown in SEQ ID NO: 11: 5'-GCCTGCTGCKTGTACATGGC-3'.

[0061] glnll gene: Amplified using primers TSglnIIf and TSglnIIr, yielding a sequence of approximately 585 bp (as shown in SEQ ID NO: 15).

[0062] The primer TSglnIIf sequence is shown in SEQ ID NO: 13: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0063] The primer TSglnIIr sequence is shown in SEQ ID NO: 14: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0064] 23S gene: Amplified using primers FGPS1490 and FGPS130, yielding a sequence of approximately 787 bp (as shown in SEQ ID NO: 18).

[0065] The primer FGPS1490 sequence is shown in SEQ ID NO: 16: 5'-TGCGGCTGGATCACCTCCTT-3';

[0066] The primer FGPS130 sequence is shown in SEQ ID NO: 17: 5'-CCGGGTTTCCCCATTCGG-3'.

[0067] Multigene phylogenetic analysis: The gene sequences of APXJTC-23-3 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 and the neighbor-joining method (bootstrap = 1000). The results are clearly shown (see...). Figure 2 APXJTC-23-3 clustered with the type strain Bradyrhizobium yuanmingense CB1024, confirming its taxonomic position as Bradyrhizobium yuanmingense.

[0068] Example 2: Functional verification of the effects of the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan on promoting peanut growth and nitrogen fixation through nodulation.

[0069] 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 indigenous rhizobia. Inoculum preparation: APXJTC-23-3 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.

[0070] Experimental treatments: APXJTC-23-3 treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned APXJTC-23-3 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.

[0071] First, growth index measurement (see...) 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). Statistical analysis was performed using an independent samples t-test, and data are expressed as mean ± standard deviation.

[0072] Plant height: The average plant height of the APXJTC-23-3 inoculated group (T) was 37.47 ± 0.53 cm, which was significantly increased by 36.4% compared with the blank control group (CK, 27.46 ± 1.45 cm) (p = 6.07 × 10⁻⁶). -10 ).

[0073] Fresh weight: The average fresh weight of group T was 82.26 ± 4.21 g, which was significantly higher than that of group CK (59.77 ± 5.99 g) by 37.6% (p = 2.45 × 10⁻⁶). -8 ).

[0074] Nodule count: The average number of root nodules in group T was 174.67 ± 18.03 per plant, which was significantly increased by 114.7% compared with group CK (81.33 ± 8.05 per plant) (p = 3.87 × 10⁻⁶). -9 ).

[0075] Nodule weight: The average fresh weight of root nodules in group T was 0.227 ± 0.034 g / plant, which was significantly increased by 51.3% compared with group CK (0.150 ± 0.033 g / plant) (p = 6.89 × 10⁻⁶). -5 ).

[0076] Conclusion: Inoculation with APXJTC-23-3 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.

[0077] Table 1: The promoting effect of APXJTC-23-3 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0078]

[0079] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0080] Inoculation with the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan Garden significantly promoted the growth and symbiotic nodulation of peanut seedlings, with the following direct phenotypic differences: Figure 3 As shown. In terms of overall morphology, the plants in the APXJTC-23-3 inoculated treatment group (T), regardless of whether they were washed ( Figure 3 B) Still in the original state ( Figure 3 A), all showed significant growth advantages: taller plants, more lush stems and leaves, and more vigorous growth. 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 (36.4%), a highly significant increase in plant fresh weight (37.6%), and highly significant increases in the number and fresh weight of root nodules (114.7% and 51.3%, respectively). Together, they constitute a complete chain of evidence that APXJTC-23-3 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0081] Second, transcriptomics validation (molecular mechanism analysis).

[0082] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the APXJTC-23-3 treatment group (T) and the blank control group (CK) (three plants were mixed for each replicate), and were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0083] 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).

[0084] Results: To elucidate the molecular basis of APXJTC-23-3's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that APXJTC-23-3 treatment specifically activated key gene networks in peanut roots related to symbiotic signal transduction and plant hormone signaling.

[0085] Evidence for key gene expression is as follows (see Figure 5 Table 2):

[0086] 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 2.21-fold and 3.18-fold, respectively. These genes are the core regulatory switches that initiate the root nodule formation process.

[0087] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 11.34-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 11.04-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0088] Conclusion: The above molecular evidence indicates that inoculation with *APXJTC-23-3*, a slow-growing rhizobium from Yuanmingyuan, 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 APXJTC-23-3 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.

[0089] Table 2: Effects of APXJTC-23-3 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0090]

[0091] Example 3: Functional verification of the inhibition of Aspergillus flavus growth and toxin production by APXJTC-23-3, a slow-growing rhizobium from Yuanmingyuan.

[0092] First, in vitro antagonism experiment (double-plate inverted method). To evaluate the antagonistic effect of *Aspergillus flavus* APXJTC-23-3 on *Aspergillus flavus*, an in vitro antagonism experiment was conducted using the double-plate inverted method.

[0093] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0094] 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.

[0095] Lower plate (90 mm in diameter): Treatment group (T) uniformly coated with 100 µL of 1×10⁻⁶ solution. 8 A fresh bacterial suspension of APXJTC-23-3 at CFU / mL 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.

[0096] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0097] Measurement indicators:

[0098] Colony diameter: The diameter of Aspergillus flavus colonies was measured using the cross-multiplication method, and the growth inhibition rate was calculated. Inhibition rate (%) = [(colonial diameter of control group – colony diameter of treatment group) / colony diameter of control group] × 100%.

[0099] 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.

[0100] 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).

[0101] The results show (see) Figure 6 (Table 3) The APXJTC-23-3 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan can effectively block the growth, reproduction, and toxin production of Aspergillus flavus, achieving highly efficient source control.

[0102] Inhibition of mycelial growth: The colony diameter in the APXJTC-23-3 treatment group (T) was 1.74 ± 0.13 cm, significantly lower than that in the blank control group (CK, 7.26 ± 0.05 cm), with an inhibition rate of 76.0% (p = 4.03 × 10⁻⁶). -13 ).

[0103] Inhibition of spore reproduction: The sporulation rate in the APXJTC-23-3 treatment group was 1.88 ± 0.15 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the blank control group (CK, 68 ± 11.45 × 10⁻⁶). 6 The inhibition rate reached 97.2% (p = 1.22 × 10⁻⁶ cells / plate). -6 ).

[0104] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the APXJTC-23-3 treatment group was 23.85 ± 7.24 ng / plate, which was significantly lower than that in the blank control group (CK, 642 ± 147.59 ng / plate), with an inhibition rate of 96.3% (p = 1.07 × 10⁻⁶). -9 ).

[0105] Table 3: Inhibitory effect of APXJTC-23-3 treatment on key life activities of Aspergillus flavus.

[0106]

[0107] Inhibition rate (%) = [(mean of CK group - mean of T group) / mean of CK group] × 100%. All data are expressed as mean ± standard deviation.

[0108] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0109] Based on the incubation apparatus, four treatments were set up. All treatments had Aspergillus flavus spore suspension (2 µL, 1×10⁻⁶) inoculated at the center of the upper plates. 5 (spores / mL). Invert the two plates together, seal with Parafilm, and incubate in the dark at 28°C for 5 days.

[0110] CK group (blank control group): The lower plate was not inoculated with APXJTC-23-3 and no activated carbon was used.

[0111] CK + Car group (activated carbon control group): The lower plate was not inoculated with APXJTC-23-3, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0112] Group T (with APXJTC-23-3, without activated carbon): The lower plate is uniformly coated with APXJTC-23-3 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0113] T + Car group (with APXJTC-23-3 and activated carbon): Half of the lower plate was coated with an equal amount of APXJTC-23-3 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0114] Results and analysis (see Figure 7 Table 4): This experiment demonstrates that the adsorption effect of activated carbon significantly weakens the antibacterial effect of APXJTC-23-3. The inhibitory effect of APXJTC-23-3 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0115] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.46 ± 0.05 cm) and the CK + Car group (7.44 ± 0.05 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0116] APXJTC-23-3 releases potent antibacterial VOCs: the colony diameter in group T (2.16 ± 0.08 cm) was significantly smaller than that in group CK, with an antibacterial rate of 71.0%.

[0117] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (4.20 ± 0.08 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 43.7%, but was still significantly lower than that of the CK group.

[0118] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0119]

[0120] The key role of volatile organic compounds (VOCs) from the slow-growing rhizobium APXJTC-23-3 of Yuanmingyuan in inhibiting the mycelial growth of Aspergillus flavus was evaluated using activated carbon adsorption blocking experiments. Data are expressed as mean ± standard deviation, and inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0121] Third, transcriptomics verification (molecular mechanism).

[0122] To elucidate the molecular mechanism by which the slow-growing rhizobium APXJTC-23-3 from the Yuanmingyuan Garden inhibits toxin production and sporulation in Aspergillus flavus, transcriptome sequencing analysis was performed on the treated Aspergillus flavus hyphae. Differential expression analysis showed (see...) Figure 8 (Table 5) APXJTC-23-3 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0123] 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 APXJTC-23-3 treatment group (T) decreased to 25.5% and 16.4% 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.

[0124] The expression of key genes regulating conidial development pathways 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 1.7% and 8.9% of those in the control group, respectively, and the downregulation was highly significant (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.

[0125] The transcriptomic evidence above demonstrates that the volatile substances produced by the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan can precisely interfere with the core life activity program 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.

[0126] Table 5: Inhibitory effect of APXJTC-23-3 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0127]

[0128] Example 4: Identification and verification of antibacterial active substances in APXJTC-23-3.

[0129] First, the collection, identification, and analysis of key differential substances in volatile compounds.

[0130] To identify the characteristic volatile compounds produced by the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan, headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) was used for untargeted metabolomics analysis of the volatile components of the strain culture. By comparing the APXJTC-23-3 group (T) with the control group (CK), a compound with extremely significant differences was identified. This compound had a retention time of 23.823 min and a retention index of 1677.05 in the chromatogram. Its mass spectrum showed a similarity of 720 to 3-methylbutyric acid (3-methyl-) in the NIST standard library, with a molecular ion peak at m / z 43, thus confirming its identification.

[0131] Quantitative and statistical analyses further confirmed the criticality of this compound (see Figure 9 Table 6):

[0132] 1) Extremely significant enrichment: The relative abundance of this compound in the APXJTC-23-3 treatment group of *Staphylococcus aureus* from Yuanmingyuan was 2049.13 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 0.0018).

[0133] 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 1.50, indicating that it is one of the core volatile markers that distinguishes APXJTC-23-3 from the blank control.

[0134] Conclusion: Comprehensive chromatographic-mass spectrometry identification and omics statistical analysis conclusively demonstrate that 3-methylbutyric acid is a characteristic volatile compound specifically and abundantly produced by APXJTC-23-3. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0135] Table 6: Identification and omics analysis results of 3-methylbutyric acid, a characteristic volatile substance in APXJTC-23-3.

[0136]

[0137] 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.

[0138] Second, purity verification experiments. To directly verify the antibacterial activity of the key volatile substance 3-methylbutyric acid, the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus was determined using the fumigation method. The results showed (see Table 7) that 3-methylbutyric acid had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0139] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.7%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 82.6%.

[0140] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 54.1%) to a 40-fold dilution (inhibition rate 5.6%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0141] Determining the activity threshold: When the dilution factor reached 40 times or more, there was no substantial difference in the colony diameter between the treatment groups and the control group (inhibition rate < 5.6%), indicating that 3-methylbutyric acid had basically lost its observable antibacterial activity at this concentration (see Table 7).

[0142] Conclusion: This pure product verification experiment conclusively demonstrates that 3-methylbutyric acid (3-methylbutyric acid) 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 (2049.13-fold) of this compound in the volatile substances of APXJTC-23-3. This strongly confirms that 3-methylbutyric acid is a key effector molecule mediating the volatile antibacterial activity of strain APXJTC-23-3.

[0143] Table 7: Inhibitory effect of pure 3-methylbutyric acid on the mycelial growth of Aspergillus flavus.

[0144]

[0145] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0146] 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 APXJTC-23-3 from the Yuanmingyuan Garden that possesses both growth-promoting and aspergillus-inhibiting functions, characterized in that... Its classification name is Bradyrhizobium yuanmingense, accession number is CCTCC M 20253052, 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 *Pseudomonas yuanmingyuanensis* APXJTC-23-3 as described in claim 1, its fermentation product, or its culture; the fermentation product includes fermentation broth, sterile supernatant, or active substances extracted from the fermentation broth or sterile supernatant; the culture includes solid culture, liquid culture, or a dried preparation thereof.

3. The application of the *Phyllostachys edulis* APXJTC-23-3 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* APXJTC-23-3 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 the use of volatile organic compounds released by the slow-growing rhizobium APXJTC-23-3 from Yuanmingyuan to achieve the inhibitory function.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the Yuanmingyuan slow-growing rhizobium APXJTC-23-3 as described in claim 1 or the microbial agent or preparation as described in claim 2 to the peanut planting environment or peanut plants.

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