Bradyrhizobium guangdongensis APHNZY-24-3 with functions of promoting growth and inhibiting aspergillus flavus and application thereof

Abstract

The invention discloses bradyrhizobium guangdongense APHNZY-24-3 which has the functions of efficiently promoting growth, promoting nodulation and inhibiting aspergillus flavus and application of the bradyrhizobium guangdongense APHNZY-24-3, and the serial number of the bradyrhizobium guangdongense APHNZY-24-3 is CCTCC (China Center for Type Culture Collection) M 2026225. According to the invention, integration of two major functions, namely a characteristic antibacterial volatile matter for producing benzothiazole and efficient symbiotic nitrogen fixation, on a single strain is realized in the bradyrhizobium guangdongensis for the first time. The action mechanism is clear: the benzothiazole produced by the strain can accurately down-regulate the expression of a toxin synthesis key gene and a spore development core gene in aspergillus flavus. Through one-time inoculation, double targets of growth promotion and nitrogen fixation and source bacteriostasis can be synchronously realized in the rhizosphere-legume microdomain of the peanuts, and particularly aiming at the biological characteristic of underground fruiting of the peanuts, a biological fumigation protection layer is constructed in soil in the critical legume development period, and aspergillus flavus infection and toxin production are continuously blocked.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Bradyrhizobium guangdongense APHNZY-24-3 that has both highly efficient growth-promoting nitrogen fixation and strong inhibition of Aspergillus flavus, and its application in the green and safe production of peanuts. Technical Background

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

[0003] Growth-promoting and yield-increasing technologies primarily rely on the application of general-purpose plant rhizosphere growth-promoting bacteria (PGPR) inoculants, such as Bacillus spp. and Pseudomonas spp. These inoculants mainly provide non-specific growth-promoting effects through pathways such as phosphorus solubilization, potassium solubilization, or hormone secretion. However, for legumes like peanuts, the most efficient yield-increasing mechanism lies in the formation of root nodules with specific symbiotic nitrogen-fixing microorganisms (rhizobia). General-purpose PGPRs cannot replace or efficiently activate this specific symbiotic system, resulting in a fundamental bottleneck in yield-increasing potential.

[0004] Aflatoxin control technologies mainly include: 1) agricultural management measures (such as crop rotation), which have passive and unstable effects; 2) physicochemical methods (such as chemical fumigation and adsorbents), which are costly and pose risks of residues and regulatory restrictions; 3) biological control methods, which mainly involve the application of antagonistic bacteria such as non-toxic Aspergillus flavus and Bacillus, which inhibit pathogens through competition or secretion of antimicrobial substances.

[0005] Systemic defects and technological gaps in existing technologies:

[0006] (1) Single function and lack of synergy: Existing growth-promoting agents (including traditional rhizobium inoculants) generally lack direct and efficient inhibition of Aspergillus flavus; conversely, existing biocontrol agents lack direct promotion of the core symbiotic nitrogen fixation process of leguminous crops. Applying them separately in the field can easily lead to interspecies competition among microorganisms, resulting in unstable effects or even mutual cancellation.

[0007] (2) Fragmented technical approaches fail to address the problem of source control: Aspergillus infection originates in the field soil. The unique habit of peanuts, which "flower above ground and bear fruit underground," means that the pods during their critical development period are buried deep in the soil, causing traditional chemical control methods to face physical obstacles during the fruiting period: the efficacy of seed coating is not long-lasting; foliar application cannot act on the underground pods; and soil chemical treatment disrupts the microecology and easily induces resistance. Existing technologies mostly focus on post-harvest treatment, and there is a serious lack of effective means to control the source of pollution in the field.

[0008] (3) Failure to integrate core biological functions: In particular, existing technologies have failed to integrate the most efficient yield-increasing mechanism of leguminous crops (symbiotic nitrogen fixation) with the most critical source control of toxins (rhizosphere inhibition) into the same biological carrier. To date, there are no publicly reported or commercially available products involving a strain of *Staphylococcus aureus* from Guangdong that possesses both highly efficient symbiotic nitrogen fixation capabilities and the ability to directly and effectively inhibit *Aspergillus flavus* by producing characteristic volatiles such as benzothiazole. This is a technological gap that urgently needs to be filled in this field.

[0009] Therefore, there is an urgent need in this field for an integrated biological solution that can simultaneously promote symbiotic nitrogen fixation and inhibit Aspergillus flavus at its source in peanuts from the same ecological niche (rhizosphere), through the same microbial carrier, and within the same growth cycle. Developing such multifunctional rhizobia is of great significance for overcoming the technical bottlenecks in the green, high-yield, and safe production of peanuts. This invention aims to provide this innovative solution. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a Guangdong slow-growing rhizobium strain APHNZY-24-3 that combines growth promotion and aspergillosis inhibition functions, along with its applications.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0012] The primary objective of this invention is to provide a slow-growing rhizobium strain from Guangdong, APHNZY-24-3, which possesses both growth-promoting and aflatoxin-inhibiting functions. Its classification name is Bradyrhizobium guangdongense, its accession number is CCTCC M 2026225, its accession date is January 23, 2026, 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.

[0013] The second objective of this invention is to provide a microbial agent or preparation comprising at least one of *A. ganciclos* APHNZY-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.

[0014] The third objective of this invention is to provide the application of *A. 24-3*, a slow-growing rhizobium from Guangdong, or a microbial agent or preparation containing *A. 24-3*, in promoting peanut growth and nodulation nitrogen fixation.

[0015] The fourth objective of this invention is to provide the application of *A. ganciclos* APHNZY-24-3, or a microbial agent or preparation containing *A. ganciclos* APHNZY-24-3, in inhibiting the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus*.

[0016] Preferably, the application specifically achieves the inhibitory function through the volatile organic compounds released by the Guangdong slow-growing rhizobium APHNZY-24-3.

[0017] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Guangdong slow-growing rhizobium APHNZY-24-3, or a microbial agent or preparation containing Guangdong slow-growing rhizobium APHNZY-24-3, to the peanut planting environment or peanut plants.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) Significant effect on promoting growth and nitrogen fixation: Guangdong slow-growing rhizobium APHNZY-24-3 can comprehensively promote peanut plant growth and nodulation nitrogen fixation. After inoculation, peanut plant height and biomass increased by 34.8% and 31.1%, respectively, and the number of root nodules and fresh weight of root nodules increased significantly by 115.3% and 18.5%, respectively. Transcriptomic analysis further confirmed that this strain can specifically activate key genes of symbiotic signaling pathways (NSP1, NSP2) and auxin signaling pathways (SAUR, AUXIN) in peanut roots, revealing the intrinsic basis of its efficient growth-promoting nitrogen fixation at the molecular mechanism level.

[0020] (2) Highly effective inhibition of Aspergillus flavus: The volatile organic compounds (VOCs) produced by the strain exhibit a strong and comprehensive inhibitory effect on Aspergillus flavus. Experiments show that its inhibition rates on Aspergillus flavus mycelial growth, spore reproduction, and aflatoxin B1 synthesis are as high as 74.5%, 97.5%, and 96.3%, respectively. Activated carbon adsorption experiments confirm that its antibacterial effect is mainly mediated by volatile substances.

[0021] (3) Key antibacterial components are clearly identified and the mechanism is well understood: Through metabolomics and pure product verification experiments, benzothiazole was identified as its key volatile antibacterial active substance. This compound was specifically enriched in the strain's metabolites (relative abundance was 2502.44 times that of the control group), and the pure product had a strong dose-dependent inhibitory effect on Aspergillus flavus (inhibition rate of 97.7% in the original solution). Molecular mechanism studies showed that the VOCs produced by the strain could 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 of Aspergillus flavus at the transcriptional level.

[0022] (4) Significant advantages of integrated innovation and application: This invention is the first to successfully integrate the dual functions of "highly efficient symbiotic nitrogen fixation" and "benzothiazole-mediated potent biocontrol" in the same strain of *Staphylococcus aureus* from Guangdong. With a single application, the dual goals of promoting nodulation and nitrogen fixation and inhibiting *Aspergillus flavus* can be achieved simultaneously in the peanut rhizosphere niche. This technology is particularly effective for the biological characteristics of peanuts, which "flower above ground and bear fruit underground," and plays a continuous protective role in the key soil microenvironment for pod development, providing an integrated and source-oriented green biotechnology solution to the industrial problem of increasing peanut yield and controlling toxins.

[0023] Cell Preservation:

[0024] This invention provides a slow-growing rhizobium strain from Guangdong, APHNZY-24-3, which has both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Bradyrhizobium guangdongense*. This slow-growing rhizobium strain APHNZY-24-3 was obtained by the inventors of this invention through screening. The accession number of this slow-growing rhizobium strain APHNZY-24-3 is CCTCC M 2026225, the accession date is January 23, 2026, and it is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. Attached Figure Description

[0025] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of Bradyrhizobium in peanut rhizosphere soil. Data in the figure are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p < 0.001).

[0026] Figure 2 This invention constructs an APHNZY-24-3 phylogenetic tree based on multiple genes (atpD, dnaK, glnII, gyrB, 23S, nirK, recA, rpoB).

[0027] Figure 3 This invention relates to the phenotypic effects of inoculation with the Guangdong slow-growing rhizobium APHNZY-24-3 on peanut seedling growth and root nodule formation; wherein, Figure 3 A shows a comparison of the phenotypes of whole plants (after washing) in the blank control group (CK) and the APHNZY-24-3 treatment group (T); Figure 3 Figure B shows a close-up comparison of the roots of plants in the blank control group (CK); Figure C shows a close-up comparison of the roots of plants in the APHNZY-24-3 treatment group (T).

[0028] Figure 4This invention relates to the effects of inoculation with the Guangdong slow-growing rhizobium APHNZY-24-3 on key phenotypic indicators of peanut seedling growth and symbiotic nodulation; among which, Figure 4 A represents the effect on plant height; Figure 4 B represents the effect on the fresh weight of the plant; Figure 4 C represents the effect on the number of root nodules; Figure 4 D represents the effect on the fresh weight of root nodules; in the figure, CK represents the blank control group, and T represents the APHNZY-24-3 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n≥3). and The numbers indicate significant differences between groups (p<0.05) and highly significant differences (p<0.001), respectively.

[0029] 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 Guangdong slow-growing rhizobium APHNZY-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 of the auxin signaling pathway gene AUXIN; in the figure, CK represents the blank control group, and T represents the APFJPT-23-4 treatment group; the column height represents the gene expression level (based on standardized counts); the error bar represents the standard deviation (n=3). , , The numbers represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0030] Figure 6 This invention evaluates the inhibitory effect of *A. guinea* slow-growing rhizobium APHNZY-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 conidia yield; Figure 6 C represents aflatoxin production. In the figure, CK represents the blank control group, and T represents the APFJPT-23-4 treatment group; quantitative data are expressed as mean ± standard deviation (n≥3); indicates highly significant differences between groups (independent samples t-test). p<0.001).

[0031] Figure 7This 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), APHNZY-24-3 treatment group (T), and APHNZY-24-3 treatment group + activated carbon (T + Car); The figure shows... This indicates that p < 0.001.

[0032] Figure 8 This invention relates to the effect of treatment with the slow-growing rhizobium bacterium APHNZY-24-3 on the expression of core regulatory genes for aflatoxin synthesis and spore development; wherein, Figure 8 A represents the expression level of aflatoxin synthesis core transcription factor gene aflR; Figure 8 B represents the expression level of the aflatoxin synthesis coactivator gene aflS; Figure 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; in the figure, CK represents the blank control group, T represents the PZ-9 treatment group; boxes represent quartile ranges, the midline represents the median, the whisker lines represent ranges, and dots represent individual cells; , , The numbers represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0033] Figure 9 This invention employs headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) to detect and compare the relative abundance of the volatile component 3-methylbutyric acid in the blank control group (CK) and the APHNZY-24-3 treatment group (T); box plots show the distribution of this compound in the two groups; in the figure, This indicates a significant difference between groups (p < 0.05). Detailed Implementation

[0034] 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.

[0035] Example 1: Isolation, identification and preservation of multifunctional slow-growing rhizobium APHNZY-24-3 from Guangdong.

[0036] The Guangdong slow-growing rhizobium APHNZY-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 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.

[0037] 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. For example... Figure 1 As shown in the bar chart, the relative abundance of *Bradyrhizobium* in rhizosphere soil samples from the control group (CK) and the ARC microbial agent treatment group (T) was obtained based on 16S rRNA amplicon sequencing analysis. Compared with the untreated control group (CK), the relative abundance of *Bradyrhizobium* in the rhizosphere soil treated with ARC microbial agent was significantly increased. Specifically, the mean relative abundance of *Bradyrhizobium* in the CK group was 0.45%, while the mean relative abundance in the ARC treatment 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 Guangdong slow-growing rhizobium APHNZY-24-3 from the rhizosphere soil of the ARC treatment group, indicating that the ARC inoculant can reshape the rhizosphere microbiome and specifically promote the colonization and enrichment of beneficial symbiotic bacteria—slow-growing rhizobium.

[0038] 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).

[0039] Spread culture: from 10 -5 10 -6 10 -7 100 μL of each of the three dilutions was spread onto YMA containing 0.0025% (w / v) Congo red (yeast extract 10.0 g / L, mannitol 10.0 g / L, KH2PO4 0.5 g / L). (NaCl 0.1 g / L, agar 15.0 g / L, pH 6.8-7.0) on a solid plate.

[0040] Colony selection and purification: After incubation in the dark at 28 ± 1℃ for 5-7 days, select typical slow-growing rhizobium single colonies that do not absorb Congo red, are milky white, raised, viscous, and have neat edges. Purify the culture by three consecutive streak plating tests to obtain a pure culture, designated APHNZY-24-3.

[0041] Liquid culture: A single colony of *A. phagnum molluscum* APHNZY-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 (OD2). 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 ≈0.8) with an equal volume of sterile 60% (v / v) glycerol protectant to achieve a final glycerol concentration of 30%, dispense into cryovials, and store in an ultra-low temperature freezer at -80°C.

[0042] Genomic DNA extraction: Genomic DNA was extracted from the APHNZY-24-3 pure culture using the TIANamp Bacteria DNA Kit according to the instructions, and its concentration and purity (A260 / A280 ratio between 1.8 and 2.0) were determined using a NanoDrop™ 2000 spectrophotometer.

[0043] 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 1254 bp (sequence shown in SEQ ID NO: 3).

[0044] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0045] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3'.

[0046] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that APHNZY-24-3 had a 16S rRNA gene similarity of >99.5% with type strains such as Bradyrhizobium guangdongense CCBAU 51649.

[0047] 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.

[0048] atpD gene: Amplified using primers TSatpDf and TSatpDr, a sequence of approximately 441 bp was obtained (the atpD sequence of APHBXY-23-1 is shown in SEQ ID NO: 6).

[0049] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0050] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0051] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 444 bp (as shown in SEQ ID NO: 9).

[0052] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0053] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0054] dnaK gene: Amplified using primers BRdnaKf and BRdnaKr, yielding a sequence of approximately 445 bp (as shown in SEQ ID NO: 12).

[0055] The primer BRdnaKf sequence is shown in SEQ ID NO: 10: 5'-TTCGACATCGACGCSAACGG-3';

[0056] The primer BRdnaKr sequence is shown in SEQ ID NO: 11: 5'-GCCTGCTGCKTGTACATGGC-3'.

[0057] glnll gene: Amplified using primers TSglnIIf and TSglnIIr, a sequence of approximately 477 bp was obtained (sequence shown in SEQ ID NO: 15).

[0058] The primer TSglnIIf sequence is shown in SEQ ID NO: 13: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0059] The primer TSglnIIr sequence is shown in SEQ ID NO: 14: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0060] 23S gene: Amplified using primers FGPS1490 and FGPS130, yielding a sequence of approximately 948 bp (as shown in SEQ ID NO: 18).

[0061] The primer FGPS1490 sequence is shown in SEQ ID NO: 16: 5'-TGCGGCTGGATCACCTCCTT-3';

[0062] The primer FGPS130 sequence is shown in SEQ ID NO: 17: 5'-CCGGGTTTCCCCATTCGG-3'.

[0063] Multigene phylogenetic analysis: The gene sequences of APHNZY-24-3 were aligned with the corresponding sequences of related slow-growing rhizobium type strains in GenBank. A phylogenetic tree was constructed using MEGA 11.0 software using the neighbor-joining method (bootstrap=1000). The results are clearly shown (see...). Figure 2 APHNZY-24-3 clustered with the type strain Bradyrhizobium guangdongense CCBAU 51649, confirming its taxonomic position as Bradyrhizobium guangdongense.

[0064] Example 2: Verification of the function of Guangdong slow-growing rhizobium APHNZY-24-3 in promoting peanut growth and nodulation nitrogen fixation.

[0065] 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: APHNZY-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.

[0066] Experimental treatments: APHNZY-24-3 treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned APHNZY-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, a day / night temperature of 28℃ / 22℃, and a relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0067] First, growth indicators were measured (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).

[0068] Plant height: The average plant height of the APHNZY-24-3 treatment group (T) was 35.56 ± 2.18 cm, which was significantly increased by 34.8% compared with the blank control group (CK, 26.39 ± 2.29 cm) (p = 3.15 × 10⁻⁶). -6 ).

[0069] Fresh weight: The average fresh weight of group T was 79.78 ± 2.79 g, which was significantly higher than that of group CK (60.86 ± 3.02 g) by 31.1% (p = 1.65 × 10⁻⁶). -8 ).

[0070] Nodule count: The average number of root nodules in group T was 172.5 ± 17.21 per plant, which was significantly increased by 115.3% compared with group CK (80.11 ± 8.25 per plant) (p = 3.11 × 10⁻⁶). -9 ).

[0071] Nodule weight: The average fresh weight of root nodules in the T group was 0.186 ± 0.021 g / plant, which was significantly increased by 18.5% compared with the CK group (0.157 ± 0.028 g / plant) (p = 0.0166).

[0072] Conclusion: Inoculation with APHNZY-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.

[0073] Table 1: The promoting effect of APHNZY-24-3 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0074]

[0075] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0076] Inoculation with the Guangdong slow-growing rhizobium APHNZY-24-3 produced a highly significant promoting effect on the growth and symbiotic nodulation of peanut seedlings, with the following direct phenotypic differences: Figure 3 As shown. In terms of overall morphology, the APHNZY-24-3 treatment group (T) plants exhibited significant growth advantages: taller plants, more abundant stems and leaves, and more vigorous growth (as shown). Figure 3 A). Most importantly, direct observation of the root system clearly shows ( Figure 3 Groups B and C), and Group T plants have more developed root systems, and the number and size of effective root nodules on their roots are far greater than those in Group CK. These striking visual evidences are highly consistent with the aforementioned measured precise quantitative data (Table 1) showing a highly significant increase in plant height (34.8%), a highly significant increase in plant fresh weight (31.1%), and highly significant increases in the number of root nodules and fresh weight (115.3% and 18.5%, respectively). Together, they constitute a complete chain of evidence that APHNZY-24-3 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0077] Second, transcriptomics validation (molecular mechanism analysis).

[0078] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the APHNZY-24-3 treatment group (T) and the blank control group (CK) (three plants were mixed for each replicate), and were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0079] 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).

[0080] Results: To elucidate the molecular basis of APHNZY-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 APHNZY-24-3 treatment specifically activated key gene networks in peanut roots related to symbiotic signal transduction and plant hormone signaling.

[0081] Evidence for key gene expression is as follows (see results) Figure 5 Table 2):

[0082] 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 2.418-fold and 2.947-fold, respectively. These genes are the core regulatory switches that initiate the root nodulation process.

[0083] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 12.14-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 17.03-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0084] Conclusion: The above molecular evidence indicates that APHNZY-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 APHNZY-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.

[0085] Table 2: Effects of APHNZY-24-3 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0086]

[0087] Example 3: Verification of the function of Guangdong slow-growing rhizobium APHNZY-24-3 in inhibiting the growth and toxin production of Aspergillus flavus.

[0088] First, in vitro antagonism experiment (double-plate method).

[0089] To evaluate the antagonistic effect of APHNZY-24-3 against Aspergillus flavus, an in vitro antagonistic experiment was conducted using the double-plate 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 (90 mm in diameter): Treatment group (T) uniformly coated with 100 µL of 1×10⁻⁶ solution. 8 A fresh bacterial suspension of APHNZY-24-3 at CFU / mL was prepared; the control group (CK) was spread onto an equal volume of sterile YMB medium. After the bacterial suspension was absorbed, the upper plate was inverted.

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

[0094] Measurement indicators (results are shown in) Figure 6 Table 3):

[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 (%) = [(Coronary 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] The results show (see) Figure 6 (Table 3) APHNZY-24-3 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by APHNZY-24-3 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus simultaneously, achieving highly efficient source control.

[0099] Inhibition of mycelial growth: The colony diameter in the treatment group (T) was 1.82 ± 0.084 cm, which was significantly lower than that in the control group (CK, 7.14 ± 0.05 cm), with an inhibition rate of 74.5% (p = 2.79 × 10⁻⁶). -14 ).

[0100] Inhibition of spore reproduction: Sporulation rate in the treatment group was 1.74 ± 0.11 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the control group (CK, 68.6 ± 11.15 × 10⁻⁶). 6 The inhibition rate reached 97.5% (p = 9.16 × 10⁻⁶ cells / plate). -7 ).

[0101] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the treatment group was 21.78 ± 7.735 ng / plate, which was significantly lower than that in the control group (CK, 583.81 ± 128.88 ng / plate), with an inhibition rate of 96.27% (p = 5.98 × 10⁻⁶). -10 ).

[0102] Table 3: Inhibitory effect of APHNZY-24-3 treatment on key life activities of Aspergillus flavus.

[0103]

[0104] Inhibition rate (%) = [(mean of CK group - mean of T group) / mean of CK group] × 100%. All data are expressed as mean ± standard deviation.

[0105] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0106] 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.

[0107] CK group (blank control): The lower plate was not inoculated with APHNZY-24-3 and no activated carbon was used.

[0108] CK+Car group (activated carbon control): The lower plate was not inoculated with APHNZY-24-3, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0109] Group T (with APHNZY-24-3, without activated carbon): The lower plate was uniformly coated with APHNZY-24-3 bacterial suspension (concentration: 1×10⁻⁶).8 (CFU / mL), no activated carbon.

[0110] T+Car group (with APHNZY-24-3 and activated carbon): Half of the lower plate was coated with an equal amount of APHNZY-24-3 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0111] Results and analysis (see Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of APHNZY-24-3. This experiment demonstrates that the inhibitory effect of APHNZY-24-3 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0112] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.14 ± 0.05 cm) and the CK+Car group (7.04 ± 0.05 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0113] APHNZY-24-3 releases potent antibacterial VOCs: the colony diameter in group T (1.82 ± 0.084 cm) was significantly smaller than that in group CK, with an antibacterial rate of 74.5%.

[0114] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T+Car group), the antibacterial effect was significantly weakened, the colony diameter (3.78 ± 0.13 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 47.1%, but was still significantly lower than that of the CK group.

[0115] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0116]

[0117] 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%.

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

[0119] To elucidate the mechanism by which APHNZY-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 APHNZY-24-3 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0120] Evidence for key gene expression is as follows (see Figure 8 Table 5):

[0121] 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 APHNZY-24-3 treatment group (T) decreased to 28.16% and 15.91% 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.

[0122] 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 1.3% and 8.62% of those in the control group, respectively, both downregulated to a highly significant level (p < 0.001). These two genes are essential for normal conidial formation and maturation, and their sharp decrease in expression directly explains the observed significant reduction in conidial yield at the molecular level.

[0123] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by APHNZY-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.

[0124] Table 5: Inhibitory effect of APHNZY-24-3 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0125]

[0126] Example 4: Identification and verification of antibacterial active substances of APHNZY-24-3, a slow-growing rhizobium from Guangdong.

[0127] To identify the characteristic volatile compounds produced by APHNZY-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 experimental group (APHNZY-24-3) with the control group (CK), a compound with extremely significant differences was identified.

[0128] The compound was clearly identified by having a retention time of 29.643 min and a retention index of 1953.94 in the chromatogram, a similarity of 778 with benzothiazole in the NIST standard library, and a molecular ion peak of m / z 135.

[0129] Quantitative and statistical analyses further confirmed the criticality of this compound (see Figure 9 Table 6):

[0130] 1) Extremely significant enrichment: The relative abundance of this compound in the APHNZY-24-3 treatment group was 2502.44 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 0.011).

[0131] 2) High model importance: In the orthogonal partial least squares discriminant analysis (OPLS-DA) model, its variable importance projection (VIP) value is as high as 1.50, indicating that it is one of the core volatile markers that distinguishes APHNZY-24-3 from the blank control.

[0132] Conclusion: Combined chromatographic-mass spectrometry identification and omics statistical analysis conclusively demonstrate that benzothiazole is a characteristic volatile compound specifically and abundantly produced by APHNZY-24-3. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0133] Table 6: Identification and omics analysis results of benzothiazole, a characteristic volatile substance in APHNZY-24-3.

[0134]

[0135] 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.

[0136] Second, purity verification experiment.

[0137] To directly verify the antibacterial activity of the key volatile compound benzothiazole, the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus was determined using the fumigation method. The results (see Table 7) showed that benzothiazole had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0138] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.71%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 79.67%.

[0139] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 54.47%) to a 40-fold dilution (inhibition rate 22.19%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0140] 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 <9%), indicating that benzothiazole has basically lost its observable antibacterial activity at this concentration.

[0141] Conclusion: This pure product validation experiment conclusively demonstrates that benzothiazole 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 (2502.44-fold) of this compound in the volatile substances of APHNZY-24-3, strongly confirming that benzothiazole is the key effector molecule mediating the volatile antibacterial effect of APHNZY-24-3.

[0142] Table 7: Effects of different dosages of benzothiazole on the growth of Aspergillus flavus mycelia.

[0143]

[0144] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0145] 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 Guangdong, APHNZY-24-3, which possesses both growth-promoting and aflatoxin-inhibiting functions, is characterized by: Its classification name is Bradyrhizobium guangdongense, accession number is CCTCC M 2026225, accession date is January 23, 2026, 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 Guangdong slow-growing rhizobium APHNZY-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 Guangdong slow-growing rhizobium APHNZY-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 Guangdong slow-growing rhizobium APHNZY-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 Guangdong slow-growing rhizobium APHNZY-24-3.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the Guangdong slow-growing rhizobium APHNZY-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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