Bradyrhizobium guangzhouensis APHBXY-23-1 with functions of promoting growth and inhibiting aspergillus flavus and application thereof

Abstract

The invention discloses a strain of bradyrhizobium guangzhouense APHBXY-23-1 with functions of efficiently promoting growth and strongly inhibiting aspergillus flavus and application of the strain of bradyrhizobium guangzhouense APHBXY-23-1. The preservation number of the strain of bradyrhizobium guangzhouense APHBXY-23-1 is CCTCC (China Center for Type Culture Collection) M 20253048. The invention not only retains the core functions of the strain as rhizobium for efficient symbiotic nitrogen fixation and crop growth promotion, but also reveals that the strain can significantly inhibit the growth of aspergillus flavus by releasing a volatile substance dimethylbutyric acid for the first time. The discovery breaks through the technical limitation that the traditional rhizobium only has a growth promoting function and lacks a biocontrol effect, the two agricultural microorganism core functions of nitrogen fixation and growth promotion and biocontrol bacteriostasis are endogenously integrated into the same microorganism carrier for the first time, and a novel rhizobium resource with double functions is successfully constructed; a new species of growth-promoting rhizobium with a biocontrol function is created, and the growth-promoting rhizobium has important innovative value in theory and application levels.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Guangzhou slow-growing rhizobium APHBXY-23-1 with both growth-promoting and aflatoxin-inhibiting functions and its applications. Background Technology

[0002] Currently, in peanut production practice, increasing yield and controlling aflatoxin contamination typically rely on two independent technical routes:

[0003] I. The main technology for increasing yield relies on plant rhizosphere growth-promoting bacteria (PGPR) inoculants. Common species include nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and Bacillus and Pseudomonas bacteria that secrete plant hormones. These inoculants promote plant growth through mechanisms such as nitrogen fixation, phosphorus release, and synthesis of growth regulators, and fall under the category of widely used biofertilizers.

[0004] II. The main technical pathways for aflatoxin control include: 1) Agricultural management measures, such as selecting resistant varieties, crop rotation, and timely harvesting, the effects of which are significantly constrained by the environment; 2) Physicochemical methods, such as irradiation, chemical fumigation, and the use of adsorbents, which are mostly used for post-harvest treatment and have problems such as high cost, residue risk, and quality impact; 3) Biological control methods, namely the application of microbial antagonistic agents, such as non-toxic Aspergillus flavus, Bacillus, Pseudomonas, Trichoderma, etc., which inhibit the growth and toxin production of Aspergillus flavus through competition, antagonism, or induction of resistance, and are currently a research hotspot.

[0005] However, the two types of technologies mentioned above have the following systemic limitations when addressing the dual needs of increasing peanut production and ensuring safety:

[0006] The existing PGPR inoculants generally lack the ability to directly inhibit Aspergillus flavus; while biocontrol inoculants do not make a substantial contribution to core yield-increasing pathways such as symbiotic nitrogen fixation in peanuts. In production, it is often necessary to apply the two types of products separately, which not only increases costs and operational complexity, but may also lead to unstable effects due to strain interactions.

[0007] The technologies are fragmented, failing to form an integrated solution. Yield enhancement and biocontrol have long been separated in terms of technological research and application, lacking a technological system capable of integrating nitrogen fixation, growth promotion, and biocontrol functions within the same microbial carrier. Simple inoculant combinations are insufficient to achieve stable functional synergy in field conditions.

[0008] There is insufficient targeting of yield-increasing mechanisms in legumes. Peanuts, as a legume, rely on efficient nitrogen fixation through symbiotic root nodules for key yield increases. General-purpose PGPR cannot replace or optimize this specific process; while traditional rhizobium inoculants generally lack the ability to control aflatoxin, resulting in a disconnect between yield increase and toxin prevention in terms of biological mechanisms.

[0009] Existing control methods have physical and regulatory limitations. The biological characteristics of peanuts, which "flower above ground and bear fruit underground," make control during the critical pod-setting stage particularly difficult: the efficacy of pesticide coatings during the sowing period is difficult to maintain until the fruiting period; foliar application during the fruiting period cannot reach the pods in the soil; drip irrigation application disrupts the soil microecology and easily leads to pesticide resistance; and physical color sorting or chemical fumigation during the storage period is costly and subject to strict food safety regulations.

[0010] Weak control at the source of contamination. Most existing technologies focus on post-harvest treatment or post-infection intervention, failing to effectively inhibit the colonization and development of Aspergillus flavus from the field source—especially the rhizosphere microenvironment. There is a lack of a microbial solution that can simultaneously promote growth, fix nitrogen, and preventatively inhibit bacteria throughout the entire peanut growth process.

[0011] Therefore, there is an urgent need in this field for a microbial strain that can both efficiently promote symbiotic nitrogen fixation in peanuts and effectively inhibit Aspergillus flavus, so as to achieve the dual goals of increasing yield and preventing poisoning in the same biological carrier, and fundamentally break through the existing technological bottlenecks. Summary of the Invention

[0012] To address the shortcomings of existing technologies, this invention provides a Guangzhou slow-growing rhizobium strain APHBXY-23-1 that combines growth promotion and aspergillosis inhibition functions, along with its applications.

[0013] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0014] The primary objective of this invention is to provide a slow-growing rhizobium strain, APHBXY-23-1, which possesses both growth-promoting and aflatoxin-inhibiting functions. Its classification name is Bradyrhizobium guangzhouense, its accession number is CCTCC M 20253048, its 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.

[0015] The second objective of this invention is to provide a microbial agent or preparation comprising at least one of the Guangzhou slow-growing rhizobium APHBXY-23-1, 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.

[0016] The third objective of this invention is to provide the application of *Phytozotropha guangnanensis* APHBXY-23-1, or a microbial agent or preparation containing *Phytozotropha guangnanensis* APHBXY-23-1, in promoting peanut growth and nodulation nitrogen fixation.

[0017] The fourth objective of this invention is to provide the application of *Aspergillus oryzae* APHBXY-23-1, or a microbial agent or preparation containing *Aspergillus oryzae* APHBXY-23-1, in inhibiting the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus*.

[0018] Preferably, the application specifically achieves the inhibitory function through the volatile organic compounds released by the Guangzhou slow-growing rhizobium APHBXY-23-1.

[0019] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the Guangzhou slow-growing rhizobium APHBXY-23-1, or a microbial agent or preparation containing Guangzhou slow-growing rhizobium APHBXY-23-1, to the peanut planting environment or peanut plants.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) Significant effect on promoting growth and nitrogen fixation: Guangzhou slow-growing rhizobium APHBXY-23-1 can comprehensively promote the growth and nitrogen fixation of peanut plants. After inoculation, the peanut plant height and biomass increased by 31.9% and 37.4% respectively, and the number of root nodules and the fresh weight of root nodules increased significantly by 110.2% and 50.7% respectively.

[0022] (2) Highly effective inhibition of Aspergillus flavus: The volatile substances released by the strain can comprehensively inhibit the growth, sporulation and aflatoxin B1 synthesis of Aspergillus flavus, with inhibition rates of 76.6%, 97.0% and 96.3%, respectively.

[0023] (3) Key antibacterial components are clearly identified: the key antibacterial active substance is 2-methylbutyric acid, which can effectively inhibit Aspergillus flavus at low concentrations, with an inhibition rate of 98.3% after treatment with the original solution.

[0024] (4) Clear mechanism of action: Molecular mechanism studies have shown that this strain can activate the symbiotic signaling (NSP1, NSP2) and auxin signaling (SAUR) pathways in peanut roots, promoting nodulation and growth; it can also specifically downregulate the expression of key genes for aflatoxin synthesis (aflR, aflS) and core genes for spore development (abaA, wetA), blocking toxin production and reproduction from the source.

[0025] Cell Preservation:

[0026] This invention provides a strain of *Bradyrhizobium guangzhouense*, which has both growth-promoting and aflatoxin-inhibiting functions. This strain was obtained by the inventors through screening. The accession number of *Bradyrhizobium guangzhouense* is CCTCC M20253048, 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

[0027] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of *Bradyrhizobium* spp. in peanut rhizosphere soil. In the figure, data are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p<0.001).

[0028] Figure 2 This invention constructs an APHBXY-23-1 phylogenetic tree based on multiple genes (16S rRNA, atpD, recA, dnaK, glnII, 23S).

[0029] Figure 3 This invention relates to the phenotypic effects of inoculation with the slow-growing rhizobium APHBXY-23-1 on peanut seedling growth and root nodule formation; wherein, Figure 3 A shows a comparison of the phenotypes of the entire plant (after washing); Figure 3 B shows a comparison of the in-situ growth status of the plants in the flowerpot; Figure 3 C shows a close-up comparison of plant roots; in the figure, CK represents the blank control group and T represents the APHBXY-23-1 treatment group.

[0030] Figure 4 This invention relates to the effects of inoculation with the slow-growing rhizobium APHBXY-23-1 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, T represents the APHBXY-23-1 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n≥3). The differences between groups were statistically significant (p<0.001).

[0031] Figure 5This is a schematic diagram illustrating the upregulation of key genes related to peanut root symbiosis and growth induced by treatment with the slow-growing rhizobium APHBXY-23-1 of this invention; 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 SAUR, an early auxin response factor gene; 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 numbers represent highly significant (p<0.01) and extremely significant (p<0.001) differences between groups, respectively.

[0032] Figure 6 This invention evaluates the inhibitory effect of the slow-growing rhizobium APHBXY-23-1 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 yield; Figure 6 C represents the yield of aflatoxin B1 (AFB1); Figure 6 D represents the overall colony morphology of the blank control group (CK); Figure 6 F is a close-up of the hyphal edge in the blank control group (CK); Figure 6 E represents the overall colony morphology of the APHBXY-23-1 treatment group (T); Figure 6 G is a close-up of the hyphal edge of the slow-growing rhizobium APHBXY-23-1 treatment group (T). The differences between groups were statistically significant (p<0.001).

[0033] Figure 7 This invention relates to the effect of treatment with the slow-growing rhizobium APHBXY-23-1 on the expression of core regulatory genes for aflatoxin synthesis and spore development; wherein, Figure 7 A represents the expression level of aflatoxin synthesis core transcription factor gene aflR; Figure 7 B represents the expression level of the aflatoxin synthesis coactivator gene aflS; Figure 7 C represents the expression level of abaA, a key regulatory gene for conidial development; Figure 7 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 APFJPT-23-4 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.

[0034] Figure 8 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), APHBXY-23-1 treatment group (T), and APHBXY-23-1 treatment group (T) + activated carbon (Car). In the figure, and The numbers indicate significant (p<0.05) and highly significant (p<0.01) differences between groups, respectively.

[0035] 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 2-methylbutyric acid in the blank control group (CK) and the APHBXY-23-1 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

[0036] 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.

[0037] Example 1: Isolation, identification and preservation of the multifunctional slow-growing rhizobium APHBXY-23-1 from Guangzhou.

[0038] The Guangzhou slow-growing rhizobium APHBXY-23-1 of this invention was isolated from peanut rhizosphere soil treated with ARC microbial agent (a compound agent whose main components are: Bacillus amyloliquefaciens, Brevibacillus laterosporus, Bacillus mucilaginosus, and Enterobacter ludwigii). The microbial agent contained ≥2 × 10⁻⁶ viable Bacillus amyloliquefaciens. 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 Enterobacter ludwig's bacterium ≥1×10⁻⁶ 10 CFU / g, effective viable count of Flavobacterium brevicus ≥2×10⁻⁶ 9 CFU / gram.

[0039] 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*, which is closely related to peanut symbiotic nitrogen fixation. Figure 1 As shown in the bar chart, the relative abundance of *Bradyrhizobium* spp. in rhizosphere soil samples from the blank control group (CK) and the ARC microbial inoculant treatment group (T), based on 16S rRNA gene amplicon sequencing analysis, is presented. Data are expressed as mean ± standard deviation. The difference between the two groups was extremely significant (p<0.001). Specifically, the average relative abundance of *Rhizobium guangnanense* 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 *Rhizobium guangnanense* strain APHBXY-23-1 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 the beneficial symbiotic bacterium *Rhizobium guangnanense*.

[0040] 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 ).

[0041] 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). , (NaCl 0.1 g / L, agar 15.0 g / L, pH 6.8-7.0) on a solid plate.

[0042] Colony selection and purification: After incubation at 28±1°C in the dark for 5-7 days, select typical single colonies of *Staphylococcus guangnanensis* 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 APHBXY-23-1.

[0043] Liquid culture: A single colony of APHBXY-23-1 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 bacterial suspension in the logarithmic growth phase (OD200). 600 Mix approximately 0.8 g of the mixture with an equal volume of sterile 60% (v / v) glycerol protectant to achieve a final glycerol concentration of 30%. Dispense the mixture into cryovials and store them in an ultra-low temperature freezer at -80°C.

[0044] Genomic DNA extraction: Genomic DNA was extracted from the APHBXY-23-1 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.

[0045] Gene sequencing and analysis: 16S rRNA gene: PCR amplification was performed using universal primers Fd1 and Rd1. The purified PCR product was sequenced, yielding a sequence of approximately 1388 bp (as shown in SEQ ID NO: 3). The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that the 16S rRNA gene similarity of APHBXY-23-1 was >99.5% with that of type strains such as Bradyrhizobium guangzhouense CCBAU 51670.

[0046] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0047] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3'.

[0048] 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.

[0049] atpD gene: Amplified using primers TSatpDf and TSatpDr, a sequence of approximately 426 bp was obtained (the atpD sequence of APHBXY-23-1 is shown in SEQ ID NO: 6).

[0050] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0051] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0052] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 348 bp (as shown in SEQ ID NO: 9).

[0053] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0054] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0055] dnaK gene: Amplified using primers BRdnaKf and BRdnaKr, yielding a sequence of approximately 445 bp (as shown in SEQ ID NO: 12).

[0056] The primer BRdnaKf sequence is shown in SEQ ID NO: 10: 5'-TTCGACATCGACGCSAACGG-3';

[0057] The primer BRdnaKr sequence is shown in SEQ ID NO: 11: 5'-GCCTGCTGCKTGTACATGGC-3'.

[0058] glnll gene: Amplified using primers TSglnIIf and TSglnIIr, a sequence of approximately 441 bp was obtained (sequence shown in SEQ ID NO: 15).

[0059] The primer TSglnIIf sequence is shown in SEQ ID NO: 13: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0060] The primer TSglnIIr sequence is shown in SEQ ID NO: 14: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0061] 23S gene: Amplified using primers FGPS1490 and FGPS130, yielding a sequence of approximately 765 bp (as shown in SEQ ID NO: 18).

[0062] The primer FGPS1490 sequence is shown in SEQ ID NO: 16: 5'-TGCGGCTGGATCACCTCCTT-3';

[0063] The primer FGPS130 sequence is shown in SEQ ID NO: 17: 5'-CCGGGTTTCCCCATTCGG-3'.

[0064] Multigene phylogenetic analysis: The gene sequences of APHBXY-23-1 were compared with the corresponding sequences of related *Syndromea gracilis* type strains from Guangzhou in GenBank. A phylogenetic tree was constructed using MEGA 11.0 software using the neighbor-joining method (bootstrap=1000). Results ( Figure 2 The results clearly show that APHBXY-23-1 clusters with the type strain Bradyrhizobium guangzhouense CCBAU 51670, confirming its taxonomic position as Bradyrhizobium guangzhouense.

[0065] Example 2: Functional verification of APHBXY-23-1 strain in promoting peanut growth and nodulation nitrogen fixation.

[0066] Test materials: The tested peanut variety was Zhonghua 28. The soil was sandy loam, sterilized twice by autoclaving at 121°C for 0.5 hours each time to eliminate the influence of indigenous rhizobia. Inoculum preparation: APHBXY-23-1 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.

[0067] Experimental treatments: Treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned APHBXY-23-1 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°C / 22°C, and a relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0068] First, growth indicators were determined: 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 above-ground 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.

[0069] Plant height: The average plant height of the APHBXY-23-1 inoculated group (T) was 37.13±2.11 cm, which was significantly increased by 31.9% compared with the blank control group (CK, 28.16±2.32 cm) (p=3.85×10⁻⁶). -6 )( Figure 3 A, Figure 4 A).

[0070] Fresh weight of plants: The average fresh weight of group T was 83.22±5.07g, which was significantly increased by 37.4% compared with group CK (60.57±7.03g) (p=1.32×10). -5 )( Figure 4 B).

[0071] Second, determination of nitrogen fixation index in nodule formation: Carefully rinse the plant roots, count the number of all root nodules (diameter ≥1mm) on each plant, and weigh the total fresh weight of the nodules using an analytical balance. Statistical analysis was performed using an independent samples t-test, and data are expressed as mean ± standard deviation.

[0072] Nodule count: The average number of nodules per plant in group T was 177.00±20.91, which was significantly increased by 110.2% compared with group CK (84.22±10.91) (p=4.14×10). -8 )( Figure 3 B, Figure 3 C, Figure 4 C).

[0073] Fresh weight of root nodules: The average fresh weight of root nodules in group T was 0.226±0.045 g / plant, which was significantly increased by 50.7% compared with group CK (0.150±0.039 g / plant) (p=7.67×10). -4 )( Figure 4 D).

[0074] Conclusion: The *APHBXY-23-1* strain from Guangzhou exhibits a highly significant and comprehensive promoting effect on peanut plant growth and nodulation nitrogen fixation. Inoculation with APHBXY-23-1 significantly increased peanut plant height and biomass by 31.9% and 37.4%, respectively; simultaneously, it fundamentally improved symbiotic nodulation efficiency, with nodule number and nodule biomass increasing significantly by 110.2% and 50.7%, respectively. These results fully demonstrate the core function of APHBXY-23-1 strain as a highly efficient symbiotic nitrogen fixation inoculant for peanuts. Phenotypic comparison photos are shown below. Figure 3 The specific data is summarized in Table 1.

[0075] Table 1: Promoting effects of Guangzhou slow-growing rhizobium APHBXY-23-1 on peanut seedling growth and nodulation phenotype.

[0076]

[0077] The data in the table are the results measured 30 days after sowing (seedling stage), expressed as mean ± standard deviation. Plant height and fresh weight reflect growth status; root nodule number and fresh weight reflect symbiotic nitrogen fixation potential. The significance of the difference between the APHBXY-23-1 inoculated treatment group (T) and the blank control group (CK) was determined using an independent samples t-test (p<0.05 was considered significant, p<0.01 was considered highly significant). The data show that APHBXY-23-1 inoculation has a significant or highly significant effect on all peanut indicators.

[0078] Inoculation with the Guangzhou slow-growing rhizobium APHBXY-23-1 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 APHBXY-23-1 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 C) 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 (31.9%), a highly significant increase in plant fresh weight (37.4%), and highly significant increases in the number and fresh weight of root nodules (110.2% and 50.7%, respectively). Together, they constitute a complete chain of evidence that the APHBXY-23-1 strain can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0079] Third, transcriptomics validation (molecular mechanism analysis).

[0080] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the APHBXY-23-1 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°C for later use.

[0081] 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-adj < 0.01 and |log2(Fold Change)| > 1).

[0082] To elucidate the molecular basis of the APHBXY-23-1 strain's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that APHBXY-23-1 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 shown in Table 2:

[0083] Symbiotic nodulation pathway: NSP1, the core transcription factor of the nodulation signaling pathway ( Figure 5 A) and NSP2 ( Figure 5 The expression levels of B) were significantly upregulated in group T, by 2.21-fold and 4.46-fold, respectively. These genes are the core regulatory switches that initiate the root nodule formation process.

[0084] Plant hormone signaling: auxin early response factor SAUR ( Figure 5 The expression level of C) was significantly upregulated by 9.10-fold, indicating that early growth programs such as auxin-mediated cell division and elongation were strongly activated.

[0085] Conclusion: The above molecular evidence indicates that inoculation with the APHBXY-23-1 strain systematically induces the co-regulation of symbiotic signaling pathways (NSP1, NSP2) and key growth hormone signaling pathways (auxin) in peanut roots. This reveals the intrinsic mechanism by which APHBXY-23-1 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.

[0086] Table 2: Effects of APHBXY-23-1 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0087]

[0088] Example 3: Verification of the function of APHBXY-23-1 strain in inhibiting the growth and toxin production of Aspergillus flavus.

[0089] First, in vitro antagonism experiment (double-plate inverted method). To evaluate the antagonistic effect of strain APHBXY-23-1 against Aspergillus flavus, an in vitro antagonism experiment was conducted using the double-plate inverted method.

[0090] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0091] 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.

[0092] Lower plate (90mm diameter): 100µL of APHBXY-23-1 treatment group (T) with a concentration of 1×10⁻⁶ was uniformly coated. 8 A fresh bacterial suspension of APHBXY-23-1 at CFU / mL was prepared; a blank control group (CK) was spread onto an equal volume of sterile YMB medium. After the bacterial suspension was absorbed, the upper plate was inverted.

[0093] Upper plate: Inoculate 2 µL of a 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0094] Measurement indicators:

[0095] Colony diameter: The diameter of Aspergillus flavus colonies was measured using the cross-hatching 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%.

[0096] 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.

[0097] 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).

[0098] Quantitative results showed that APHBXY-23-1 treatment produced a highly significant and comprehensive inhibition of key life activities of Aspergillus flavus.

[0099] Inhibition of mycelial growth: The colony diameter in the APHBXY-23-1 treatment group (T) was significantly reduced to 1.72±0.22 cm, with an inhibition rate of 76.6% compared to the blank control group (CK, 7.34±0.11 cm) (p=2.31×10⁻⁶). -11 )( Figure 6 A).

[0100] Inhibition of spore reproduction: The sporulation rate in the APHBXY-23-1 treatment group was significantly reduced to 1.94±0.21×10⁻⁶. 6 The number of samples per dish was 65 ± 13.06 × 10⁻⁶ compared to the control group (CK). 6 The percentage of samples per dish decreased by 97.0% (p = 4.77 × 10⁻⁶). -6 )( Figure 6 B).

[0101] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the APHBXY-23-1 treatment group was significantly reduced to 23.59±6.52 ng / plate, a 96.3% reduction compared to the blank control group (CK, 641.78±147.98 ng / plate) (p=1.11×10⁻⁶). -9 )( Figure 6 C).

[0102] Phenotypic observation ( Figure 6 The D, E, F, G values ​​were highly consistent with the quantitative data, and the CK group ( Figure 6 D,F) and T group ( Figure 6 E,G) colonies are sparse, hyphae are abnormal, and sporulation structures are severely damaged.

[0103] Conclusion: The APHBXY-23-1 strain can substantially inhibit the growth, reproduction and toxin production of Aspergillus flavus, and almost completely block its toxin synthesis at the source.

[0104] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0105] Based on the inverted culture device, four treatments were set up. For all treatments, the upper plates were inoculated with Aspergillus flavus spore suspension (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.

[0106] CK group (blank control): The lower plate was not inoculated with APHBXY-23-1 and no activated carbon was used.

[0107] CK+Car group (activated carbon control): The lower plate was not inoculated with APHBXY-23-1, but a layer of about 5g of high-temperature sterilized granular activated carbon was spread on it.

[0108] APHBXY-23-1 group: APHBXY-23-1 bacterial suspension was evenly spread on the lower plate (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0109] APHBXY-23-1+Car group: Half of the lower plate was coated with an equal amount of APHBXY-23-1 bacterial suspension, and the other half was covered with 5g of sterilized activated carbon.

[0110] Quantitative results showed that the adsorption effect of activated carbon significantly affected the antibacterial effect of APHBXY-23-1 (Table 3, Figure 7 ).

[0111] Activated carbon itself had no effect: there was no significant difference in colony size between the CK group (colon diameter: 7.40±0.07cm) and the CK+Car group (7.28±0.04cm) (p=0.22), indicating that activated carbon itself did not have non-specific interference with the growth of Aspergillus flavus. Figure 7 ).

[0112] APHBXY-23-1 produces a potent volatile antibacterial substance: the colony diameter in the APHBXY-23-1 group (0.18±0.11cm) was significantly smaller than that in the CK group (p=6.2×10⁻⁶). -10 ) and CK+Car group (p=7.2×10 -10 With an antibacterial rate as high as 97.6%, it directly proves that the APHBXY-23-1 culture can produce potent antibacterial VOCs.

[0113] Activated carbon partially adsorbs antibacterial substances: When activated carbon was added to the APHBXY-23-1 treatment (APHBXY-23-1+Car group), its antibacterial effect was partially but significantly weakened, and the colony diameter (4.40±0.07cm) was significantly larger than that of the APHBXY-23-1 group (p=2.0×10⁻⁶). -8 The result was still significantly smaller than that of the CK group (p=1.5×10). -9 ) and CK+Car group (p=2.3×10 -9 This indicates that activated carbon adsorbed some of the key volatile antibacterial components, causing the antibacterial effect to decrease from 97.6% to 40.5%.

[0114] This experiment definitively demonstrates that the inhibitory effect of strain APHBXY-23-1 on Aspergillus flavus mainly depends on the release of volatile substances that can be adsorbed by activated carbon. This discovery clearly points to volatile metabolites as the material basis of antibacterial activity, providing crucial mechanistic evidence for the subsequent identification of specific volatile active ingredients (such as 2-methylbutyric acid).

[0115] Table 3: Effect of activated carbon adsorption on the volatile antibacterial activity of APHBXY-23-1.

[0116]

[0117] The key role of volatile organic compounds (VOCs) in inhibiting Aspergillus flavus mycelial growth by activated carbon adsorption blocking experiments was evaluated. Data are expressed as mean ± standard deviation. Inhibition rate (%) = [(mean diameter of CK group - mean diameter of treatment group) / mean diameter of CK group] × 100%. Statistical significance was calculated using a two-sample t-test, comparing with the CK group: The difference in ns was significant.

[0118] Third, transcriptomics verification (molecular mechanism).

[0119] To elucidate the molecular mechanism by which the APHBXY-23-1 strain inhibits the growth, sporulation, and toxin production of Aspergillus flavus, we performed transcriptome sequencing analysis on Aspergillus flavus hyphae cultured in pairs (blank control group CK and APHBXY-23-1 treatment group T). High-quality sequences were aligned to the Aspergillus flavus reference genome and differential expression analysis was performed.

[0120] aflR is a core transcriptional regulator of aflatoxin biosynthesis. aflS is a coactivator of aflatoxin synthesis, co-regulating the transcription of toxin gene clusters with AflR. abaA is a key transcription factor regulating conidiophore development and is crucial for sporulation. wetA is a key transcription factor regulating conidial wall formation and maturation.

[0121] The analysis focused on the core regulatory network of Aspergillus flavus development and toxin synthesis, finding that APHBXY-23-1 treatment specifically and significantly inhibited the expression of several key regulatory genes. Compared with the CK group, in the T group:

[0122] 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 aflatoxin biosynthesis were significantly downregulated by 3.93-fold (Log2FC=1.97, Figure 8 A) and 6.07 times (Log2FC=2.60, Figure 8 B). The aflR / aflS complex is the master switch for activating the entire toxin synthesis gene cluster, and its significant inhibition directly leads to the shutdown of downstream toxin synthesis pathways.

[0123] The expression of key genes regulating conidial development pathways was strongly suppressed: the expression of the core transcription factor genes abaA and wetA, which regulate conidial development, was extremely suppressed, downregulated by 57.72-fold (Log2FC=5.85, ...). Figure 8 C) and 11.31 times (Log2FC=3.50, Figure 8 D). These two genes are essential for normal spore formation and maturation, and the sharp decrease in their expression levels directly explains the observed phenotype of significantly reduced sporulation at the molecular level.

[0124] The downregulation of the expression of the aforementioned key genes in group T was statistically significant (p<0.05). To visually demonstrate this inhibitory effect, we plotted box plots of the expression levels of the key genes. Figure 8 The figure confirms that the expression levels of the four genes aflR, aflS, abaA, and wetA were all significantly downregulated in group T, and the biologically reproducible dataset showed good reproducibility.

[0125] The aforementioned transcriptomic evidence conclusively demonstrates at the gene expression regulation level that the volatile substances produced by strain APHBXY-23-1 can precisely interfere with the core life processes 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." This, together with the previously observed phenotypic data of inhibited colony growth, drastic reduction in sporulation, and sharp decrease in toxin production, forms a complete "molecular mechanism-macrophenotypic" evidence chain, fundamentally elucidating the biocontrol mechanism of strain APHBXY-23-1. Key gene expression data for Aspergillus flavus are shown in Table 4.

[0126] Table 4: Effects of APHBXY-23-1 treatment on the expression of genes regulating core development and toxin production in Aspergillus flavus.

[0127]

[0128] Example 4: Identification and verification of antibacterial active substances in APHBXY-23-1.

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

[0130] To identify the main volatile components produced by strain APHBXY-23-1, we employed headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME / GC-MS) for non-targeted metabolomics analysis. By comparing the volatile components of the experimental and control groups, we identified a compound with a highly significant difference. This compound exhibited a retention time of 23.665 min and a retention index of 1670.2 in the chromatogram. Its mass spectrum showed a high match (882) with 2-methylbutanoic acid (2-methyl-) in the NIST standard library, with a molecular ion peak at m / z 74, thus confirming its identification. Figure 9 ).

[0131] Quantitative and statistical results further confirmed the criticality of this compound:

[0132] 1) Abundance difference: The relative abundance of this compound in the APHBXY-23-1 treatment group was 1.26 × 10⁻⁶ compared to the blank control group. 5 times.

[0133] 2) Statistical significance: The difference between groups reached a highly significant level (p=0.0046).

[0134] 3) Model importance: In the orthogonal partial least squares discriminant analysis (OPLS-DA) model, its variable importance projection (VIP) value is 1.49, indicating that it is one of the key volatile markers that distinguishes APHBXY-23-1 strain from the blank control.

[0135] Conclusion: Through comprehensive chromatographic-mass spectrometry identification and omics statistical analysis, we conclusively demonstrate that 2-methylbutyric acid is a characteristic volatile acid compound specifically and abundantly produced by strain APHBXY-23-1. This discovery provides a clear chemical target for further investigation into the microbiological function of this compound.

[0136] Table 5: GC-MS identification and abundance analysis of 2-methylbutyric acid, a characteristic volatile compound of strain APHBXY-23-1.

[0137]

[0138] 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.

[0139] Second, purity verification experiment. To evaluate the in vitro antibacterial activity of 2-methylbutanoic acid against Aspergillus flavus, the p-incubation method was used. The specific steps were as follows: 2-methylbutanoic acid was dissolved in anhydrous ethanol as the stock solution and serially diluted. Anhydrous ethanol was used as a blank control (CK). A 5mm diameter sterile filter paper was placed in the center of the inside of the lid of a sterile culture dish, and 20µL of the corresponding concentration of reagent was accurately added. A PDA plate was prepared at the bottom of the culture dish, and 2µL of a 1×10⁻⁶ solution was inoculated at the center of the plate. 4 A suspension of Aspergillus flavus spores per mL was prepared. The bottom plate was then inverted onto the lid, ensuring the filter paper and colonies were in relative but not in contact, and incubated at 28°C. The antibacterial effect was assessed by measuring colony diameter (cm). Each treatment was performed in triplicate. Data are expressed as mean ± standard deviation.

[0140] Experimental results showed that 2-methylbutyric acid had a significant and concentration-dependent inhibitory effect on the mycelial growth of Aspergillus flavus (Table 6):

[0141] The inhibitory effect is extremely strong at high concentrations: the colony diameter of the original solution treatment group (solvent is anhydrous ethanol) is only 0.12±0.01cm, which is 98.3% higher than that of the solvent control CK group (7.17±0.06cm) (p<0.001), indicating that it can almost completely inhibit the growth of Aspergillus flavus mycelium in the undiluted state.

[0142] Antibacterial activity decreased with dilution gradient: As the compound concentration decreased, the antibacterial effect showed a regular decreasing trend. After a 1.6-fold dilution, the colony diameter increased to 0.87±0.05 cm, with an inhibition rate of 87.9% (p<0.001); when diluted 8 times, the average colony diameter increased to 2.82±0.32 cm, and the inhibition rate decreased to 60.7% (p<0.01). When diluted 40 times, the colony diameter (6.64±0.19 cm) was similar to that of the CK group, and the inhibition rate was only 7.4%, indicating that its effective antibacterial concentration was close to the critical point.

[0143] Conclusion: This study demonstrated, using a standardized cross-linking culture method, that 2-methylbutyric acid (2-methylbutyric acid) exhibits a strong and concentration-dependent inhibitory effect on Aspergillus flavus mycelial growth in vitro. Its stock solution and low-level dilutions (≤8 times) showed extremely strong to significant inhibitory effects, while the activity rapidly disappeared after high-level dilutions (≥40 times). These results clarify the effective concentration range of this compound for inhibiting Aspergillus flavus mycelial growth, providing crucial dosimetric evidence for its potential as a volatile antifungal agent for controlling Aspergillus flavus contamination.

[0144] Table 6: Effects of different dosages of 2-methylbutyric acid on the growth of Aspergillus flavus mycelia (including dosage, inhibition rate and significance).

[0145]

[0146] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%. Statistical significance: , , There was no significant difference in ns.

[0147] 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 slow-growing rhizobium strain from Guangzhou, APHBXY-23-1, which possesses both growth-promoting and aflatoxin-inhibiting functions, characterized in that... Its classification name is Bradyrhizobium guangzhouense, accession number is CCTCC M 20253048, 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 Guangzhou slow-growing rhizobium APHBXY-23-1 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 its dried preparation.

3. The application of the Guangzhou slow-growing rhizobium APHBXY-23-1 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 Guangzhou slow-growing rhizobium APHBXY-23-1 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 Guangzhou slow-growing rhizobium APHBXY-23-1.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the Guangzhou slow-growing rhizobium APHBXY-23-1 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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