Bradyrhizobium angergii ASHLJNJ-23-2 with functions of promoting growth and inhibiting aspergillus flavus and application of bradyrhizobium angergii ASHLJNJ-23-2

Abstract

The invention discloses bradyrhizobium elkanii ASHLJNJ-23-2 which has the functions of efficiently promoting growth and inhibiting aspergillus flavus and application of the bradyrhizobium elkanii ASHLJNJ-23-2. The preservation number of the bradyrhizobium elkanii ASHLJNJ-23-2 is CCTCC M 2026227. According to the invention, the integration of the two functions of generating a characteristic antibacterial volatile matter of 2-tridecanone and efficiently symbiotic nitrogen fixation on a single strain is realized in the bradyrhizobium angularis for the first time. The 2-tridecanone 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, growth promotion, nitrogen fixation and source bacteriostasis can be synchronously realized 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 can be 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 Bradyrhizobium elkanii (ASHLJNJ-23-2) that has both 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 and increasing farmers' income. However, its production continues to face two core challenges: increasing yield and controlling aflatoxin contamination. Aflatoxins (AFs) are potent carcinogens produced by Aspergillus flavus, which 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 *Staphylococcus aureus*.

[0005] For legumes like peanuts, the most fundamental and efficient way to increase yield lies in establishing a specific symbiotic relationship with rhizobia, thereby achieving biological nitrogen fixation through the formation of root nodules. Bradyrhizobium elkanii is a known rhizobia capable of highly efficient symbiosis with legumes such as peanuts, and it has significant application value in promoting plant nodulation and nitrogen supply. However, traditional rhizobium agents focus on nitrogen fixation and growth promotion, generally lacking direct antagonistic ability against soil-borne pathogens such as Aspergillus flavus, thus failing to meet the dual needs of "increased yield" and "safety" in peanut production.

[0006] III. Limitations of Existing Aflatoxin Control Technologies

[0007] Currently, the control of aflatoxin mainly relies on three types of technologies: agricultural management measures (with passive and unstable effects), physicochemical methods (high cost and risk of residue), and biological control, primarily using Bacillus spp. and Trichoderma spp. Existing biological control strains mostly focus on inhibiting bacteria and lack direct contribution to promoting the core symbiotic nitrogen fixation process in legumes. Particularly noteworthy is the unique growth habit of peanuts—flowering above ground and fruiting underground—which makes traditional aboveground chemical or biological control methods ineffective against the soil-borne fruit during the critical stages of pod development and infection, creating a significant obstacle to physical control.

[0008] IV. Systemic Defects of Existing Technological Approaches "Increasing production" and "preventing poisoning" are disconnected in the existing technological system, leading to the following systemic defects:

[0009] (1) Limited Function and Lack of Biological Synergy: Existing growth-promoting microbial agents (including traditional rhizobium inoculants) are mainly designed to promote growth through mechanisms such as nitrogen fixation, phosphorus solubilization, or secretion of plant hormones, and generally lack direct and efficient inhibitory capabilities against specific pathogenic fungi such as Aspergillus flavus. Meanwhile, the selection criteria for existing biocontrol agents mainly focus on antagonistic activity against pathogens, failing to directly contribute to the crucial symbiotic nitrogen fixation process in legumes. Furthermore, their metabolites may even interfere with the colonization and function of rhizobia. When these two different types of microorganisms are simultaneously introduced into the complex rhizosphere niche, they not only fail to achieve synergistic effects but may also lead to unstable, unpredictable, or even mutually destructive field effects due to competition for nutrients and space or the production of antagonistic metabolites.

[0010] (2) Cumbersome operation and significantly increased overall costs: In actual production applications, farmers need to purchase, store, prepare and apply two different types of microbial products separately. This application mode not only directly increases the material costs of seed treatment or field operations, but also significantly increases the costs of labor input, equipment use and time management. The complicated operation process reduces the operability and user acceptance of the technology, especially in large-scale and intensive planting models, becoming an important obstacle to the promotion of the technology.

[0011] (3) Insufficient adaptation to the special biological characteristics of peanuts and weak source control: Aspergillus flavus infection of peanuts begins in the field soil and infects the pods buried deep in the soil through the pegs or cracks during the critical period of pod development and enlargement. The unique habit of peanuts to "flower above ground and bear fruit underground" makes traditional above-ground chemical or biological control methods almost completely ineffective during the critical control window of the fruiting period. Seed treatment agents applied at the sowing time have an effective period that is difficult to cover the pod development period of several months; while applying large amounts of chemical fungicides to the soil through the irrigation system will indiscriminately disrupt the balance of the soil microbial community, easily induce pathogens to develop drug resistance, and bring environmental pollution and pesticide residue risks. The existing separation technology system cannot provide an integrated solution that can simultaneously perform the dual functions of "nutrient supply (nitrogen fixation)" and "biological protection (bacteriostasis)" in situ and continuously from the roots in the soil microenvironment during pod development, resulting in serious inadequacy in the control of toxin pollution sources.

[0012] V. The Innovative Positioning and Technological Gaps of this Invention

[0013] 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 *Syntrophus esculenta* possesses a multifunctional strain capable of effectively inhibiting *Aspergillus flavus* by producing characteristic antibacterial volatiles such as 2-tetranone, while maintaining highly efficient symbiotic nitrogen fixation capabilities.

[0014] This invention aims to fill this gap by screening and developing a novel *Ashlilleria echocarpa* strain, ASHLJNJ-23-2, 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-function 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

[0015] To address the shortcomings of existing technologies, this invention provides a slow-growing rhizobium strain ASHLJNJ-23-2 that combines growth promotion and aspergillosis inhibition functions, along with its applications.

[0016] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0017] The first objective of this invention is to provide a slow-growing rhizobium strain ASHLJNJ-23-2 that has both growth-promoting and aflatoxin-inhibiting functions. Its classification name is Bradyrhizobium elkanii, its accession number is CCTCC M 2026227, its accession date is January 23, 2026, and it is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University.

[0018] A second objective of this invention is to provide a microbial agent or preparation comprising at least one of *Syntrophus esculenta* ASHLJNJ-23-2, its fermentation product, or a culture thereof; the fermentation product comprises fermentation broth, sterile supernatant, or an active substance extracted from the fermentation broth or sterile supernatant; the culture comprises a solid culture, a liquid culture, or a dried preparation thereof.

[0019] The third objective of this invention is to provide the application of *Syntrophus esculenta* ASHLJNJ-23-2, or a microbial agent or preparation containing *Syntrophus esculenta* ASHLJNJ-23-2, in promoting peanut growth and nodulation nitrogen fixation.

[0020] The fourth objective of this invention is to provide the application of *Aspergillus oryzae* ASHLJNJ-23-2, or a microbial agent or preparation containing *Aspergillus oryzae* ASHLJNJ-23-2, in inhibiting the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus*.

[0021] Preferably, the application specifically achieves the inhibitory function through the volatile organic compounds released by the *Syntrophus esculenta* ASHLJNJ-23-2.

[0022] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Aspergillus esculenta ASHLJNJ-23-2, or a microbial agent or preparation containing Aspergillus esculenta ASHLJNJ-23-2, to the peanut growing environment or peanut plants.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Significant effect on promoting growth and nitrogen fixation: ASHLJNJ-23-2, a slow-growing rhizobium of Ehrlich, can comprehensively promote the growth and nitrogen fixation of peanut plants. After inoculation, the peanut plant height and biomass increased by 35.7% and 30.4%, respectively, and the number of root nodules and the fresh weight of root nodules increased significantly by 111.0% and 19.1%, 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.

[0025] (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 79.0%, 97.6%, and 96.4%, respectively. Activated carbon adsorption experiments confirmed that this antibacterial effect is mainly mediated by volatile substances.

[0026] (3) Key antibacterial components are clearly identified and their mechanisms are well understood: Through metabolomics and pure product verification experiments, 2-tetanetanone was confirmed as the key volatile antibacterial active substance. This compound was specifically enriched in the strain's metabolites (relative abundance was 8535.85 times that of the control group), and the pure product had 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 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.

[0027] (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 "2-tetranone-mediated potent biocontrol" in the same strain of *Syntrophus esculenta*. 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-based green biotechnology solution to the industrial problem of increasing peanut yield and controlling toxins.

[0028] This invention provides a Bradyrhizobium elkanii strain ASHLJNJ-23-2, which has both growth-promoting and Aspergillus-inhibiting functions. This strain was obtained by the inventors through screening. The accession number for this strain is CCTCC M 2026227, the 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. Attached Figure Description

[0029] 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).

[0030] Figure 2 This invention constructs a phylogenetic tree of ASHLJNJ-23-2 based on multiple genes (16S, atpD, recA, dnaK, glnII, 23S).

[0031] Figure 3 Effects of inoculation with ASHLJNJ-23-2 on growth and nodulation of potted peanut plants. Figure 3 A represents a comparison of the growth status of potted plants in the blank control group (CK) and the ASHLJNJ-23-2 inoculation treatment group (T); Figure 3 B represents a comparison of the number of root nodules between the blank control group (CK) and the ASHLJNJ-23-2 inoculation treatment group (T); Figure 3 C is a close-up of root nodules in group T; Figure 3 D is a close-up of root nodules in the CK group.

[0032] Figure 4This invention relates to the effects of inoculation with the slow-growing rhizobium ASHLJNJ-23-2 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 ASHLJNJ-23-2 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n ≥ 3). and "p" indicates that the differences between groups were significant (p < 0.05) and extremely significant (p < 0.001), respectively.

[0033] 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 slow-growing rhizobium ASHLJNJ-23-2 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 The values ​​represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[0034] Figure 6 This invention evaluates the inhibitory effect of *Aspergillus oryzae* ASHLJNJ-23-2 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) / ASHLJNJ-23-2 treatment group (T); Figure 6 B represents the sporulation rate of the blank control group (CK) / the ASHLJNJ-23-2 treatment group (T); Figure 6 C represents the aflatoxin production of the blank control group (CK) / the ASHLJNJ-23-2 treatment group (T); Figure 6 D represents the overall colony morphology of the blank control group (CK); Figure 6E represents the overall colony morphology of the ASHLJNJ-23-2 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).

[0035] 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), ASHLJNJ-23-2 treatment group (T), and ASHLJNJ-23-2 treatment group + activated carbon (T + Car); The figure shows... The differences between groups were statistically significant (p < 0.001).

[0036] Figure 8 This invention relates to the effect of treatment with the chronic rhizobium ASHLJNJ-23-2 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.

[0037] 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-tetaneone in the blank control group (CK) and the ASHLJNJ-23-2 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 < 0.05). Detailed Implementation

[0038] 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.

[0039] Example 1: Isolation, identification and preservation of the multifunctional slow-growing rhizobium ASHLJNJ-23-2.

[0040] The *A. amyloliquefaciens* var. *e.* of this invention was isolated from peanut rhizosphere soil treated with an 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.

[0041] 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* spp., which is closely related to peanut symbiotic nitrogen fixation. For example... Figure 1 As shown, compared with the untreated control group (CK), the relative abundance of *Syntrophus* spp. in the rhizosphere soil treated with ARC microbial inoculant was significantly increased. Specifically, the average relative abundance of *Syntrophus* spp. in the CK group was 0.36%, while the average relative abundance in the ARC-treated group (T group) increased to 0.47%, a significant increase of 30.6% (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 *Syntrophus esculentus* ASHLJNJ-23-2 from the rhizosphere soil of the ARC-treated group, indicating that ARC inoculant can reshape the rhizosphere microbiota and specifically promote the colonization and enrichment of beneficial symbiotic bacteria—*Syntrophus esculentus*.

[0042] 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 ).

[0043] 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.

[0044] 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 using three consecutive streak plating tests to obtain a pure culture, designated ASHLJNJ-23-2.

[0045] Liquid culture: A single colony of *Syntrophus esculenta* ASHLJNJ-23-2 was inoculated into 5 mL of YMB liquid medium and cultured at 28°C with shaking at 180 rpm for 72 hours until the late logarithmic growth phase (OD2). 600 (Values ​​are between 0.8 and 1.0). Short-term storage: Streak the above bacterial suspension on fresh YMA plates and store at 4°C, subculturing monthly. Long-term storage: Take the 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.

[0046] Genomic DNA Extraction: Genomic DNA was extracted from the pure culture of ASHLJNJ-23-2 using the TIANamp Bacteria DNA Kit according to the instructions. Its concentration and purity were measured using a spectrophotometer (A260 / A280 ratio between 1.8 and 2.0).

[0047] 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 1401 bp (as shown in SEQ ID NO: 3).

[0048] The primer Fd1 sequence is shown in SEQ ID NO: 1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0049] The primer Rd1 sequence is shown in SEQ ID NO: 2: 5'-AAGGAGGTGATCCAGCC-3').

[0050] The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that ASHLJNJ-23-2 had a 16S gene similarity of >99.5% with type strains such as Bradyrhizobium elkanii USDA 61.

[0051] 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.

[0052] atpD gene: Amplified using primers TSatpDf and TSatpDr, yielding a sequence of approximately 476 bp (the atpD sequence of APHBXY-23-1 is shown in SEQ ID NO: 6).

[0053] The primer TSatpDf sequence is shown in SEQ ID NO: 4: 5'-TCTGGTCCGYGGCCAGGAAG-3';

[0054] The primer TSatpDr sequence is shown in SEQ ID NO: 5: 5'-CGACACTTCCGARCCSGCCTG-3'.

[0055] recA gene: Amplified using primers TSrecAf and TSrecAr, yielding a sequence of approximately 488 bp (as shown in SEQ ID NO: 9).

[0056] The primer TSrecAf sequence is shown in SEQ ID NO: 7: 5'- CAACTGCMYTGCGTATCGTCGAAGG-3';

[0057] The primer TSrecAr sequence is shown in SEQ ID NO: 8: 5'-CGGATCTGGTTGATGAAGATCACCATG-3'.

[0058] dnaK gene: Amplified using primers BRdnaKf and BRdnaKr, yielding a sequence of approximately 603 bp (as shown in SEQ ID NO: 12).

[0059] The primer BRdnaKf sequence is shown in SEQ ID NO: 10: 5'- TTCGACATCGACGCSAACGG-3';

[0060] The primer BRdnaKr sequence is shown in SEQ ID NO: 11: 5'-GCCTGCTGCKTGTACATGGC-3'.

[0061] glnll gene: Amplified using primers TSglnIIf and TSglnIIr, a sequence of approximately 644 bp was obtained (sequence shown in SEQ ID NO: 15).

[0062] The primer TSglnIIf sequence is shown in SEQ ID NO: 13: 5'-AAGCTCGAGTACATCTGGCTCGACGG-3';

[0063] The primer TSglnIIr sequence is shown in SEQ ID NO: 14: 5'-SGAGCCGTTCCAGTCGGTGTCG-3'.

[0064] 23S gene: Amplified using primers FGPS1490 and FGPS130, yielding a sequence of approximately 639 bp (as shown in SEQ ID NO: 18).

[0065] The primer FGPS1490 sequence is shown in SEQ ID NO: 16: 5'-TGCGGCTGGATCACCTCCTT-3';

[0066] The primer FGPS130 sequence is shown in SEQ ID NO: 17: 5'-CCGGGTTTCCCCATTCGG-3'.

[0067] Multigene phylogenetic analysis: The gene sequences of ASHLJNJ-23-2 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 ASHLJNJ-23-2 clustered with the type strain Bradyrhizobium elkanii USDA 61, confirming its taxonomic status as Bradyrhizobium elkanii.

[0068] Example 2: Verification of the function of ASHLJNJ-23-2 in promoting peanut growth and nitrogen fixation.

[0069] 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: ASHLJNJ-23-2 was cultured in YMB until mid-log (OD2). 600 (≈ 0.6), collect bacterial cells by centrifugation at 5000 rpm for 5 minutes, resuspend in sterile 0.85% NaCl solution and adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL (calibrated by plate count) was used as the inoculum suspension.

[0070] Experimental treatments: ASHLJNJ-23-2 treatment group (T): Plump and uniform peanut seeds were selected and evenly mixed with the ASHLJNJ-23-2 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, day / night temperature set at 28℃ / 22℃, and relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0071] 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).

[0072] Plant height: The average plant height of the ASHLJNJ-23-2 treatment group (T) was 36.22 ± 1.64 cm, which was significantly increased by 35.7% compared with the blank control group (CK, 26.69 ± 1.92 cm) (p = 1.86 × 10⁻⁶). -7 ).

[0073] Fresh weight: The average fresh weight of group T was 78.25 ± 3.73 g, which was significantly higher than that of group CK (60.03 ± 3.48 g) by 30.4% (p = 2.70 × 10⁻⁶). -7 ).

[0074] Nodule count: The average number of root nodules in group T was 169.67 ± 14.42 per plant, which was significantly increased by 111.0% compared with group CK (80.44 ± 8.46 per plant) (p = 1.17 × 10⁻⁶). -9 ).

[0075] Nodule weight: The average fresh weight of root nodules in the T group was 0.187 ± 0.029 g / plant, which was significantly increased by 19.1% compared with the CK group (0.157 ± 0.031 g / plant) (p = 0.035).

[0076] Conclusion: Inoculation with ASHLJNJ-23-2 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.

[0077] Table 1: The promoting effect of ASHLJNJ-23-2 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0078]

[0079] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0080] Inoculation with the slow-growing rhizobium ASHLJNJ-23-2 significantly promoted the growth and symbiotic nodulation of peanut seedlings, as evidenced by the obvious phenotypic differences. Figure 3 As shown. The results indicate that, as Figure 3 As shown in A, the potted plants in group T are growing much better than those in group CK. Figure 3 B further showed that the number of root nodules in group T was significantly higher than that in group CK. Combined with… Figure 3 C and Figure 3 Close-up images of root nodules in group D show that the treatment group inoculated with ASHLJNJ-23-2 exhibited superior performance in terms of plant height, leaf color, nodule number, and root development, demonstrating the significant promoting effect of this strain on peanut growth and nodule formation, and possessing good potential for promoting plant growth and enhancing nodule formation. This strong visual evidence is highly consistent with the aforementioned precise quantitative data (Table 1) showing a highly significant increase in plant height (35.7%), a highly significant increase in plant fresh weight (30.4%), and highly significant increases in nodule number and fresh weight (111% and 19.1%, respectively), together forming a complete chain of evidence that ASHLJNJ-23-2 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen-fixing ability.

[0081] Second, transcriptomics validation (molecular mechanism analysis).

[0082] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the ASHLJNJ-23-2 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.

[0083] 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).

[0084] Results: To elucidate the molecular basis of ASHLJNJ-23-2's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that ASHLJNJ-23-2 treatment specifically activated a network of key genes in peanut roots related to symbiotic signal transduction and plant hormone signaling. Evidence for key gene expression is as follows (see...). Figure 5 Table 2):

[0085] Symbiotic nodulation pathway: The expression levels of NSP1 and NSP2, the core transcription factors of the nodulation signaling pathway, were significantly upregulated in group T, by 2.324-fold and 2.252-fold, respectively. These genes are the core regulatory switches that initiate the root nodulation process.

[0086] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 10.826-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 19.116-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0087] Conclusion: The above molecular evidence indicates that inoculation with ASHLJNJ-23-2 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 ASHLJNJ-23-2 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.

[0088] Table 2: Effects of ASHLJNJ-23-2 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0089]

[0090] Example 3: Verification of the function of ASHLJNJ-23-2 in inhibiting the growth and toxin production of Aspergillus flavus.

[0091] First, in vitro antagonism experiment (double-plate method).

[0092] To evaluate the antagonistic effect of ASHLJNJ-23-2 against Aspergillus flavus, an in vitro antagonistic experiment was conducted using the double-plate method.

[0093] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0094] 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.

[0095] 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 ASHLJNJ-23-2 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.

[0096] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0097] Measurement indicators:

[0098] 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%.

[0099] 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.

[0100] 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).

[0101] The results show (see) Figure 6 (Table 3) The ASHLJNJ-23-2 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by ASHLJNJ-23-2 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus, achieving efficient source control.

[0102] Inhibition of mycelial growth: The colony diameter in the ASHLJNJ-23-2 treatment group (T) was 1.52 ± 0.084 cm, significantly lower than that in the blank control group (CK, 7.22 ± 0.084 cm), with an inhibition rate of 78.95% (p = 6.16 × 10⁻⁶). -14 ).

[0103] Inhibition of spore reproduction: The sporulation rate in the ASHLJNJ-23-2 treatment group was 1.66 ± 0.17 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the blank control group (CK, 69.6 ± 10.01 × 10⁻⁶). 6 The inhibition rate reached 97.6% (p = 3.54 × 10⁻⁶ cells / plate). -7 ).

[0104] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the ASHLJNJ-23-2 treatment group was 21.05 ± 7.7 ng / plate, which was significantly lower than that in the blank control group (CK, 583.56 ± 144.67 ng / plate), with an inhibition rate of 96.4% (p = 3.16 × 10⁻⁶). -9 ).

[0105] Table 3: Inhibitory effect of ASHLJNJ-23-2 treatment on key life activities of Aspergillus flavus.

[0106]

[0107] Inhibition rate (%) = [(mean of CK group - mean of T group) / mean of CK group] × 100%. All data are expressed as mean ± standard deviation.

[0108] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0109] 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.

[0110] CK group (blank control): The lower plate was not inoculated with ASHLJNJ-23-2 and no activated carbon was used.

[0111] CK + Car group (activated carbon control): The lower plate was not inoculated with ASHLJNJ-23-2, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0112] Group T (with ASHLJNJ-23-2, without activated carbon): The lower plate was evenly coated with ASHLJNJ-23-2 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0113] T + Car group (with ASHLJNJ-23-2 and activated carbon): Half of the lower plate was coated with an equal amount of ASHLJNJ-23-2 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0114] Results and analysis (see Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of ASHLJNJ-23-2. This experiment proves that the inhibitory effect of ASHLJNJ-23-2 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0115] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.22 ± 0.084 cm) and the CK + Car group (7.06 ± 0.11 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0116] ASHLJNJ-23-2 releases potent antibacterial VOCs: The colony diameter in group T (1.52 ± 0.084 cm) was significantly smaller than that in group CK, with an antibacterial rate of 79.1%.

[0117] 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.

[0118] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0119]

[0120] The key role of volatile organic compounds (VOCs) in inhibiting the mycelial growth of Aspergillus flavus was evaluated by 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%.

[0121] Third, transcriptomics verification (molecular mechanism).

[0122] To elucidate the mechanism by which ASHLJNJ-23-2 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 ASHLJNJ-23-2 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0123] Evidence for key gene expression is as follows (see Figure 8 Table 5):

[0124] 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 ASHLJNJ-23-2 treatment group (T) decreased to 24.00% and 16.34% 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.

[0125] 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 1.34% and 8.50% 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.

[0126] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by ASHLJNJ-23-2 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.

[0127] Table 5: Inhibitory effect of ASHLJNJ-23-2 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0128]

[0129] Example 4: Identification and verification of antibacterial active substances of ASHLJNJ-23-2, a slow-growing rhizobium of Ehrlich.

[0130] To identify the characteristic volatile compounds produced by ASHLJNJ-23-2, 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 experimental group (ASHLJNJ-23-2) with the control group (CK), a compound with highly significant differences was identified. This compound had a retention time of 23.127 min and a retention index of 1646.925 in the chromatogram. Its mass spectrum showed a similarity of 779 to 2-tridecanone in the NIST standard library, with a molecular ion peak at m / z 58, thus confirming its identification.

[0131] Quantitative and statistical analyses further confirmed the criticality of this compound (see Figure 9 Table 6):

[0132] 1) Extremely significant enrichment: The relative abundance of this compound in the ASHLJNJ-23-2 treatment group was 8535.854 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 0.024).

[0133] 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 1.489, indicating that it is one of the core volatile markers that distinguishes ASHLJNJ-23-2 from the blank control.

[0134] Conclusion: Through comprehensive chromatographic-mass spectrometry identification and omics statistical analysis, we conclusively demonstrate that 2-tetanetanone is a characteristic volatile compound specifically and abundantly produced by ASHLJNJ-23-2. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0135] Table 6: Identification and omics analysis results of 2-tetaneone, a characteristic volatile substance of ASHLJNJ-23-2.

[0136]

[0137] 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.

[0138] Second, purity verification experiment.

[0139] To directly verify the antibacterial activity of the key volatile substance 2-tridecanetone, we used the fumigation method to determine the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus. The results showed (see Table 7) that 2-tridecanetone had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0140] 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 79.9%.

[0141] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 52.9%) to a 40-fold dilution (inhibition rate 21.3%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0142] 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 < 10%), indicating that 2-tetaneone has basically lost its observable antibacterial activity at this concentration.

[0143] Conclusion: This pure product verification experiment conclusively demonstrates that 2-tridecaneone itself is a potent inhibitor of Aspergillus flavus mycelial growth. It exerts a significant antibacterial effect even at low concentrations, a characteristic consistent with the highly specific and significant enrichment (2502.44-fold) of this compound in the volatile substances of ASHLJNJ-23-2, strongly confirming that 2-tridecaneone is the key effector molecule mediating the volatile antibacterial effect of ASHLJNJ-23-2.

[0144] Table 7: Effects of different dosages of 2-tetaneone on the growth of Aspergillus flavus mycelia.

[0145]

[0146] Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

[0147] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A slow-growing rhizobium strain ASHLJNJ-23-2 that combines growth-promoting and aflatoxin-inhibiting functions, characterized in that, Its classification name is Bradyrhizobium elkanii, accession number is CCTCC M 2026227, 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, The product comprises at least one of the following: ASHLJNJ-23-2, a fermentation product thereof, or a culture thereof, as described in claim 1; the fermentation product comprises fermentation broth, sterile supernatant, or an active substance extracted from the fermentation broth or sterile supernatant; the culture comprises a solid culture, a liquid culture, or a dried preparation thereof.

3. The application of the *Syntrophus esculenta* ASHLJNJ-23-2 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 use of the *Aspergillus oryzae* ASHLJNJ-23-2 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 *Syntrophus esculenta* ASHLJNJ-23-2.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the *Syntrophus esculenta* ASHLJNJ-23-2 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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