Strain of sinorhizobium meliloti ASZJHZ25-4 with functions of promoting growth and inhibiting aspergillus flavus and application of sinorhizobium meliloti ASZJHZ25-4

Abstract

The invention discloses sinorhizobium meliloti ASZJHZ25-4 with functions of efficiently promoting growth and inhibiting aspergillus flavus and application of the sinorhizobium meliloti ASZJHZ25-4, and the preservation number of the sinorhizobium meliloti ASZJHZ25-4 is CCTCC M 2026230. According to the invention, the integration of two functions of generating a characteristic antibacterial volatile matter of 2-methylbutyric acid and efficiently symbiotic nitrogen fixation on a single strain is realized in the sinorhizobium meliloti for the first time. The 2-methylbutyric acid volatile matter released by the strain can accurately down-regulate expression of toxin synthesis key genes and spore development core genes in aspergillus flavus, so that efficient bacteriostasis is realized on the molecular level. Through one-time inoculation, the dual purposes of growth promotion and nitrogen fixation and source bacteriostasis can be synchronously achieved at the rhizosphere ecological niche of the peanuts. Especially aiming at the biological characteristics of overground flowering and underground fruiting of peanuts, a lasting biological fumigation protection layer is formed in a soil microenvironment in a legume development critical period.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of *Sinorhizobium meliloti* ASZJHZ25-4 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. Background Technology

[0002] I. The Importance and Core Challenges of the Peanut Industry

[0003] As a globally important oilseed and cash crop, the safe production of peanuts is of strategic significance for ensuring food and oil supply, increasing farmers' income, and maintaining trade stability. However, sustainable peanut production faces two core challenges: increasing yield and controlling aflatoxin contamination. Aflatoxins (AFs), potent carcinogens produced by Aspergillus flavus, not only seriously endanger food safety and consumer health but also frequently trigger international trade technical barriers, becoming a key bottleneck restricting the high-quality development of the peanut industry.

[0004] II. The core mechanism of increased yield in leguminous crops and the role of Rhizobium sinense

[0005] For legumes like peanuts, the most efficient way to increase yield lies in establishing a specific symbiotic relationship with rhizobia, forming root nodules, and converting atmospheric nitrogen into a usable nitrogen source for the plant. *Sinorhizobium meliloti* is a known group of rhizobia capable of highly efficient symbiosis with various legumes, and it has significant potential for promoting nodulation and nitrogen fixation. However, traditional rhizobium agents have limited functions, primarily focusing on nitrogen fixation and growth promotion, and generally lack direct antagonistic ability against soil-borne pathogens such as *Aspergillus flavus*.

[0006] III. Limitations of Existing Aflatoxin Control Technologies

[0007] Currently, aflatoxin control mainly relies on agricultural management measures, physicochemical methods, and biological control primarily using Bacillus spp. and Trichoderma spp. These methods are either passively effective and unstable, or costly and pose a risk of chemical residues. Crucially, peanuts' unique growth habit of "flowering above ground and fruiting underground" makes traditional above-ground chemical or biological control methods ineffective against the soil-borne fruits during the critical stages of pod development and infection, creating a significant obstacle to physical control.

[0008] IV. Systemic Defects of Existing Technological Approaches

[0009] In current peanut production, "increasing yield" and "preventing pesticide poisoning" are separated into two parallel but independent systems in terms of technological approaches. This structural separation has led to the following deep-seated systemic defects:

[0010] (1) Lack of functional singularity and biological synergy

[0011] The core design of existing growth-promoting microbial agents (including traditional rhizobium inoculants) mainly revolves around growth-promoting physiological mechanisms such as nitrogen fixation, phosphorus solubilization, and hormone production. Their functional positioning is singular, and they generally lack direct and efficient inhibition of specific soil-borne pathogens such as Aspergillus flavus. Conversely, the breeding and application of existing biocontrol agents focus on antagonizing pathogens. Their metabolic pathways and mechanisms of action are often not directly related to promoting plant growth (especially the symbiotic nitrogen fixation process unique to leguminous crops), and may even interfere with rhizobium colonization and symbiotic establishment due to nutrient competition, space occupation, or the secretion of inhibitory metabolites. Introducing these two types of single-function microorganisms simultaneously into the complex rhizosphere ecosystem not only makes it difficult to achieve functional complementarity and synergistic effects, but may also lead to unstable and unpredictable field effects due to interspecific competition or antagonism, or even mutual weakening, creating a technical dilemma of "1+1<2".

[0012] (2) The operation is more complicated and the overall cost is significantly increased.

[0013] In actual production, farmers need to purchase, store, prepare, and apply two different types of microbial agents separately. This "dual-product, dual-process" application model not only directly increases the material costs of seed treatment or field operations but also significantly increases labor input, equipment usage, and time management costs. The complex operating procedures reduce the operability and user acceptance of the technology, especially in large-scale, intensive planting models, becoming a major obstacle to technology promotion. In addition, the storage conditions, application timing, and methods of the two types of products may differ, further increasing the difficulty and risk of technology implementation.

[0014] (3) Insufficient adaptation to the unique biological characteristics of peanuts and weak control of pollution sources

[0015] Aspergillus flavus infection of peanuts begins in the field soil and infects the deeply buried pods through the pegs or cracks during the critical period of pod development and enlargement. The unique growth habit of peanuts—flowering above ground and fruiting underground—makes traditional above-ground chemical or biological control methods almost completely ineffective during the crucial fruiting period. Seed treatments applied at sowing cannot cover the months-long pod development period; foliar sprays cannot reach the underground pods; and large-scale application of chemical fungicides through irrigation systems indiscriminately disrupts the soil microbial community balance, easily inducing pathogen resistance and leading to environmental pollution and pesticide residue risks. Existing separation technologies lack an integrated solution that can simultaneously implement "nutrient supply (nitrogen fixation)" and "biological protection (antibacterial activity)" within the same ecological niche (rhizosphere) and the same growth cycle, using the same biological carrier in the soil microenvironment during pod development. This results in a serious deficiency in field-level control of aflatoxin.

[0016] V. The Innovative Positioning and Technological Gaps of this Invention

[0017] Based on the aforementioned industry bottlenecks, there is a clear technological gap in this field: to date, no publicly reported or commercially available products have demonstrated that *Rhizobium sinense* of Osmanthus fragrans possesses a multifunctional strain capable of strongly inhibiting *Aspergillus flavus* by producing characteristic antibacterial volatiles such as 2-methylbutyric acid, while maintaining highly efficient symbiotic nitrogen fixation capabilities.

[0018] This invention aims to fill this gap by screening and developing *Sinomenium adenophorum* ASZJHZ25-4, which possesses both of the aforementioned dual functions, to achieve synergistic regulation of peanut yield increase and toxin control on a single microbial carrier. This strain, through a single inoculation, can simultaneously establish a dual-functional system of "nitrogen fixation and supply" and "bio-inhibition" in the peanut rhizosphere microdomain. It provides a source-oriented, integrated, and environmentally friendly innovative biological solution, particularly addressing the traditional control challenges posed by peanut "underground fruiting," and is of great significance for promoting green and safe peanut production. Summary of the Invention

[0019] To address the shortcomings of existing technologies, this invention provides a *Rhizobium sinense* strain ASZJHZ25-4 that combines growth promotion and aspergillosis inhibition functions, along with its applications.

[0020] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0021] The first objective of this invention is to provide a strain of *Sinorhizobium meliloti* ASZJHZ25-4 that has both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Sinorhizobium meliloti*, accession number is CCTCC M 2026230, 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.

[0022] A second objective of this invention is to provide a microbial agent or preparation comprising at least one of *Rhizobium sinense* ASZJHZ25-4, 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.

[0023] The third objective of this invention is to provide the application of *Rhizobium sinense* ASZJHZ25-4, or a microbial agent or preparation containing *Rhizobium sinense* ASZJHZ25-4, in promoting peanut growth and nodulation nitrogen fixation.

[0024] The fourth objective of this invention is to provide the application of *Rhizobium sinense* ASZJHZ25-4, or a microbial agent or preparation containing *Rhizobium sinense* ASZJHZ25-4, in inhibiting the growth, sporulation, and synthesis of aflatoxin by *Aspergillus flavus*.

[0025] Preferably, the application specifically achieves the inhibitory function through the volatile organic compounds released by the *Rhizobium sinense* ASZJHZ25-4.

[0026] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Rhizobium sinense ASZJHZ25-4, or a microbial agent or preparation containing Rhizobium sinense ASZJHZ25-4, to the peanut growing environment or peanut plants.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) Significant effect on promoting growth and nitrogen fixation: Rhizobium sinense ASZJHZ25-4 can comprehensively promote the growth and nitrogen fixation of peanut plants. After inoculation, the peanut plant height and biomass increased by 36.4% and 29.6%, respectively, and the number of root nodules and the fresh weight of root nodules increased significantly by 110.4% and 32.4%, respectively. Transcriptome analysis further confirmed that this strain can specifically upregulate the expression of key genes in the nodulation signaling (NSP1, NSP2) and auxin signaling (SAUR, AUXIN) pathways in peanut roots, revealing its efficient growth-promoting nitrogen fixation mechanism at the molecular level.

[0029] (2) Highly effective inhibition of Aspergillus flavus: The volatile organic compounds (VOCs) produced by the strain have a strong and comprehensive 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 79.7%, 97.8%, and 96.4%, respectively. Activated carbon adsorption experiments confirmed that this antibacterial effect is mainly mediated by volatile substances.

[0030] (3) Key antibacterial components are clearly identified and their mechanisms are well understood: Through metabolomics and pure product verification experiments, 2-methylbutyric acid was confirmed as the key volatile antibacterial active substance. This compound was specifically enriched in the strain's metabolites (relative abundance was 171,200 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 at the transcriptional level.

[0031] (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 "highly effective biocontrol mediated by 2-methylbutyric acid" into the same strain of *Rhizobium sinense*. 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.

[0032] This invention provides a *Sinorhizobium meliloti* strain ASZJHZ25-4, which possesses both growth-promoting and aflatoxin-inhibiting functions. This strain was obtained by the inventors through screening. The *Sinorhizobium meliloti* strain ASZJHZ25-4 has the accession number CCTCC M2026230, was deposited on January 23, 2026, at the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. Attached Figure Description

[0033] Figure 1This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of *Sinorhizobium meliloti* in peanut rhizosphere soil. In the figure, CK represents the blank control group, and T represents the ASZJHZ25-4 treatment group. Statistical analysis was performed using an independent samples t-test, and data are expressed as mean ± standard deviation. This indicates that the difference between the two groups is extremely significant (p < 0.001).

[0034] Figure 2 This invention constructs the ASZJHZ25-4 phylogenetic tree based on multiple genes (16S, atpD, recA, glnII, nifH).

[0035] Figure 3 This invention relates to the effects of inoculation with *Rhizobium sinense* ASZJHZ25-4 on peanut growth, nodulation, and pod development. (A) Comparison of whole plant phenotypes between the inoculated group and the control group (uninoculated); (B) Close-up of roots and nodules in the treated group; (C) Close-up of roots and nodules in the control group; (D) Comparison of mature pod morphology between the inoculated group and the control group (uninoculated). As shown in the figures, inoculation with ASZJHZ25-4 significantly promoted peanut plant growth, nodule formation, and pod development, demonstrating the comprehensive potential of this strain in promoting symbiotic nitrogen fixation and yield formation in peanuts.

[0036] Figure 4 This invention relates to the effects of inoculation with *Rhizobium sinense* ASZJHZ25-4 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 ASZJHZ25-4 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n ≥ 3). and "p < 0.01" and "p < 0.001" respectively indicate extremely significant differences between groups.

[0037] Figure 5 This is a schematic diagram illustrating the upregulation of key genes related to peanut root symbiosis and growth induced by treatment with *Rhizobium sinense* ASZJHZ25-4 of this invention; wherein, Figure 4 A represents the expression level of NSP1, a core transcription factor gene in the nodule signaling pathway; Figure 4 B represents the expression level of NSP2, a co-regulatory factor gene of the nodule signaling pathway; Figure 4 C represents the expression level of the auxin early response factor gene SAUR; Figure 4 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 treatment group; the column height represents the gene expression level (based on standardized counts); the error bar represents the standard deviation (n=3). , "p < 0.01" and "p < 0.001" respectively indicate that the differences between groups are highly significant (p < 0.01) and highly significant (p < 0.001).

[0038] Figure 6 This invention evaluates the inhibitory effect of *Rhizobium sinense* ASZJHZ25-4 on the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus* using a double-plate method; wherein... Figure 6 A represents the diameter of Aspergillus colonies in the blank control group (CK) / ASZJHZ25-4 treatment group (T); Figure 6 B represents the sporulation rate of the blank control group (CK) / ASZJHZ25-4 treatment group (T); Figure 6 C represents the aflatoxin yield in the blank control group (CK) / ASZJHZ25-4 treatment group (T). In the figure, quantitative data are expressed as mean ± standard deviation (n ≥ 3). This indicates that the difference between groups is extremely significant (independent samples t-test, p < 0.001).

[0039] Figure 7 This is a bar chart showing the quantitative statistical results of Aspergillus colony diameter in the blank control group (CK), blank control group + activated carbon (CK + Car), ASZJHZ25-4 treatment group (T), and ASZJHZ25-4 treatment group + activated carbon (T + Car); The figure shows... The differences between groups were statistically significant (p < 0.001).

[0040] Figure 8 This invention relates to the effect of treatment with *Rhizobium sinense* ASZJHZ25-4 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. , The numbers indicate significant differences between groups (p < 0.05) and highly significant differences (p < 0.001), respectively.

[0041] Figure 9 This invention employs headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) to detect and compare the relative abundance of the volatile component 2-methylbutyric acid in the blank control group (CK) and the ASZJHZ25-4 treatment group (T); box plots show the distribution of this compound in the two groups; in the figure, This indicates that the difference between the two groups is extremely significant (independent samples t-test, p < 0.005). Detailed Implementation

[0042] 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.

[0043] Example 1: Isolation, identification and preservation of the multifunctional herb *Sinomenium sinense* ASZJHZ25-4.

[0044] The *Osmanthus fragrans* rhizobium ASZJHZ25-4 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 was ≥ 2 × 10⁻⁶. 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 Ludwig's Enterobacter ≥ 1×10⁻⁶ 10 CFU / g, effective viable count of Flavobacterium breve ≥ 2×10⁻⁶ 9 CFU / gram.

[0045] 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 *Sinorhizobium* spp., which is closely related to peanut symbiotic nitrogen fixation. For example... Figure 1As shown, the average relative abundance of *Rhizobium sinense* in the CK group was 0.037%, while the average relative abundance in the ARC-treated group (T group) increased to 0.048%, a significant increase of 29.7% (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 functionally enhanced *Rhizobium sinense* ASZJHZ25-4 from the rhizosphere soil of the ARC-treated group, indicating that the ARC inoculant can reshape the rhizosphere microbiota and specifically promote the colonization and enrichment of the beneficial symbiotic bacterium *Rhizobium sinense*.

[0046] 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 ).

[0047] 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. , On a solid plate containing 0.1 g / L NaCl, 15.0 g / L agar, pH 6.8 - 7.0.

[0048] Colony selection and purification: After incubation in the dark at 28 ± 1℃ for 5-7 days, select typical single colonies of *Rhizobium sinense* that do not absorb Congo red, are milky white, raised, viscous, and have neat edges. Purify the culture using three consecutive streak plating tests to obtain a pure culture, designated ASZJHZ25-4.

[0049] Liquid culture: A single colony of *Rhizobium sinense* ASZJHZ25-4 was inoculated into 5 mL of YMB liquid medium and cultured at 28°C with shaking at 180 rpm for 72 hours until the late logarithmic growth phase (OD200). 600 (Values ​​are between 0.8 and 1.0). Short-term storage: Streak the above bacterial suspension on fresh YMA plates and store at 4°C, subculturing monthly. Long-term storage: Take the logarithmic growth phase bacterial suspension (OD... 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.

[0050] 1.4 Molecular biological identification

[0051] Genomic DNA extraction: Genomic DNA was extracted from the ASZJHZ25-4 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.

[0052] 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 1480 bp (sequence shown in SEQ ID NO: 3).

[0053] The primer Fd1 sequence is shown in SEQ ID NO: 3 as 5'-AGAGTTTGATCCTGGCTCAG-3';

[0054] The primer Rd1 sequence is shown in SEQ ID NO: 3 as 5'-AAGGAGGTGATCCAGCC-3'.

[0055] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that ASZJHZ25-4 had a 16S gene similarity of >99.5% with type strains such as Sinorhizobium meliloti IAM 12611.

[0056] Housekeeping gene identification: To accurately identify its species classification, partial sequences of the atpD, recA, glnII, and nifH housekeeping genes were further amplified and determined.

[0057] atpD gene: Amplified using primers TSatpDf and TSatpDr, yielding a sequence of approximately 531 bp (atpD sequence as shown in SEQ ID NO: 6).

[0058] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0059] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0060] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 539 bp (as shown in SEQ ID NO: 9).

[0061] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0062] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0063] glnII gene: Amplified using primers TSglnIIf and TSglnIIr, a sequence of approximately 646 bp was obtained (sequence shown in SEQ ID NO: 12).

[0064] The primer TSglnIIf sequence is shown in SEQ ID NO: 10: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0065] The primer TSglnIIr sequence is shown in SEQ ID NO: 11: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0066] nifH gene: Amplified using primers TSnifHf and TSnifHr, yielding a sequence of approximately 478 bp (as shown in SEQ ID NO: 15).

[0067] The primer TSnifHf sequence is shown in SEQ ID NO: 13: 5′-AAAGGYGGWATCGGYAARTCCACCAC-3′;

[0068] The primer TSnifHr sequence is shown in SEQ ID NO: 14: 5′-TTGTTSGCSGCRTACATSGCCATCAT-3′.

[0069] Multigene phylogenetic analysis: The gene sequences of ASZJHZ25-4 were aligned with the corresponding sequences of the related *Rhizobium sinense* 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 2ASZJHZ25-4 clustered with the type strain Sinorhizobium meliloti IAM 12611, confirming its taxonomic position as Sinorhizobium meliloti.

[0070] Example 2: Functional verification of the effects of Osmanthus fragrans rhizobium ASZJHZ25-4 on peanut growth, nodulation, and nitrogen fixation.

[0071] 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: ASZJHZ25-4 was cultured in YMB until mid-log (OD25). 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.

[0072] Experimental treatments: ASZJHZ25-4 treatment group (T): Plump and uniform peanut seeds were selected and evenly coated with the ASZJHZ25-4 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 coated 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, day / night temperature set at 28℃ / 22℃, and relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0073] 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).

[0074] Plant height: The average plant height of the ASZJHZ25-4 treatment group (T) was 36.05 ± 1.42 cm, which was significantly increased by 36.4% compared with the blank control group (CK, 26.44 ± 1.63 cm) (p = 2.72 × 10⁻⁶). -8 ).

[0075] Fresh weight: The average fresh weight of group T was 78.07 ± 2.42 g, which was significantly higher than that of group CK (60.23 ± 3.67 g) by 29.6% (p = 1.09 × 10⁻⁶). -7 ).

[0076] Nodule count: The average number of root nodules in group T was 171.83 ± 9.79 / plant, which was significantly increased by 110.4% compared with group CK (81.67 ± 7.65 / plant) (p = 3.69 × 10⁻⁶). -11 ).

[0077] Nodule weight: The average fresh weight of root nodules in the T group was 0.229 ± 0.032 g / plant, which was significantly increased by 32.4% compared with the CK group (0.173 ± 0.037 g / plant) (p = 0.00201).

[0078] Conclusion: Inoculation with ASZJHZ25-4 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.

[0079] Table 1: The promoting effect of ASZJHZ25-4 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0080]

[0081] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0082] Inoculation with *Rhizobium sinense* ASZJHZ25-4 from *Osmanthus fragrans* 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 plants in the treatment group (T) inoculated with Rhizobium sinense ASZJHZ25-4, regardless of whether they were washed ( Figure 3 A) In the in-situ condition, both showed significant growth advantages: taller plants, more lush stems and leaves, and more vigorous growth. Figure 3 A). Crucially, direct observation of the root system is essential. Figure 3 (B, 3C) clearly show that the plants in group T have a more developed root system. Figure 3 B), and the number and size of effective root nodules growing on the roots far exceeded those of the blank control group (CK). Figure 3 C). Furthermore, in terms of pod phenotype, the T group plants exhibited a significantly increased number of pods, cleaner pod surfaces, and a significantly higher commercial value. Figure 3D). These striking visual evidences are highly consistent with the aforementioned measured precise quantitative data (Table 1) showing a highly significant increase in plant height (36.4%), a highly significant increase in plant fresh weight (29.6%), and highly significant increases in root nodule number and fresh weight (110.4% and 32.4%, respectively). Together, they constitute a complete chain of evidence that ASZJHZ25-4 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0083] Second, transcriptomics validation (molecular mechanism analysis).

[0084] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the ASZJHZ25-4 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.

[0085] 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).

[0086] Results: To elucidate the molecular basis of ASZJHZ25-4's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that ASZJHZ25-4 treatment specifically activated key gene networks in peanut roots related to symbiotic signal transduction and plant hormone signaling.

[0087] Evidence for key gene expression is as follows (see Figure 5 Table 2):

[0088] 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 1.836-fold and 3.937-fold, respectively. These genes are the core regulatory switches that initiate the root nodule formation process.

[0089] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 11.577-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 16.018-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0090] Conclusion: The above molecular evidence indicates that ASZJHZ25-4 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 ASZJHZ25-4 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.

[0091] Table 2: Effects of ASZJHZ25-4 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0092]

[0093] Example 3: Verification of the function of Rhizobium sinense ASZJHZ25-4 in inhibiting Aspergillus flavus growth and toxin production.

[0094] First, in vitro antagonism experiment (double-plate inverted method). To evaluate the antagonistic effect of ASZJHZ25-4 on Aspergillus flavus, an in vitro antagonism experiment was conducted using the double-plate inverted method.

[0095] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0096] 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.

[0097] 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 ASZJHZ25-4 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.

[0098] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0099] Measurement indicators:

[0100] 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%.

[0101] 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.

[0102] 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).

[0103] The results show (see) Figure 6 (Table 3) The ASZJHZ25-4 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by ASZJHZ25-4 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus, achieving efficient source control.

[0104] Inhibition of mycelial growth: The colony diameter in the treatment group (T) was 1.46 ± 0.24 cm, which was significantly lower than that in the control group (CK, 7.18 ± 0.13 cm), with an inhibition rate of 79.7% (p = 4.88 × 10⁻⁶). -11 ).

[0105] Inhibition of spore reproduction: Sporulation yield in the treatment group was 1.48 ± 0.26 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the control group (CK, 67.8 ± 6.83 × 10⁻⁶). 6 The inhibition rate reached 97.8% (p = 2.16 × 10⁻⁶). -8 ).

[0106] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the treatment group was 19.57 ± 4.85 ng / plate, which was significantly lower than that in the control group (CK, 546.36 ± 58.5 ng / plate), with an inhibition rate of 96.4% (p = 9.39 × 10⁻⁶). -15 ).

[0107] Table 3: Inhibitory effect of ASZJHZ25-4 treatment on key life activities of Aspergillus flavus.

[0108]

[0109] Note: Inhibition rate (%) = [(CK group mean - T group mean) / CK group mean] × 100%. All data are expressed as mean ± standard deviation.

[0110] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0111] Based on the inverted culture device, four treatment groups were set up. In all treatment groups, the upper plate was 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.

[0112] CK group (blank control): The lower plate was not inoculated with ASZJHZ25-4 and no activated carbon was used.

[0113] CK + Car group (activated carbon control): The lower plate was not inoculated with ASZJHZ25-4, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0114] Group T (with ASZJHZ25-4, without activated carbon): The lower plate is uniformly coated with ASZJHZ25-4 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0115] T + Car group (with ASZJHZ25-4 and activated carbon): Spread an equal amount of ASZJHZ25-4 bacterial suspension on half of the lower plate, and spread 5 g of sterilized activated carbon on the other half.

[0116] Results and analysis (see Figure 7 Table 4): The adsorption effect of activated carbon significantly weakened the antibacterial effect of ASZJHZ25-4 (Table X). This experiment proves that the inhibitory effect of ASZJHZ25-4 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0117] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.18 ± 0.13 cm) and the CK + Car group (7 ± 0.16 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0118] ASZJHZ25-4 releases potent antibacterial VOCs: the colony diameter in group T (1.46 ± 0.24 cm) was significantly smaller than that in group CK, with an antibacterial rate of 79.7%.

[0119] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (3.66 ± 0.17 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 49.0%, but was still significantly lower than that of the CK group.

[0120] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0121]

[0122] 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%.

[0123] Third, transcriptomics verification (molecular mechanism).

[0124] To elucidate the mechanism by which ASZJHZ25-4 inhibits toxin production and sporulation in Aspergillus flavus at the molecular level, we performed transcriptome sequencing analysis on the treated Aspergillus flavus hyphae. Differential expression analysis showed that ASZJHZ25-4 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0125] Evidence for key gene expression is as follows (see Figure 8 Table 5):

[0126] 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 ASZJHZ25-4 treatment group (T) decreased to 28.38% and 16.90% 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.

[0127] The expression of key genes regulating conidial development 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 1.05% and 8.88% 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.

[0128] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by ASZJHZ25-4 can precisely interfere with the core life activity program of Aspergillus flavus, namely, by strongly inhibiting the core regulatory hubs of toxin production (aflR / aflS) and sporulation (abaA / wetA), thereby achieving the dual effects of "antibacterial" and "toxicity reduction".

[0129] Table 5: Inhibitory effect of ASZJHZ25-4 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0130]

[0131] Example 4: Identification and verification of antibacterial active substances of Rhizobium sinense ASZJHZ25-4 from Osmanthus fragrans.

[0132] To identify the characteristic volatile compounds produced by ASZJHZ25-4, we performed untargeted metabolomics analysis of the volatile components of the strain culture using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS). By comparing the ASZJHZ25-4 treatment group (T) with the control group (CK), a compound with highly significant differences was identified. This compound had a retention time of 23.665 min and a retention index of 1670.2 in the chromatogram. Its mass spectrum showed a similarity of 882 to 2-methylbutyric acid (2-methyl-) in the NIST standard library, with a molecular ion peak at m / z 74, thus confirming its identification.

[0133] Quantitative and statistical analyses further confirmed the criticality of this compound (see Figure 9 Table 6):

[0134] 1) Extremely significant enrichment: The relative abundance of this compound in the ASZJHZ25-4 treatment group was 171,180.1 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 0.004649).

[0135] 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 1.490, indicating that it is one of the core volatile markers that distinguishes ASZJHZ25-4 from the blank control.

[0136] Conclusion: Through comprehensive chromatographic-mass spectrometry identification and omics statistical analysis, we conclusively demonstrate that 2-methylbutyric acid is a characteristic volatile compound specifically and abundantly produced by ASZJHZ25-4. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0137] Table 6: Identification and omics analysis results of 2-methylbutyric acid, a characteristic volatile substance in ASZJHZ25-4.

[0138]

[0139] 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.

[0140] Second, purity verification experiment.

[0141] To directly verify the antibacterial activity of the key volatile substance 2-methylbutyric acid, we used the fumigation method to determine the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus (Table 7). The results showed that 2-methylbutyric acid had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0142] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.6%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 80.9%.

[0143] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 53.3%) to a 40-fold dilution (inhibition rate 19.7%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0144] Determining the activity threshold: When the dilution factor reaches 40 times or more, there is no substantial difference in the colony diameter between each treatment group and the control group (inhibition rate < 5%), indicating that 2-methylbutyric acid has basically lost its observable antibacterial activity at this concentration.

[0145] Conclusion: This pure product verification experiment conclusively demonstrates that 2-methylbutyric acid (2-methylbutyric acid) 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 (80.9641-fold) of this compound in the volatile substances of ASZJHZ25-4, strongly confirming that 2-methylbutyric acid is the key effector molecule mediating the volatile antibacterial effect of ASZJHZ25-4.

[0146] Table 7: Effects of different dosages of 2-methylbutyric acid on the growth of Aspergillus flavus mycelia.

[0147]

[0148] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0149] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A *Rhizobium sinense* strain ASZJHZ25-4, which possesses both growth-promoting and aflatoxin-inhibiting functions, characterized in that... Its classification name is Sinorhizobium meliloti, accession number is CCTCC M 2026230, 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 contains at least one of the following: *Rhizobium sinense* ASZJHZ25-4 as described in claim 1, its fermentation product, or its culture; the fermentation product includes fermentation broth, sterile supernatant, or active substances extracted from the fermentation broth or sterile supernatant; the culture includes solid culture, liquid culture, or a dried preparation thereof.

3. The application of the *Rhizobium sinense* ASZJHZ25-4 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 *Rhizobium sinense* ASZJHZ25-4 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 *Rhizobium sinense* ASZJHZ25-4.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the *Rhizobium sinense* ASZJHZ25-4 as described in claim 1 or the microbial agent or preparation as described in claim 2 to the peanut growing environment or peanut plants.

Images