Rhizobium anhuiense ASHBQJ-24-3 with functions of promoting growth and inhibiting aspergillus flavus and application of rhizobium anhuiense ASHBQJ-24-3

Abstract

The invention discloses a rhizobium anhuiense ASHBQJ-24-3 with functions of promoting growth and inhibiting aspergillus flavus and application of the rhizobium anhuiense ASHBQJ-24-3. The rhizobium anhuiense ASHBQJ-24-3 is classified and named as Rhizobium anhuiense, and the preservation number of the rhizobium anhuiense ASHBQJ-24-3 is CCTCC M 20253051. According to the invention, the integration of two major functions of a characteristic antibacterial volatile matter of phenol production and efficient symbiotic nitrogen fixation on a single strain is realized in the rhizobium anhuiense for the first time. The action mechanism is clear, and the phenol volatile matter produced by the strain can accurately down-regulate the expression of a toxin synthesis key gene (aflR / aflS) and a spore development core gene (abaA / wetA) in aspergillus flavus. Through one-time inoculation, the dual purposes of growth promotion and nitrogen fixation and source bacteriostasis can be synchronously achieved in the rhizosphere-legume microdomain of the peanuts, and especially for the biological characteristic of underground fruiting of the peanuts, a lasting biological fumigation protection layer is constructed in soil in the critical legume development period.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of agricultural microbiology and plant protection, specifically involving a strain of Rhizobium anhuiense ASHBQJ-24-3 that has both efficient growth-promoting and nitrogen-fixing functions and strong inhibition of Aspergillus flavus, and its application in the green and safe production of peanuts.

[0002] Research Background

[0003] I. The growth-promoting potential and limitations of rhizobia

[0004] Rhizobia are key symbiotic nitrogen-fixing partners in legumes, forming root nodules with the host to efficiently convert atmospheric nitrogen into plant-usable nitrogen—the core yield-increasing mechanism for legumes. In peanut production, inoculation with highly effective rhizobium agents is a crucial green technology for increasing yield and reducing reliance on chemical fertilizers. However, traditional rhizobia have relatively limited functions, primarily focusing on symbiotic nitrogen fixation, and generally lack direct biocontrol capabilities against major soil-borne diseases of peanuts, especially Aspergillus flavus.

[0005] II. Aflatoxin contamination and existing control bottlenecks

[0006] Aflatoxins (AFs) produced by Aspergillus flavus infection of peanuts are potent carcinogens, posing a serious threat to food safety and the economic value of the industry. The contamination begins in the field soil, infecting the underground pods through the pegs or cracks during the pod development stage. The unique characteristic of peanuts—flowering above ground and fruiting underground—makes traditional chemical control methods (such as foliar spraying) ineffective against the pods in the soil during the critical infection period, creating obstacles to physical control. Existing field and storage control technologies, including chemical fumigation, physical adsorption, and biocontrol applications using non-toxic aflatoxin-producing strains, either focus on post-harvest treatment or have limited effectiveness, high costs, and are subject to pesticide residues and regulatory restrictions, making it difficult to effectively curb toxin contamination at its source.

[0007] III. Systemic Defects and Innovation Directions of Existing Technologies

[0008] Currently, peanut yield increase and toxin control rely on two parallel but separate technical paths: one is general-purpose plant rhizosphere growth promoters (PGPR) or single-function rhizobium inoculants, which focus on promoting growth or nitrogen fixation but lack efficient antibacterial capabilities; the other is antagonistic agents, mainly Bacillus spp. and Pseudomonas spp., which focus only on disease control and contribute very little to the core symbiotic nitrogen fixation process of leguminous crops.

[0009] This single function necessitates the separate application of two or more products in agricultural production, which not only increases costs and labor complexity but also leads to unstable or even mutually destructive field effects due to potential competition or antagonism between microorganisms. Therefore, a critical technological gap exists in this field: the lack of a biotechnology solution that can integrate the most efficient yield-increasing mechanism (symbiotic nitrogen fixation) of leguminous crops with the most critical toxin source control (targeted inhibition of Aspergillus flavus) from the same ecological niche (rhizosphere) on a single microbial carrier.

[0010] IV. Innovative Positioning of the Invention

[0011] Based on the aforementioned bottlenecks, this invention aims to provide an innovative solution. By screening and developing a novel multifunctional rhizobium strain that simultaneously possesses highly efficient symbiotic nitrogen fixation capabilities and directly and potently inhibits Aspergillus flavus by producing specific antibacterial volatiles (phenols), the goal is to achieve the dual objectives of "promoting growth and nitrogen fixation" and "antibacterial and antiviral" in the rhizosphere-pod microdomain within the same peanut growth cycle through a single inoculation. This fundamentally overcomes the shortcomings of existing technologies that are functionally fragmented and only address the symptoms, providing revolutionary microbial resources and technical support for green, high-yield, and safe peanut production. Summary of the Invention

[0012] To address the shortcomings of existing technologies, this invention provides an Anhui rhizobium strain ASHBQJ-24-3 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 strain of *Rhizobium anhuiense* ASHBQJ-24-3 that possesses both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Rhizobium anhuiense*, accession number is CCTCC M 20253051, 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 *Rhizobium huinanensis* ASHBQJ-24-3, its fermentation product, or a culture thereof; the fermentation product includes fermentation broth, sterile supernatant, or an active substance extracted from the fermentation broth or sterile supernatant; the culture includes a solid culture, a liquid culture, or a dried preparation thereof.

[0016] The third objective of this invention is to provide the application of *Rhizobium ASHBQJ-24-3* from Anhui Province, or a microbial agent or preparation containing *Rhizobium ASHBQJ-24-3* from Anhui Province, in promoting peanut growth and nodulation nitrogen fixation.

[0017] The fourth objective of this invention is to provide the application of *Rhizobium anaconda* ASHBQJ-24-3, or a microbial agent or preparation containing *Rhizobium anaconda* ASHBQJ-24-3, 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 Anhui rhizobium ASHBQJ-24-3.

[0019] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Anhui Rhizobium ASHBQJ-24-3, or a microbial agent or preparation containing Anhui Rhizobium ASHBQJ-24-3, 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: Anhui rhizobium ASHBQJ-24-3 can comprehensively promote peanut plant growth and nodulation nitrogen fixation. After inoculation, peanut plant height and biomass increased by 41.4% and 31.4%, respectively, and the number of root nodules and fresh weight of root nodules increased significantly by 111.2% and 29.5%, respectively. Transcriptome analysis further confirmed that this strain can specifically upregulate the expression of key genes in the nodulation signaling (NSP1, NSP2) and auxin signaling (SAUR, AUXIN) pathways in peanut roots, revealing its efficient growth-promoting nitrogen fixation mechanism at the molecular level.

[0022] (2) Highly effective inhibition of Aspergillus flavus: The volatile organic compounds (VOCs) produced 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 76.2%, 97.7%, and 96.3%, respectively. Activated carbon adsorption experiments confirmed that this antibacterial effect is mainly mediated by volatile substances.

[0023] (3) Key antibacterial components are clearly identified and the mechanism is well understood: Through metabolomics and pure product verification experiments, phenol was identified as the key volatile antibacterial active substance. This compound is specifically enriched in the strain's metabolites, and the pure product has a strong dose-dependent inhibitory effect on Aspergillus flavus (the inhibition rate of the original solution reached 97.7%). Molecular mechanism studies have shown that the VOCs produced by the strain can precisely and significantly downregulate the expression of key regulatory genes (aflR, aflS) for aflatoxin synthesis and core genes (abaA, wetA) for spore development, blocking the toxin production and reproduction process at the transcriptional level.

[0024] (4) Significant advantages of integrated innovation and application: This invention is the first to successfully integrate the two major functions of "highly efficient symbiotic nitrogen fixation" and "phenol-mediated potent biocontrol" in the same strain of Rhizobium husk from Anhui. With a single application, the dual goals of promoting nodulation and nitrogen fixation and inhibiting Aspergillus flavus can be achieved simultaneously in the peanut rhizosphere niche. This technology is particularly effective for the biological characteristics of peanuts, which "flower above ground and bear fruit underground," and plays a continuous protective role in the key soil microenvironment for pod development, providing an integrated and source-oriented green biotechnology solution for solving the industrial problem of increasing peanut yield and controlling toxins.

[0025] This invention provides a *Rhizobium anhuiense* strain ASHBQJ-24-3, which possesses both growth-promoting and aflatoxin-inhibiting functions. The strain is classified as *Rhizobium anhuiense*. ASHBQJ-24-3 was obtained by the inventors through screening. Its accession number is CCTCC M 20253051, and its accession date is December 30, 2025. 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

[0026] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of Rhizobium spp. in peanut rhizosphere soil. The independent samples t-test was used for statistical analysis in the figure, and the data are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p < 0.01).

[0027] Figure 2 This invention constructs an ASHBQJ-24-3 phylogenetic tree based on multiple genes (16S, atpD, recA, glnII).

[0028] Figure 3 The effects of inoculation with Anhui rhizobium ASHBQJ-24-3 on peanut seedling growth and root nodule formation were demonstrated. Figure 3 A shows the phenotypic comparison of the whole plant after washing between the blank control group (CK) and the ASHBQJ-24-3 treatment group (T); Figure 3 B shows a close-up comparison of the root system of the blank control group (CK). Figure 3 C represents a close-up comparison of the root systems of the ASHBQJ-24-3 treatment group (T).

[0029] Figure 4 This invention relates to the effects of inoculation with *A. ASHBQJ-24-3* rhizobium from Anhui Province 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 ASHBQJ-24-3 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n ≥ 3). This indicates that the difference between groups is extremely significant (independent samples t-test, p < 0.001).

[0030] 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 Anhui rhizobium ASHBQJ-24-3 of this invention; wherein, Figure 5 A represents the expression level of NSP1, a core transcription factor gene in the nodule signaling pathway; Figure 5 B represents the expression level of NSP2, a co-regulatory factor gene of the nodule signaling pathway; Figure 5 C represents the expression level of the auxin early response factor gene SAUR; Figure 5 D represents the expression level of the auxin early response factor gene AUXIN. In the figure, CK represents the blank control group, and T represents the ASHBQJ-24-3 treatment group; the column height represents the gene expression level (based on standardized counts); the error bar represents the standard deviation (n=3). , , The numbers represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0031] Figure 6 This invention evaluates the inhibitory effect of *Rhizobium huinanense* ASHBQJ-24-3 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 the sporulation rate; 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). This indicates that the difference between groups is extremely significant (independent samples t-test, p < 0.001).

[0032] Figure 7 This is a bar chart showing the quantitative statistical results of Aspergillus colony diameter in the blank control group (CK), blank control group + activated carbon (CK + Car), ASHBQJ-24-3 treatment group (T), and ASHBQJ-24-3 treatment group + activated carbon (T + Car); The figure shows... The differences between groups were statistically significant (p < 0.001).

[0033] Figure 8 This invention relates to the effect of treatment with *Rhizobium ASHBQJ-24-3* from Anhui Province on the expression of core regulatory genes for aflatoxin synthesis and spore development; wherein, Figure 8 A represents the expression level of aflatoxin synthesis core transcription factor gene aflR; Figure 8 B represents the expression level of the aflatoxin synthesis coactivator gene aflS; Figure 8 C represents the expression level of abaA, a key regulatory gene for conidial development; Figure 8 D represents the expression level of wetA, a key regulatory gene for conidial maturation; boxes represent quartile ranges, the midline represents the median, the whisker lines represent ranges, and dots represent individual cells. , "p < 0.01" and "p < 0.001" respectively indicate extremely significant differences between groups.

[0034] 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 phenol, a volatile component, in the blank control group (CK) and the ASHBQJ-24-3 treatment group (T). Box plots show the distribution of this compound in the two groups. (The figure is incomplete in the original text.) This indicates that the difference between the two groups is extremely significant (independent samples t-test, p < 0.001). Detailed Implementation

[0035] 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.

[0036] Example 1: Isolation, identification and preservation of multifunctional Anhui rhizobium ASHBQJ-24-3.

[0037] The Anhui rhizobium ASHBQJ-24-3 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 subtilis ≥ 1×1010 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.

[0038] 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 *Rhizobium*, which is closely related to symbiotic nitrogen fixation in peanuts. For example... Figure 1 As shown, the average relative abundance of *Rhizobium* in the CK group was 0.0397%, while the average relative abundance in the ARC-treated group (T group) increased to 0.0573%, a significant increase of 44.5% (p < 0.01). 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 functionally enhanced *Rhizobium* ASHBQJ-24-3 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 the beneficial symbiotic bacteria—*Rhizobium*.

[0039] 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 ).

[0040] Spread culture: from 10 -5 10 -6 10 -7 100 μL of each of the three dilutions was spread onto YMA (yeast extract 10.0 g / L, mannitol 10.0 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.

[0041] Colony selection and purification: After incubation in the dark at 28 ± 1℃ for 5-7 days, select typical single colonies of *Rhizobium anhuiense* that do not absorb Congo red, are milky white, raised, viscous, and have neat edges. Purify the culture using three consecutive streak plating tests to obtain a pure culture, designated ASHBQJ-24-3.

[0042] Liquid culture: A single colony of ASHBQJ-24-3 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 ≈ 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.

[0043] Genomic DNA extraction: Genomic DNA was extracted from the ASHBQJ-24-3 pure culture using the TIANamp Bacteria DNA Kit according to the instructions. Its concentration and purity were measured using a spectrophotometer (A260 / A280 ratio between 1.8 and 2.0).

[0044] 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 1312 bp (sequence shown in SEQ ID NO: 3).

[0045] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3');

[0046] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3'.

[0047] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that ASHBQJ-24-3 had a 16S rRNA gene similarity of >99.5% with type strains such as Rhizobium anhuiense YIC11270.

[0048] Housekeeping gene identification: To accurately identify its species classification, partial sequences of atpD, recA, glnII, and housekeeping genes were further amplified and determined.

[0049] atpD gene: Amplified using primers atpD 255F and atpD 782R, yielding a sequence of approximately 350 bp (the atpD sequence of ASHBQJ-24-3 is shown in SEQ ID NO: 6).

[0050] The primer atpD 255F sequence is shown in SEQ ID NO: 4: 5'- GCTSGGCCGCATCMTSAACGTC-3';

[0051] The primer atpD 782R sequence is shown in SEQ ID NO: 5: 5'-GCCGACACTTCMGAACCNGCCTG-3'.

[0052] recA gene: Amplified using primers recA1 and recA2, yielding a sequence of approximately 342 bp (as shown in SEQ ID NO: 9).

[0053] The primer recA1 sequence is shown in SEQ ID NO: 7: 5'-CATGCRCTGGATCCGGTCTATGC-3';

[0054] The primer recA2 sequence is shown in SEQ ID NO: 8: 5'- CTTGTTCTTGTCGACCTTGACGCG-3'.

[0055] glnII gene: Amplified using primers glnII 12F and glnII 689R, yielding a sequence of approximately 468 bp (as shown in SEQ ID NO: 12).

[0056] The primer glnII 12F sequence is shown in SEQ ID NO: 10: 5'- YAAGCTCGAGTACATYTGGCT -3';

[0057] The primer glnII 689R sequence is shown in SEQ ID NO: 11: 5'-TGCATGCCSGAGCCGTTCCA-3'.

[0058] Multigene phylogenetic analysis: The gene sequences of ASHBQJ-24-3 were aligned with the corresponding sequences of related *Rhizobium anhuiense* type strains in GenBank. A phylogenetic tree was constructed using MEGA 11.0 software using the neighbor-joining method (bootstrap=1000). The results are clearly shown (see...). Figure 2 ASHBQJ-24-3 clustered with the type strain Rhizobium anhuiense YIC11270, confirming its taxonomic position as Rhizobium anhuiense.

[0059] Example 2: Verification of the function of Anhui rhizobium ASHBQJ-24-3 in promoting peanut growth and nodulation nitrogen fixation.

[0060] 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: ASHBQJ-24-3 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.

[0061] Experimental treatments: ASHBQJ-24-3 treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned ASHBQJ-24-3 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 per group (i.e., 12 pots). 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, day / night temperature set at 28℃ / 22℃, and relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0062] First, growth index measurement (results are shown in...) Figure 4 Table 1): Plants were harvested 30 days after sowing (seedling stage). Plant height was measured using a ruler (accuracy: 1 mm); the total fresh weight of the aboveground and underground parts of the plant was weighed using an analytical balance (accuracy: 0.01 g).

[0063] Plant height: The average plant height of the ASHBQJ-24-3 treatment group (T) was 36.67 ± 0.85 cm, which was significantly increased by 41.4% compared with the blank control group (CK, 25.93 ± 1.56 cm) (p = 1.12 × 10⁻⁶). -9 ).

[0064] Fresh weight: The average fresh weight of group T was 80.53 ± 1.25 g, which was significantly higher than that of group CK (61.31 ± 2.56 g) by 31.4% (p = 3.11 × 10⁻⁶). -10 ).

[0065] Nodule count: The average number of root nodules in group T was 172.5 ± 14.46 / plant, which was significantly increased by 111.2% compared with group CK (81.67 ± 5.36 / plant) (p = 2.19 × 10⁻⁶).-10 ).

[0066] Nodule weight: The average fresh weight of root nodules in group T was 0.228 ± 0.03 g / plant, which was significantly increased by 29.5% compared with group CK (0.176 ± 0.025 g / plant) (p = 5.03 × 10⁻⁶). -4 ).

[0067] Conclusion: Inoculation with ASHBQJ-24-3 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.

[0068] Table 1: The promoting effect of ASHBQJ-24-3 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0069]

[0070] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0071] Inoculation with the Anhui rhizobium ASHBQJ-24-3 significantly promoted the growth and symbiotic nodulation of peanut seedlings, as evidenced by the obvious phenotypic differences. Figure 3 As shown. In terms of overall morphology, the plants in the ASHBQJ-24-3 treatment group (T) exhibited significant growth advantages: taller plants, more lush stems and leaves, and more vigorous growth. Figure 3 A). Most importantly, direct observation of the root system ( Figure 3 (B and C) clearly show 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 (41.4%), a highly significant increase in plant fresh weight (31.4%), and highly significant increases in the number and fresh weight of root nodules (111.2% and 29.5%, respectively). Together, they constitute a complete chain of evidence that ASHBQJ-24-3 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0072] Second, transcriptomics validation (molecular mechanism analysis).

[0073] Sample collection and processing: 30 days after sowing, root samples were randomly collected from 3 biological replicates of the ASHBQJ-24-3 treatment group (T) and the blank control group (CK) (3 plants were mixed for each replicate), and were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0074] 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).

[0075] Results: To elucidate the molecular basis of ASHBQJ-24-3's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that ASHBQJ-24-3 treatment specifically activated a network of key genes in peanut roots related to symbiotic signal transduction and plant hormone signaling. Evidence for key gene expression is as follows (see...). Figure 5 Table 2):

[0076] Symbiotic nodulation pathway: The expression levels of NSP1 and NSP2, the core transcription factors of the nodulation signaling pathway, were significantly upregulated in the T group, by 2.325-fold and 2.42-fold, respectively. These genes are the core regulatory switches that initiate the root nodulation process.

[0077] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 12.146-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 20.703-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0078] Conclusion: The above molecular evidence indicates that ASHBQJ-24-3 inoculation systematically induces the co-regulation of key genes in the symbiotic signaling (NSP1, NSP2) and auxin signaling (SAUR, AUXIN) pathways in peanut roots. This reveals the intrinsic mechanism by which ASHBQJ-24-3 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.

[0079] Table 2: Effects of ASHBQJ-24-3 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0080]

[0081] Example 3: Verification of the function of Anhui Rhizobium ASHBQJ-24-3 in inhibiting Aspergillus flavus growth and toxin production.

[0082] First, in vitro antagonism experiment (double-plate method).

[0083] To evaluate the antagonistic effect of ASHBQJ-24-3 against Aspergillus flavus, an in vitro antagonistic experiment was conducted using the double-plate method.

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

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

[0086] Lower plate (90 mm in diameter): 100 µL of ASHBQJ-24-3 treatment group (T) with a concentration of 1×10⁻⁶ was uniformly coated. 8 Fresh ASHBQJ-24-3 bacterial suspension (CFU / mL); control group (CK) plated with an equal volume of sterile YMB medium. After the bacterial suspension was absorbed, the upper plate was inverted.

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

[0088] Measurement indicators:

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

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

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

[0092] The results show (see) Figure 6 (Table 3) The ASHBQJ-24-3 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus.

[0093] Inhibition of mycelial growth: The colony diameter in the ASHBQJ-24-3 treatment group (T) was 1.7 ± 0.1 cm, significantly lower than that in the blank control group (CK, 7.14 ± 0.05 cm), with an inhibition rate of 76.2% (p = 6.66 × 10⁻⁶). -14 ).

[0094] Inhibition of spore reproduction: The spore production in the ASHBQJ-24-3 treatment group was 1.7 ± 0.12 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the blank control group (CK, 73.6 ± 6.31 × 10⁻⁶). 6 The inhibition rate reached 97.69% (p = 6.03 × 10⁻⁶). -9 ).

[0095] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the ASHBQJ-24-3 treatment group was 22.07 ± 7.28 ng / plate, which was significantly lower than that in the blank control group (CK, 588.48 ± 94.71 ng / plate), with an inhibition rate of 96.3% (p = 5.30 × 10⁻⁶). -12 ).

[0096] Conclusion: The volatile substances produced by ASHBQJ-24-3 can effectively block the growth, reproduction and toxin production of Aspergillus flavus, achieving efficient source control.

[0097] Table 3: Inhibitory effect of ASHBQJ-24-3 treatment on key life activities of Aspergillus flavus.

[0098]

[0099] Inhibition rate (%) = [(CK group mean - T group mean) / CK group mean] × 100%. All data are expressed as mean ± standard deviation.

[0100] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0101] Based on the incubation apparatus, four treatment groups were set up. In all treatment groups, the upper plates were inoculated with a suspension of Aspergillus flavus spores (2 µL, 1×10⁻⁶) at the center point. 5 (spores / mL). Invert the two plates together, seal with Parafilm, and incubate in the dark at 28°C for 5 days.

[0102] CK group (blank control): The lower plate was not inoculated with ASHBQJ-24-3 and no activated carbon was used.

[0103] CK + Car group (without ASHBQJ-24-3, with activated carbon): The lower plate was not inoculated with ASHBQJ-24-3, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0104] Group T (with ASHBQJ-24-3, without activated carbon): The lower plate is uniformly coated with ASHBQJ-24-3 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0105] T + Car group (with ASHBQJ-24-3 and activated carbon): Spread an equal amount of ASHBQJ-24-3 bacterial suspension on half of the lower plate area, and spread 5 g of sterilized activated carbon on the other half area.

[0106] Results and analysis (see Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of ASHBQJ-24-3. This experiment demonstrates that the inhibitory effect of ASHBQJ-24-3 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0107] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.26 ± 0.05 cm) and the CK + Car group (7.12 ± 0.04 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0108] ASHBQJ-24-3 releases potent antibacterial VOCs: The colony diameter in group T (1.7 ± 0.1 cm) was significantly smaller than that in group CK, with an antibacterial rate of 76.6%.

[0109] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (3.64 ± 0.11 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 49.9%, but was still significantly lower than that of the CK group.

[0110] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0111]

[0112] The key role of volatile organic compounds (VOCs) in inhibiting the mycelial growth of Aspergillus flavus was evaluated using activated carbon adsorption blocking experiments. Data are expressed as mean ± standard deviation. Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0113] Third, transcriptomics verification (molecular mechanism).

[0114] To elucidate the mechanism by which ASHBQJ-24-3 inhibits toxin production and sporulation in Aspergillus flavus at the molecular level, transcriptome sequencing analysis was performed on the treated Aspergillus flavus hyphae. Differential expression analysis showed that ASHBQJ-24-3 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0115] Evidence for key gene expression is as follows (see Figure 8 Table 5):

[0116] 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 ASHBQJ-24-3 treatment group (T) decreased to 28.03% and 17.80% 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.

[0117] 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 1.08% and 8.19% of the control group, respectively, both downregulated to a highly significant level (p < 0.001). These two genes are essential for normal conidial formation and maturation, and their sharp decrease in expression directly explains the observed significant reduction in conidial yield at the molecular level.

[0118] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by ASHBQJ-24-3 can precisely interfere with the core life activity programs of Aspergillus flavus, namely, by strongly inhibiting the core regulatory hubs of toxin production (aflR / aflS) and sporulation (abaA / wetA), thereby achieving the dual effects of antibacterial and toxicity reduction.

[0119] Table 5: Inhibitory effect of ASHBQJ-24-3 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0120]

[0121] Example 4: Identification and verification of antibacterial active substances of Rhizobium ASHBQJ-24-3 from Anhui.

[0122] To identify the characteristic volatile substances produced by ASHBQJ-24-3, headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) was used to perform untargeted metabolomics analysis on the volatile components of the bacterial culture. By comparing the ASHBQJ-24-3 treatment group (T) with the control group (CK), a compound with extremely significant differences was identified. This compound had a retention time of 30.664 min and a retention index of 2006.143 in the chromatogram. Its mass spectrum showed a similarity of 892 to phenol in the NIST standard library, with a molecular ion peak at m / z 94, thus confirming its identification.

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

[0124] 1) Extremely significant enrichment: The relative abundance of this compound in the ASHBQJ-24-3 treatment group was 2.05 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 1.22 × 10⁻⁶). -4 ).

[0125] 2) High model importance: In the orthogonal partial least squares discriminant analysis (OPLS-DA) model, its variable importance projection (VIP) value is as high as 1.33, indicating that it is one of the core volatile markers that distinguish ASHBQJ-24-3 from the blank control.

[0126] Conclusion: Comprehensive chromatographic-mass spectrometry identification and omics statistical analysis conclusively demonstrate that phenol is a characteristic volatile compound specifically and abundantly produced by ASHBQJ-24-3. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0127] Table 6: Identification and omics analysis results of phenol, a characteristic volatile substance in ASHBQJ-24-3.

[0128]

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

[0130] Second, purity verification experiment.

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

[0132] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.7%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 81.5%.

[0133] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 54.4%) to a 40-fold dilution (inhibition rate 19.8%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0134] Determining the activity threshold: When the dilution factor reaches 40 times or more, the colony diameter of each treatment group is not substantially different from that of the control group (inhibition rate < 4%), indicating that phenol has basically lost its observable antibacterial activity at this concentration.

[0135] Conclusion: This pure product verification experiment conclusively demonstrates that phenol itself is a potent inhibitor of Aspergillus flavus mycelial growth. It exerts a significant antibacterial effect even at low concentrations, a characteristic consistent with the highly specific and significant enrichment (2.05-fold) of this compound in the volatile substances of ASHBQJ-24-3, strongly confirming that phenol is the key effector molecule mediating the volatile antibacterial effect of ASHBQJ-24-3.

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

[0137]

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

[0139] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A *Rhizobium anamensis* strain ASHBQJ-24-3 from Anhui Province that possesses both growth-promoting and aflatoxin-inhibiting functions, characterized in that... Its classification name is Rhizobium anhuiense, accession number is CCTCC M 20253051, 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 Anhui rhizobium ASHBQJ-24-3 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 its dried preparation.

3. The application of the Anhui rhizobium ASHBQJ-24-3 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 Anhui Rhizobium ASHBQJ-24-3 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 achieving the inhibitory function through the volatile organic compounds released by the Anhui rhizobium ASHBQJ-24-3.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the Anhui rhizobium ASHBQJ-24-3 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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