Bradyrhizobium japonicum ASNMZFT-24-1 with functions of promoting growth and inhibiting aspergillus flavus and application of bradyrhizobium japonicum ASNMZFT-24-1

Abstract

The invention discloses bradyrhizobium japonicum ASNMZFT-24-1 with functions of efficiently promoting growth, promoting nodulation and inhibiting aspergillus flavus and application of the bradyrhizobium japonicum ASNMZFT-24-1, and the preservation number of the bradyrhizobium japonicum ASNMZFT-24-1 is CCTCC (China Center for Type Culture Collection) M 20253049. According to the invention, coupling of two major functions of producing (E)-cinnamyl aldehyde, namely a specific bacteriostatic volatile matter, and high-efficiency symbiotic nitrogen fixation on a single strain is realized in bradyrhizobium japonicum for the first time. The action mechanism is clear: the (E)-cinnamyl aldehyde generated by the strain can accurately down-regulate expression of toxin synthesis key genes and spore development core genes in the 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 particularly aiming at the biological characteristic of underground fruiting of the peanuts, a biological fumigation protection layer is constructed in the soil in the critical legume development period, and aspergillus flavus infection is continuously blocked.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of agricultural microbiology and plant protection, specifically involving a soybean slow-growing rhizobium (Bradyrhizobium japonicum) ASNMZFT-24-1 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 has long faced two mutually exclusive challenges: increasing yield and controlling toxin contamination. 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 legumes (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 soybean slow-growing rhizobium that possesses both highly efficient symbiotic nitrogen fixation capabilities and the ability to directly and effectively inhibit Aspergillus flavus by producing specific volatiles. 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 soybean slow-growing rhizobium strain ASNMZFT-24-1 that combines growth promotion and aflatoxin inhibition functions, along with its applications.

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

[0012] The first objective of this invention is to provide a slow-growing rhizobium of soybean, ASNMZFT-24-1, which has both growth-promoting and aflatoxin-inhibiting functions. Its classification name is Bradyrhizobium japonicum, its accession number is CCTCC M 20253049, its accession date is December 30, 2025, and it is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University.

[0013] A second objective of this invention is to provide a microbial agent or preparation comprising at least one of soybean slow-growing rhizobium ASNMZFT-24-1, 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 soybean slow-growing rhizobium ASNMZFT-24-1, or a microbial agent or preparation containing soybean slow-growing rhizobium ASNMZFT-24-1, in promoting peanut growth and nodulation nitrogen fixation.

[0015] The fourth objective of this invention is to provide the application of soybean slow-growing rhizobium ASNMZFT-24-1, or a microbial agent or preparation containing soybean slow-growing rhizobium ASNMZFT-24-1, 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 soybean slow-growing rhizobium ASNMZFT-24-1.

[0017] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the soybean slow-growing rhizobium ASNMZFT-24-1, or a microbial agent or preparation containing soybean slow-growing rhizobium ASNMZFT-24-1, to the peanut growing 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: Soybean slow-growing rhizobium ASNMZFT-24-1 can effectively promote peanut plant growth and nodulation nitrogen fixation. After 30 days of inoculation, peanut plant height and biomass increased significantly by 42.2% and 28.9% respectively compared with the control; at the same time, the number of root nodules and the fresh weight of root nodules increased significantly by 101.3% and 31.1% respectively. Transcriptome analysis further revealed 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, confirming its synergistic mechanism of promoting symbiosis and growth at the molecular level.

[0020] (2) Comprehensive inhibition of Aspergillus flavus activity: The volatile organic compounds (VOCs) released by the strain have a strong and broad-spectrum inhibitory effect on Aspergillus flavus. In vitro fumigation experiments showed that the inhibition rates on Aspergillus flavus mycelial growth, spore production, and aflatoxin B1 synthesis were as high as 78.0%, 97.5%, and 96.5%, respectively. Activated carbon adsorption experiments confirmed that this antibacterial effect is mainly mediated by volatile substances.

[0021] (3) The antibacterial mechanism is clear and the key components are identified: The characteristic volatile (E)-cinnamaldehyde produced by the strain was identified as the key antibacterial molecule. It was specifically enriched in the strain's metabolites, and the pure product showed a strong dose-dependent inhibitory effect on Aspergillus flavus (inhibition rate of 97.9% in the original solution). Molecular mechanism studies showed that the VOCs of this strain can accurately and significantly downregulate the expression of the core regulatory genes (aflR, aflS) for aflatoxin synthesis and the key genes (abaA, wetA) for spore development, thereby blocking its toxin production and reproduction at the transcriptional level.

[0022] (4) Integrated Functions and Strong Field Applicability: This invention is the first to achieve an innovative coupling of two major functions in a single strain of soybean slow-growing rhizobium: "highly efficient symbiotic nitrogen fixation" and "(E)-cinnamaldehyde-mediated potent antibacterial activity". With a single inoculation, it can simultaneously promote growth and increase yield while preventing the spread of toxins at the source in the same ecological niche of peanut rhizosphere. In particular, it provides a green and efficient integrated biological solution to address the traditional chemical control obstacles caused by peanut's "flowering above ground and fruiting underground".

[0023] This invention provides a slow-growing rhizobium strain of soybean, ASNMZFT-24-1, which has both growth-promoting and aflatoxin-inhibiting functions. Its classification name is Bradyrhizobium japonicum. This slow-growing rhizobium strain ASNMZFT-24-1 was obtained by the inventors of this invention through screening. The accession number of ASNMZFT-24-1 is CCTCC M 20253049, the accession date is December 30, 2025, and it is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. Attached Figure Description

[0024] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of Bradyrhizobium spp. in peanut rhizosphere soil. The independent samples t-test was used for statistical analysis, and the data are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p < 0.001).

[0025] Figure 2 This invention constructs an ASNMZFT-24-1 phylogenetic tree based on multiple genes (16S, atpD, recA, dnaK, glnII, 23S).

[0026] Figure 3 This invention relates to the phenotypic effects of inoculation with the soybean slow-growing rhizobium ASNMZFT-24-1 on peanut seedling growth and root nodule formation; wherein, Figure 3 A shows a comparison of the phenotypes of the entire plant (after washing); Figure 3 B shows a close-up comparison of peanut pods; in the figure, CK represents the blank control group, and T represents the ASNMZFT-24-1 inoculation treatment group.

[0027] Figure 4 This invention relates to the effects of soybean slow-growing rhizobium ASNMZFT-24-1 inoculation on key phenotypic indicators of peanut seedling growth and symbiotic nodulation; wherein, Figure 4 A represents the effect on plant height; Figure 4 B represents the effect on the fresh weight of the plant; Figure 4C 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 ASNMZFT-24-1 inoculation treatment group; the column height represents the mean value of each indicator, and the error bar represents the standard deviation (n ≥ 3). The differences between groups were statistically significant (p < 0.001).

[0028] 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 soybean slow-growing rhizobium ASNMZFT-24-1 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 in the nodule signaling pathway; Figure 5 C represents the expression level of the auxin early response factor gene SAUR; Figure 5 D represents the expression level of the auxin early response factor gene AUXIN. In the figure, CK represents the blank control group, and T represents the APFJPT-23-4 inoculation treatment group; the column height represents the gene expression level (based on standardized counts); the error bar represents the standard deviation (n=3). and "p < 0.05" and "p < 0.001" respectively indicate significant differences between groups.

[0029] Figure 6 This invention evaluates the inhibitory effect of soybean slow-growing rhizobium ASNMZFT-24-1 on the growth, sporulation, and aflatoxin synthesis of Aspergillus flavus using a double-plate method; wherein... Figure 6 A represents the diameter of Aspergillus flavus colonies; Figure 6 B represents conidia yield; Figure 6 C represents aflatoxin production. In the figure, CK represents the blank control group, and T represents the ASNMZFT-24-1 treatment group; quantitative data are expressed as mean ± standard deviation (n ≥ 3); indicates highly significant differences between groups (independent samples t-test). ).

[0030] Figure 7 This is a bar chart showing the quantitative statistical results of Aspergillus flavus colony diameter in the blank control group (CK), blank control group (CK) + activated carbon (Car), Bradyrhizobium japonicum ASNMZFT-24-1 treatment group (T), and slow-growing rhizobium ASNMZFT-24-1 treatment group (T) + activated carbon (Car); In the figure, The differences between groups were statistically significant (p < 0.001).

[0031] Figure 8 This invention relates to the effect of treatment with the soybean slow-growing rhizobium ASNMZFT-24-1 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, boxes represent quartile ranges, the midline represents the median, the whisker lines represent ranges, and dots represent individual cells. , and The values ​​represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0032] 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 (E) cinnamaldehyde in the blank control group (CK) and the ASNMZFT-24-1 treated group (T); box plots show the distribution of this compound in the two groups; in the figure, This indicates that the difference between the two groups is extremely significant (independent samples t-test, p < 0.001). Detailed Implementation

[0033] 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.

[0034] Example 1: Isolation, identification and preservation of ASNMZFT-24-1, a multifunctional slow-growing rhizobium of soybean.

[0035] The slow-growing rhizobium ASNMZFT-24-1 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 9CFU / g, Bacillus subtilis ≥ 1×10 10 CFU / g, effective viable count of Ludwig's Enterobacter ≥ 1×10⁻⁶ 10 CFU / g, effective viable count of Flavobacterium breve ≥ 2×10⁻⁶ 9 CFU / gram.

[0036] 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-treated group (T) was obtained based on 16S rRNA amplicon sequencing analysis. Specifically, the average relative abundance of *Bradyrhizobium* in the CK group was 0.45%, while the average relative abundance in the ARC-treated group (T group) increased to 0.55%, a significant increase of 24.3% (p < 0.001). Statistical analysis showed that this enrichment effect was extremely significant (p < 0.001). This result provides direct ecological evidence for the subsequent targeted isolation and screening of the functionally enhanced soybean *Bradyrhizobium* ASNMZFT-24-1 from the rhizosphere soil of the ARC-treated group, indicating that the ARC inoculant can reshape the rhizosphere microbiota and specifically promote the colonization and enrichment of the beneficial symbiotic bacterium—*Bradyrhizobium*.

[0037] 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. Gradient dilution: Take 1.0 mL of the mother suspension and perform seven consecutive 10-fold serial dilutions (10... -1 Up to 10 -7 ).

[0038] 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.

[0039] 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 ASNMZFT-24-1.

[0040] Liquid culture: A single colony of soybean slow-growing rhizobium ASNMZFT-24-1 was inoculated into 5 mL of YMB liquid medium and cultured at 28℃ with shaking at 180 rpm for 72 hours until the late logarithmic growth phase (OD200). 600 (Values ​​are between 0.8 and 1.0). Short-term storage: Streak the above bacterial suspension on fresh YMA plates and store at 4°C, subculturing monthly. Long-term storage: Take the bacterial suspension in the logarithmic growth phase (OD200). 600 ≈ 0.8) is mixed with an equal volume of sterile 60% (v / v) glycerol protectant to make a final glycerol concentration of 30%, dispensed into cryovials, and stored in an ultra-low temperature freezer at -80 °C.

[0041] Genomic DNA extraction: Genomic DNA was extracted from the ASNMZFT-24-1 pure culture using the TIANamp Bacteria DNA Kit according to the instructions, and its concentration and purity (A260 / A280 ratio between 1.8 and 2.0) were determined using a NanoDrop™ 2000 spectrophotometer.

[0042] 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 1441 bp (as shown in SEQ ID NO: 3).

[0043] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0044] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3.

[0045] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that ASNMZFT-24-1 had a 16S rRNA gene similarity of >99.5% with type strains such as Bradyrhizobium japonicum ACCC 15027.

[0046] 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.

[0047] atpD gene: Amplified using primers TSatpDf and TSatpDr, yielding a sequence of approximately 488 bp (the atpD sequence of ASNMZFT-24-1 is shown in SEQ ID NO: 6).

[0048] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0049] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0050] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 481 bp (as shown in SEQ ID NO: 9).

[0051] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0052] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0053] dnaK gene: Amplified using primers BRdnaKf and BRdnaKr, yielding a sequence of approximately 594 bp (as shown in SEQ ID NO: 12).

[0054] The primer BRdnaKf sequence is shown in SEQ ID NO: 10: 5'- TTCGACATCGACGCSAACGG-3';

[0055] The primer BRdnaKr sequence is shown in SEQ ID NO: 11: 5'-GCCTGCTGCKTGTACATGGC-3'.

[0056] glnll gene: Amplified using primers TSglnIIf and TSglnIIr, yielding a sequence of approximately 587 bp (as shown in SEQ ID NO: 15).

[0057] The primer TSglnIIf sequence is shown in SEQ ID NO: 13: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0058] The primer TSglnIIr sequence is shown in SEQ ID NO: 14: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0059] 23S gene: Amplified using primers FGPS1490 and FGPS130, yielding a sequence of approximately 850 bp (as shown in SEQ ID NO: 18).

[0060] The primer FGPS1490 sequence is shown in SEQ ID NO: 16: 5'-TGCGGCTGGATCACCTCCTT-3';

[0061] The primer FGPS130 sequence is shown in SEQ ID NO: 17: 5'-CCGGGTTTCCCCATTCGG-3'.

[0062] Multigene phylogenetic analysis: The gene sequences of ASNMZFT-24-1 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 with the neighbor-joining method (bootstrap = 1000). The results are clearly shown (see...). Figure 2 ASNMZFT-24-1 clustered with the type strain Bradyrhizobium japonicum ACCC 15027, confirming its taxonomic position as Bradyrhizobium japonicum.

[0063] Example 2: Functional verification of soybean slow-growing rhizobium ASNMZFT-24-1 in promoting peanut growth and nodulation nitrogen fixation.

[0064] Test materials: The peanut variety used was Zhonghua 28. The soil was sandy loam, sterilized twice by autoclaving at 121℃ for 0.5 hours each time to eliminate the influence of native rhizobia. Inoculum preparation: ASNMZFT-24-1 was cultured in YMB until mid-log (OD600 ≈ 0.6), and the cells were collected by centrifugation at 5000 rpm for 5 minutes. The cells were resuspended in sterile 0.85% NaCl solution and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL (calibrated by plate count) was used as the inoculum suspension.

[0065] Experimental treatments: ASNMZFT-24-1 treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned ASNMZFT-24-1 bacterial suspension at a ratio of 10% (v / w) of seed weight. After slightly drying, the seeds were sown in pots. Blank control group (CK): Seeds were mixed with an equal volume of sterile 0.85% NaCl solution, and the remaining operations were the same as the treatment group. Experimental setup: A completely randomized block design was adopted, with 12 replicates (i.e., 12 pots) in each group. 8 seeds were sown in each pot, and 3 strong seedlings were retained after thinning. Culture conditions: Cultured in an artificial climate chamber with a photoperiod of 16 hours light / 8 hours dark, a day / night temperature of 28℃ / 22℃, and a relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0066] 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).

[0067] Plant height: The average plant height of the ASNMZFT-24-1 inoculated group (T) was 37.3 ± 0.58 cm, which was significantly increased by 42.2% compared with the blank control group (CK, 26.23 ± 1.81 cm) (p = 2.45 × 10⁻⁶). -9 ).

[0068] Fresh weight: The average fresh weight of group T was 78.37 ± 0.73 g, which was significantly higher than that of group CK (60.78 ± 1.85 g) by 28.9% (p = 1.91 × 10⁻⁶). -11 ).

[0069] Nodule count: The average number of root nodules in group T was 176 ± 7.43 / plant, which was significantly increased by 101.3% compared with group CK (87.44 ± 5.13 / plant) (p = 6.70 × 10⁻⁶). -13 ).

[0070] Nodule weight: The average fresh weight of root nodules in group T was 0.211 ± 0.019 g / plant, which was significantly increased by 31.1% compared with group CK (0.161 ± 0.013 g / plant) (p = 2.03 × 10⁻⁶). -6 ).

[0071] Conclusion: Inoculation with ASNMZFT-24-1 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.

[0072] Table 1: The promoting effect of ASNMZFT-24-1 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0073]

[0074] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0075] Inoculation with the soybean slow-growing rhizobium ASNMZFT-24-1 significantly promoted the growth and symbiotic nodulation of peanut seedlings, with the following direct phenotypic differences: Figure 3 As shown. In terms of overall morphology, the plants in the treatment group (T) inoculated with ASNMZFT-24-1, after washing ( Figure 3 A), exhibiting significant growth advantages: taller plants, more lush foliage, and more vigorous growth. Most importantly, direct observation of the peanut pods ( Figure 3 B) Clearly, group T has more peanut pods, cleaner pod surfaces, and significantly more effective root nodules in both number and size than group CK. Figure 3 A). These striking visual evidences, along with the aforementioned highly significant quantitative data (Table 1) showing a 42.2% increase in plant height, a 28.9% increase in plant fresh weight, and a 101.3% and 31.1% increase in root nodule number and fresh weight, respectively, together constitute a complete chain of evidence demonstrating that ASNMZFT-24-1 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation capacity.

[0076] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the ASNMZFT-24-1 treatment group (T) and the blank control group (CK) (three plants were mixed for each replicate), and were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0077] 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).

[0078] Results: To elucidate the molecular basis of ASNMZFT-24-1's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that ASNMZFT-24-1 treatment specifically activated a network of key genes in peanut roots related to symbiotic signal transduction and plant hormone signaling. Evidence for key gene expression is as follows (see results). Figure 5 Table 2):

[0079] 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 1.98-fold and 3.06-fold, respectively. These genes are the core regulatory switches that initiate the root nodule formation process.

[0080] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 10.91-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 18.54-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0081] Conclusion: The above molecular evidence indicates that ASNMZFT-24-1 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 ASNMZFT-24-1 promotes nodulation and growth at the transcriptional level: by mimicking or enhancing natural symbiotic dialogue signals, it initiates and accelerates the host's symbiotic developmental program and growth response.

[0082] Table 2: Effects of ASNMZFT-24-1 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0083]

[0084] Example 3: Functional verification of soybean slow-growing rhizobium ASNMZFT-24-1 in inhibiting Aspergillus flavus growth and toxin production.

[0085] First, in vitro antagonism experiment (double-plate inverted method). To evaluate the antagonistic effect of ASNMZFT-24-1 against Aspergillus flavus, an in vitro antagonism experiment was conducted using the double-plate inverted method.

[0086] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0087] 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.

[0088] Lower plate (90 mm in diameter): ASNMZFT-24-1 treatment group (T) uniformly coated with 100 µL of 1×10⁻⁶ solution. 8 A fresh bacterial suspension of ASNMZFT-24-1 at CFU / mL; a blank control group (CK) was spread onto an equal volume of sterile YMB medium. After the bacterial suspension was absorbed, the upper plate was inverted.

[0089] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0090] Measurement indicators:

[0091] 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%.

[0092] 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.

[0093] 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).

[0094] The results show (see) Figure 6 (Table 3) The ASNMZFT-24-1 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by ASNMZFT-24-1 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus, achieving efficient source control.

[0095] Inhibition of mycelial growth: The colony diameter in the ASNMZFT-24-1 treatment group (T) was 1.6 ± 0.07 cm, significantly lower than that in the blank control group (CK, 7.26 ± 0.05 cm), with an inhibition rate of 77.96% (p = 6.96 × 10⁻⁶). -15 ).

[0096] Inhibition of spore reproduction: The spore yield in the ASNMZFT-24-1 treatment group was 1.74 ± 0.11 × 10⁻⁶. 6The number of samples per dish was significantly lower than that of the blank control group (CK, 70.6 ± 6.88 × 10⁻⁶). 6 The inhibition rate reached 97.53% (p = 1.68 × 10⁻⁶). -8 ).

[0097] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the treatment group was 20.67 ± 6.08 ng / plate, which was significantly lower than that in the control group (CK, 590 ± 66.93 ng / plate), with an inhibition rate of 96.5% (p = 2.31 × 10⁻⁶). -14 ).

[0098] Table 3: Inhibitory effect of ASNMZFT-24-1 treatment on key life activities of Aspergillus flavus.

[0099]

[0100] Note: Inhibition rate (%) = [(CK group mean - T group mean) / CK group mean] × 100%. All data are expressed as mean ± standard deviation.

[0101] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0102] To confirm that the antibacterial effect of ASNMZFT-24-1 is mainly mediated by the volatile organic compounds (VOCs) it releases, an activated carbon adsorption blocking experiment was conducted.

[0103] 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.

[0104] CK group (blank control): The lower plate was not inoculated with ASNMZFT-24-1 and no activated carbon was used.

[0105] CK + Car group (activated carbon control): The lower plate was not inoculated with ASNMZFT-24-1, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0106] Group T (with ASNMZFT-24-1, without activated carbon): The lower plate is uniformly coated with ASNMZFT-24-1 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0107] T + Car group (with ASNMZFT-24-1 and activated carbon): Half of the lower plate was coated with an equal amount of ASNMZFT-24-1 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0108] Results and analysis (see Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of ASNMZFT-24-1. This experiment demonstrates that the inhibitory effect of ASNMZFT-24-1 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0109] 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 ± 0.08 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0110] ASNMZFT-24-1 releases potent antibacterial VOCs: the colony diameter in group T (1.6 ± 0.07 cm) was significantly smaller than that in group CK, with an antibacterial rate of 77.96%.

[0111] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (3.54 ± 0.11 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 51.2%, but was still significantly lower than that of the CK group.

[0112] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0113]

[0114] The key role of ASNMZFT-24-1 in inhibiting the growth of Aspergillus flavus mycelia 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%.

[0115] Third, transcriptomics verification (molecular mechanism).

[0116] To elucidate the mechanism by which ASNMZFT-24-1 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 ASNMZFT-24-1 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0117] Evidence for key gene expression is as follows (see results) Figure 8 Table 5):

[0118] 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 ASNMZFT-24-1 treatment group (T) decreased to 30.04% and 13.84% 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.

[0119] 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 1.24% and 8.76% 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.

[0120] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by ASNMZFT-24-1 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.

[0121] Table 5: Inhibitory effect of ASNMZFT-24-1 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0122]

[0123] Example 4: Identification and verification of antibacterial active substances of soybean slow-growing rhizobium ASNMZFT-24-1.

[0124] To identify the characteristic volatile compounds produced by the soybean slow-growing rhizobium ASNMZFT-24-1, headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) was used for untargeted metabolomics analysis of the volatile components of the strain culture. By comparing the ASNMZFT-24-1 treatment group (T) with the control group (CK), a compound with extremely significant differences was identified. This compound had a retention time of 27.724 min and a retention index of 1858.5 in the chromatogram. Its mass spectrum showed a similarity of 793 to (E)-cinnamaldehyde ((E)-) in the NIST standard library, with a molecular ion peak at m / z 132, thus confirming its identification.

[0125] Quantitative and statistical analyses further confirmed the criticality of this compound (see Figure 9 Table 6):

[0126] 1) Extremely significant enrichment: The relative abundance of this compound in the ASNMZFT-24-1 treated group was 2.5 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 9.36 × 10⁻⁶). -5 ).

[0127] 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 1.34, indicating that it is one of the core volatile markers that distinguishes ASNMZFT-24-1 from the blank control.

[0128] Conclusion: Combined chromatographic-mass spectrometry identification and omics statistical analysis conclusively demonstrate that (E)-cinnamaldehyde is a characteristic volatile compound specifically and abundantly produced by the soybean slow-growing rhizobium ASNMZFT-24-1. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0129] Table 6: Identification and omics analysis results of characteristic volatile substance (E)-cinnamaldehyde in ASNMZFT-24-1.

[0130]

[0131] 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.

[0132] Second, purity verification experiment.

[0133] To directly verify the antibacterial activity of the key volatile substance (E)-cinnamaldehyde, the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus was determined using the fumigation method. The results showed (see Table 7) that (E)-cinnamaldehyde had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0134] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.9%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 81.4%.

[0135] 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 22.4%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0136] 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 < 8%), indicating that (E)-cinnamaldehyde has basically lost its observable antibacterial activity at this concentration.

[0137] Conclusion: This pure product verification experiment conclusively demonstrates that (E)-cinnamaldehyde itself is a potent inhibitor of Aspergillus flavus mycelial growth. It exerts a significant antibacterial effect even at low concentrations, a characteristic consistent with the highly specific and significant enrichment (64,000-fold) of this compound in the volatile substances of ASNMZFT-24-1, strongly confirming that (E)-cinnamaldehyde is the key effector molecule mediating the volatile antibacterial effect of ASNMZFT-24-1.

[0138] Table 7: Effects of different dosages of (E)-cinnamaldehyde on the growth of Aspergillus flavus mycelia.

[0139]

[0140] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0141] 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 of soybean, ASNMZFT-24-1, which has both growth-promoting and aflatoxin-inhibiting functions, is characterized by, Its classification name is Bradyrhizobium japonicum, accession number is CCTCC M 20253049, 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 soybean slow-growing rhizobium ASNMZFT-24-1 as described in claim 1, its fermentation product or culture thereof; 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 dried preparation thereof.

3. The application of the soybean slow-growing rhizobium ASNMZFT-24-1 as described in claim 1 or the microbial agent or preparation as described in claim 2 in promoting peanut growth and nodulation nitrogen fixation.

4. The application of the soybean slow-growing rhizobium ASNMZFT-24-1 as described in claim 1 or the microbial agent or preparation as described in claim 2 in inhibiting the growth, sporulation, and aflatoxin synthesis of Aspergillus flavus.

5. The application according to claim 4, characterized in that, The specific application involves achieving the inhibitory function through the volatile organic compounds released by the soybean slow-growing rhizobium ASNMZFT-24-1.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the soybean slow-growing rhizobium ASNMZFT-24-1 as described in claim 1 or the microbial agent or preparation as described in claim 2 to the peanut growing environment or peanut plants.

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