Sinkiang sinorhizobium sp. APXJHT24-1 with functions of promoting growth and inhibiting aspergillus flavus and application of Sinkiang sinorhizobium sp. APXJHT24-1

Abstract

The invention discloses sinorhizium xinjiangense APXJHT24-1 which has the functions of promoting growth and inhibiting aspergillus flavus, and the preservation number of the sinorhizium xinjiangense APXJHT24-1 is CCTCC (China Center for Type Culture Collection) M 2026229. According to the invention, the integration of two major functions of'generation of a characteristic antibacterial volatile matter of dimethyl disulfide 'and'efficient symbiotic nitrogen fixation' on a single strain is realized in Sinkiang Sinkiang rhizobium for the first time. The dimethyl disulfide released by the strain can accurately down-regulate expression of toxin synthesis key genes and spore development core genes in the aspergillus flavus, so that efficient bacteriostasis is realized on the molecular level. Through one-time inoculation, the strain can synchronously achieve the dual purposes of growth promotion and nitrogen fixation and source bacteriostasis 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 *Sinorhizobium xinjiangense* APXJHT24-1 that combines efficient growth promotion and nitrogen fixation with 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] Peanuts, as an important oilseed and cash crop, play a vital role in ensuring food and oil supply and the agricultural economy through stable production. However, peanut production continues to face two key challenges: increasing yield and controlling aflatoxin contamination. Aflatoxins (AFs) are potent carcinogens produced by Aspergillus flavus, posing a direct threat to food safety and becoming a major bottleneck restricting the sustainable development of the industry.

[0004] II. Core Mechanisms of Leguminous Crop Yield Increase and the Role of Rhizobium sinense

[0005] As a legume, peanuts rely most efficiently on specific symbiosis with rhizobia for yield enhancement, using root nodules for biological nitrogen fixation. *Sinorhizobium xinjiangense* is a known group of rhizobia capable of highly efficient symbiosis with legumes, showing significant potential in promoting nodulation and nitrogen fixation. However, traditional rhizobium agents have limited functionality, primarily focusing on nitrogen fixation and growth promotion, and generally lack direct antagonistic effects 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, physicochemical methods, and biological control primarily using Bacillus spp. and Trichoderma spp. These methods are either inconsistent in effectiveness, costly, or pose a risk of chemical residues. Crucially, peanuts' growth habit of "flowering above ground and fruiting underground" makes traditional above-ground control methods ineffective against the pods in the soil during the critical period of pod development, creating a significant obstacle to physical control.

[0008] IV. Systemic Defects of Existing Technological Approaches

[0009] Currently, "increasing yield" and "preventing poisoning" in peanut production are separated into two independent systems in terms of technological approaches. This separation leads to the following systemic defects:

[0010] (1) Limited Function Leads to 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 nutrient competition, spatial competition, or the production of antagonistic metabolites.

[0011] (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 "two-step" application model 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 complex operation process reduces the operability and user acceptance of the technology, especially under large-scale planting conditions, becoming an important obstacle to the promotion of the technology.

[0012] (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 chemical or biological control methods based on the above-ground parts 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 disrupt the balance of the soil microbial community, easily induce pathogens to develop drug resistance, and bring environmental pollution and residue risks. The existing separation technology system cannot provide an integrated solution that can simultaneously perform the dual functions of "nutrient supply" and "biological protection" in situ and continuously from the roots in the soil microenvironment during pod development.

[0013] V. The Innovative Positioning and Technological Gaps of this Invention

[0014] Based on the aforementioned bottlenecks, a clear technological gap exists in this field: to date, no publicly available reports have demonstrated that *Rhizobium sinense* strains from Xinjiang possess the ability to effectively inhibit *Aspergillus flavus* by producing characteristic antibacterial volatiles such as dimethyl disulfide, while simultaneously maintaining highly efficient symbiotic nitrogen fixation capabilities. This invention aims to overcome this gap by screening and developing *Rhizobium sinense* strain APXJHT24-1, which possesses both of these dual functions, to achieve synergistic regulation of peanut yield increase and toxin control on a single microbial carrier. With a single application, this strain can simultaneously establish a dual-functional system of "nitrogen fixation and supply" and "bio-antibacterial activity" 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 Xinjiang rhizobium APXJHT24-1 strain 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 primary objective of this invention is to provide a *Sinorhizobium xinjiangense* strain APXJHT24-1, which possesses both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Sinorhizobium xinjiangense*, accession number is CCTCC M 2026229, 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.

[0018] The second objective of this invention is to provide a microbial agent or preparation comprising at least one of *Rhizobium sinense* APXJHT24-1, 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.

[0019] The third objective of this invention is to provide the application of *Rhizobium sinense* APXJHT24-1, or a microbial agent or preparation containing *Rhizobium sinense* APXJHT24-1, in promoting peanut growth and nodulation nitrogen fixation.

[0020] The fourth objective of this invention is to provide the application of *Rhizobium sinense* APXJHT24-1, or a microbial agent or preparation containing *Rhizobium sinense* APXJHT24-1, 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 Xinjiang rhizobium APXJHT24-1.

[0022] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Rhizobium sinense APXJHT24-1, or a microbial agent or preparation containing Rhizobium sinense APXJHT24-1, 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: The Xinjiang rhizobium APXJHT24-1 can comprehensively promote the growth and nitrogen fixation of peanut plants. After inoculation, the peanut plant height and biomass increased by 40.9% and 28.8%, respectively, and the number of root nodules and the fresh weight of root nodules increased significantly by 102.4% and 37.7%, 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 79.2%, 97.7%, and 96.7%, 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, dimethyl disulfide was identified as the key volatile antibacterial active substance. This compound was specifically enriched in the strain's metabolites (relative abundance was 80.96 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.

[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 "dimethyl disulfide-mediated potent biocontrol" in the same strain of *Rhizobium sinense* from Xinjiang. 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.

[0028] This invention provides a *Sinorhizobium xinjiangense* strain APXJHT24-1 that possesses both growth-promoting and aflatoxin-inhibiting functions. This strain was obtained by the inventors through screening. The accession number for *Sinorhizobium xinjiangense* is CCTCC M 2026229, the accession date is January 23, 2026, and it is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0029] Figure 1 This invention relates to the effect of ARC microbial inoculant treatment on the relative abundance of *Sinorhizobium 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 < 0.01).

[0030] Figure 2 This invention constructs the APXJHT24-1 phylogenetic tree based on multiple genes (16S, atpD, glnII, nifH, recA).

[0031] Figure 3 The promoting effect of *Rhizobium sinense* APXJHT24-1 on peanut growth and nodulation. (A) Comparison of growth phenotypes of potted peanuts; (B) Comparison of overall morphology of peanut plants after washing; (C) Close-up comparison of root system and root nodules. In the figure, CK represents the blank control group, and T represents the APXJHT24-1 treatment group.

[0032] Figure 4 This invention relates to the effects of inoculation with *Rhizobium sinense* APXJHT24-1 from Xinjiang 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 4C represents the effect on the number of root nodules; Figure 4 D represents the effect on the fresh weight of root nodules; in the figure, CK represents the blank control group, and T represents the APXJHT24-1 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n ≥ 3). This indicates that the difference between groups is highly significant (p < 0.001).

[0033] Figure 5 This is a schematic diagram illustrating the upregulation of key genes related to peanut root symbiosis and growth induced by the treatment of *Rhizobium sinense* APXJHT24-1 in Xinjiang; 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 APXJHT24-1 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).

[0034] Figure 6 This invention evaluates the inhibitory effect of *Rhizobium sinense* APXJHT24-1 on the growth, sporulation, and aflatoxin synthesis of *Aspergillus flavus* using a double-plate method; wherein... Figure 6 A represents the diameter of Aspergillus flavus colonies; Figure 6 B represents conidia production; Figure 6 C represents the yield of aflatoxin B1 (AFB1); 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), APXJHT24-1 treatment group (T), and APXJHT24-1 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 *Rhizobium sinense* APXJHT24-1 from Xinjiang on the expression of core regulatory genes for aflatoxin synthesis and spore development; wherein, Figure 8 A represents the expression level of aflatoxin synthesis core transcription factor gene aflR; Figure 8 B represents the expression level of the aflatoxin synthesis coactivator gene aflS; Figure 8 C represents the expression level of abaA, a key regulatory gene for conidial development; Figure 8 D represents the expression level of wetA, a key regulatory gene for conidial maturation; in the figure, CK represents the blank control group, T represents the APXJHT24-1 treatment group; boxes represent quartile ranges, the midline represents the median, the whisker lines represent ranges, and dots represent individual cells; , , The numbers represent significant (p < 0.05), highly significant (p < 0.01), and extremely significant (p < 0.001) differences between groups, respectively.

[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 dimethyl disulfide, a volatile component, in the blank control group (CK) and the APXJHT24-1 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 < 0.001). 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 multifunctional Xinjiang rhizobium APXJHT24-1.

[0040] The *Xinjiang rhizobium* APXJHT24-1 of this invention was isolated from peanut rhizosphere soil treated with ARC microbial agent (a compound agent mainly composed of *Bacillus amyloliquefaciens*, *Brevibacillus laterosporus*, *Bacillus mucilaginosus*, and *Enterobacter ludwigii*). The effective viable count of *Bacillus amyloliquefaciens* in the microbial agent is ≥ 2 × 10⁻⁶. 9 CFU / g, effective viable count of Bacillus laterosporus brevis ≥ 2×10⁻⁶ 9 CFU / g, Bacillus subtilis ≥ 1×10 10 CFU / g, effective viable count of Ludwig's Enterobacter ≥ 1×10⁻⁶10 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 *Sinorhizobium* 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 *Rhizobium sinense* in the rhizosphere soil treated with ARC microbial inoculant was significantly increased. Specifically, the average relative abundance of *Rhizobium sinense* in the CK group was 0.044%, while the average relative abundance in the ARC-treated group (T group) increased to 0.0653%, a significant increase of 48.5% (p < 0.01). Statistical analysis showed that this enrichment effect was extremely significant (p < 0.01). This result provides direct ecological evidence for the subsequent targeted isolation and screening of functionally enhanced *Rhizobium sinense* APXJHT24-1 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 the beneficial symbiotic bacterium *Rhizobium sinense*.

[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. , (NaCl 0.1 g / L, agar 15.0 g / L, pH 6.8 - 7.0) on a solid plate.

[0044] Colony selection and purification: After incubation in the dark at 28 ± 1℃ for 5-7 days, select typical single colonies of *Rhizobium sinense* from Xinjiang 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 APXJHT24-1.

[0045] Liquid culture: A single colony of APXJHT24-1 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 Mix approximately 0.8 g of the product with an equal volume of sterile 60% (v / v) glycerol protectant to achieve a final glycerol concentration of 30%. Dispense the mixture into cryovials and store them in an ultra-low temperature freezer at -80°C.

[0046] Genomic DNA extraction: Genomic DNA was extracted from the APXJHT24-1 pure culture using the TIANamp Bacteria DNA Kit according to the instructions, and its concentration and purity (A260 / A280 ratio between 1.8 and 2.0) were determined using a NanoDrop™ 2000 spectrophotometer.

[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 (sequence 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 APXJHT24-1 had a 16S rRNA gene similarity of >99.5% with type strains such as Sinorhizobium xinjiangense CCBAU110.

[0051] 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.

[0052] atpD gene: Amplified using primers atpD 255F and atpD 782R, yielding a sequence of approximately 500 bp (the atpD sequence of ASHBQJ-24-3 is shown in SEQ ID NO: 6).

[0053] The primer atpD 255F sequence is shown in SEQ ID NO: 4: 5'- GCTSGGCCGCATCMTSAACGTC-3';

[0054] The primer atpD 782R sequence is shown in SEQ ID NO: 5: 5'-GCCGACACTTCMGAACCNGCCTG-3'.

[0055] recA gene: Amplified using primers recA1 and recA2, yielding a sequence of approximately 450 bp (as shown in SEQ ID NO: 9).

[0056] The primer recA1 sequence is shown in SEQ ID NO: 7: 5'-CATGCRCTGGATCCGGTCTATGC-3';

[0057] The primer recA2 sequence is shown in SEQ ID NO: 8: 5'- CTTGTTCTTGTCGACCTTGACGCG-3'.

[0058] glnII gene: Amplified using primers glnII 12F and glnII 689R, yielding a sequence of approximately 613 bp (as shown in SEQ ID NO: 12).

[0059] The primer glnII 12F sequence is shown in SEQ ID NO: 10: 5'- YAAGCTCGAGTACATYTGGCT -3';

[0060] The primer glnII 689R sequence is shown in SEQ ID NO: 11: 5'-TGCATGCCSGAGCCGTTCCA-3'.

[0061] nifH gene: Amplified using primers nifH PolF and nifH PolR, yielding a sequence of approximately 687 bp (as shown in SEQ ID NO: 15).

[0062] The primer nifH PolF sequence is shown in SEQ ID NO: 13: 5′-TGCGAYCCSAARGCBGACTC-3′;

[0063] The primer nifH PolR sequence is shown in SEQ ID NO: 14: 5′-ATSGCCATCATYTCRCCGGA-3′.

[0064] Multigene phylogenetic analysis: The gene sequences of APXJHT24-1 were aligned with the corresponding sequences of related *Rhizobium sinense* type strains from Xinjiang in GenBank. A phylogenetic tree was constructed using MEGA 11.0 software using the neighbor-joining method (bootstrap = 1000). The results are clearly shown (see...). Figure 2 APXJHT24-1 clustered with the type strain Sinorhizobium xinjiangense CCBAU110, confirming its taxonomic position as Sinorhizobium xinjiangense.

[0065] Example 2: Functional verification of the Xinjiang rhizobium APXJHT24-1 in promoting peanut growth and nitrogen fixation.

[0066] 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: APXJHT24-1 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.

[0067] Experimental treatments: APXJHT24-1 treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the above-mentioned APXJHT24-1 bacterial suspension at a ratio of 10% (v / w) of seed weight. After slightly drying, the seeds were sown in pots. Blank control group (CK): Seeds were mixed with an equal volume of sterile 0.85% NaCl solution, and the remaining operations were the same as the treatment group. Experimental setup: A completely randomized block design was adopted, with 12 replicates (i.e., 12 pots) in each group. 8 seeds were sown in each pot, and 3 strong seedlings were retained after thinning. Culture conditions: Cultured in an artificial climate chamber with a photoperiod of 16 hours light / 8 hours dark, day / night temperature set at 28℃ / 22℃, and relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0068] First, growth index measurement (results are shown in...) Figure 4Table 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).

[0069] Plant height: The average plant height of the APXJHT24-1 inoculated group (T) was 36.37 ± 0.99 cm, which was significantly increased by 40.9% compared with the blank control group (CK, 25.82 ± 1.82 cm) (p = 9.12 × 10⁻⁶). -9 ).

[0070] Fresh weight: The average fresh weight of group T was 76.37 ± 3.76 g, which was significantly higher than that of group CK (59.28 ± 4.21 g) by 28.8% (p = 2.19 × 10⁻⁶). -6 ).

[0071] Nodule count: The average number of root nodules in group T was 162.83 ± 14.77 per plant, which was significantly increased by 102.4% compared with group CK (80.44 ± 8.41 per plant) (p = 3.69 × 10⁻⁶). -9 ).

[0072] Nodule weight: The average fresh weight of root nodules in the T group was 0.201 ± 0.028 g / plant, which was significantly increased by 37.7% compared with the CK group (0.146 ± 0.025 g / plant) (p = 0.00021).

[0073] Conclusion: Inoculation with APXJHT24-1 significantly and comprehensively promoted the growth (plant height, fresh weight) and symbiotic nodulation ability (number of root nodules, root nodule weight) of peanut plants, indicating that this strain is a highly efficient peanut growth-promoting and symbiotic nitrogen-fixing strain.

[0074] Table 1: The promoting effect of APXJHT24-1 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0075]

[0076] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0077] This study systematically evaluated the effects of *Phytobacter spp.* APXJHT24-1 on peanut growth, development, nodulation, and nitrogen fixation through pot experiments. Figure 3As shown, compared with the uninoculated control group (CK), the peanut plants in the inoculated group (T) showed significant improvements in multiple growth indicators. Specifically, the plants in the inoculated group were significantly taller, and the leaves had a darker green color. Figure 3 A); The overall biomass accumulation of the plant is more complete, and the root system structure is more developed ( Figure 3 B); Notably, the number of root nodules on the roots of the inoculated group was significantly greater than that of the control group, indicating a stronger potential for nodulation and symbiotic nitrogen fixation (B). Figure 3 C). Based on the above phenotypic analysis results, *Rhizobium sinense* APXJHT24-1 from Xinjiang can effectively promote peanut plant growth and significantly enhance its nodulation ability, providing direct experimental evidence for its development into a microbial agent with both growth-promoting and nitrogen-fixing functions. This strong visual evidence is highly consistent with the aforementioned precise quantitative data (Table 1) showing a highly significant increase in plant height (40.9%), a highly significant increase in plant fresh weight (28.8%), and highly significant increases in nodule number and fresh weight (102.4% and 37.7%, respectively), together forming a complete chain of evidence that APXJHT24-1 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen-fixing ability.

[0078] Second, transcriptomics validation (molecular mechanism analysis).

[0079] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the APXJHT24-1 treatment group (T) and the blank control group (CK) (three plants were mixed for each replicate), and were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0080] 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).

[0081] Results: To elucidate the molecular basis of APXJHT24-1's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that APXJHT24-1 treatment specifically activated key gene networks in peanut roots related to symbiotic signal transduction and plant hormone signaling.

[0082] Evidence for key gene expression is as follows (see Figure 5 Table 2):

[0083] 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 2.444-fold and 2.667-fold, respectively. These genes are the core regulatory switches that initiate the root nodulation process.

[0084] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 9.588-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 24.308-fold. This indicates that early growth programs such as auxin-mediated cell division and elongation are strongly and extensively activated.

[0085] Conclusion: The above molecular evidence indicates that APXJHT24-1 inoculation systematically induces the co-regulation of key genes in the symbiotic signaling (NSP1, NSP2) and auxin signaling (SAUR, AUXIN) pathways in peanut roots. This reveals the intrinsic mechanism by which APXJHT24-1 promotes nodulation and growth at the transcriptional level: by mimicking or enhancing natural symbiotic dialogue signals, it initiates and accelerates the host's symbiotic developmental program and growth response.

[0086] Table 2: Effects of APXJHT24-1 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0087]

[0088] Example 3: Functional verification of the inhibition of Aspergillus flavus growth and toxin production by Xinjiang Rhizobium APXJHT24-1.

[0089] First, in vitro antagonism experiment (double-plate method). To evaluate the antagonistic effect of APXJHT24-1 against Aspergillus flavus, an in vitro antagonism experiment was conducted using the double-plate method.

[0090] Experimental setup:

[0091] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0092] Method: A double-layer plate interlocking method was used. The upper and lower plates were interlocked, the edges were sealed with Parafilm sealing film, and the plates were incubated in the dark at 28 °C for 5 days.

[0093] Lower plate (90 mm in diameter): 100 µL of APXJHT24-1 treatment group (T) with a concentration of 1×10⁻⁶ was uniformly coated. 8 A fresh bacterial suspension of APXJHT24-1 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.

[0094] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores per mL.

[0095] Measurement indicators:

[0096] 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%.

[0097] 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.

[0098] 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).

[0099] The results show (see) Figure 6 (Table 3) APXJHT24-1 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by APXJHT24-1 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus simultaneously, achieving highly efficient source control.

[0100] Inhibition of mycelial growth: The colony diameter in the APXJHT24-1 treatment group (T) was 1.5 ± 0.071 cm, significantly lower than that in the blank control group (CK, 7.22 ± 0.11 cm), with an inhibition rate of 79.22% (p = 1.30 × 10⁻⁶). -13 ).

[0101] Inhibition of spore reproduction: The sporulation rate in the APXJHT24-1 treatment group was 1.62 ± 0.19 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the blank control group (CK, 69.8 ± 6.14 × 10⁻⁶). 6 The inhibition rate reached 97.7% (p = 7.43 × 10⁻⁶ cells / plate). -9 ).

[0102] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the APXJHT24-1 treatment group was 19.84 ± 6.12 ng / plate, which was significantly lower than that in the blank control group (CK, 592.12 ± 131.42 ng / plate), with an inhibition rate of 96.7% (p = 6.05 × 10⁻⁶). -10 ).

[0103] Table 3: Inhibitory effect of APXJHT24-1 treatment on key life activities of Aspergillus flavus.

[0104]

[0105] Inhibition rate (%) = [(mean of CK group - mean of T group) / mean of CK group] × 100%. All data are expressed as mean ± standard deviation.

[0106] Second, activated carbon adsorption experiments (proving that the antibacterial activity originates from volatile substances).

[0107] 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.

[0108] CK group (blank control): The lower plate was not inoculated with APXJHT24-1 and no activated carbon was used.

[0109] CK + Car group (activated carbon control): The lower plate was not inoculated with APXJHT24-1, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0110] Group T (with APXJHT24-1, without activated carbon): The lower plate was uniformly coated with APXJHT24-1 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0111] T + Car group (with APXJHT24-1 and activated carbon): Half of the lower plate was coated with an equal amount of APXJHT24-1 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0112] Results and analysis (see Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of APXJHT24-1. This experiment demonstrates that the inhibitory effect of APXJHT24-1 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

[0113] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.22 ± 0.11 cm) and the CK + Car group (7.04 ± 0.15 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0114] APXJHT24-1 releases potent antibacterial VOCs: the colony diameter in group T (1.5 ± 0.071 cm) was significantly smaller than that in group CK, with an antibacterial rate of 79.2%.

[0115] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (3.56 ± 0.11 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 50.7%, but was still significantly lower than that of the CK group.

[0116] Table 4: Effect of activated carbon adsorption blocking experiment on Aspergillus flavus mycelial growth.

[0117]

[0118] 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%.

[0119] Third, transcriptomics verification (molecular mechanism).

[0120] To elucidate the molecular mechanism by which APXJHT24-1 inhibits toxin production and sporulation in Aspergillus flavus, transcriptome sequencing analysis was performed on treated Aspergillus flavus hyphae. Differential expression analysis showed that APXJHT24-1 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

[0121] Evidence for key gene expression is as follows (see Figure 8 Table 5):

[0122] 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 APXJHT24-1 treatment group (T) decreased to 27.56% and 21.33% 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.

[0123] 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 1.31% and 8.16% 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.

[0124] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by APXJHT24-1 can precisely interfere with the core life activity programs of Aspergillus flavus, namely, by strongly inhibiting the core regulatory hubs of toxin production (aflR / aflS) and sporulation (abaA / wetA), thereby achieving the dual effects of antibacterial and toxicity reduction.

[0125] Table 5: Inhibitory effect of APXJHT24-1 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0126]

[0127] Example 4: Identification and verification of antibacterial active substances in *Rhizobium sinense* APXJHT24-1 from Xinjiang.

[0128] To identify the characteristic volatile substances produced by APXJHT24-1, headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) was used to perform untargeted metabolomics analysis on the volatile components of the strain culture. By comparing the experimental group (APXJHT24-1) with the control group (CK), a compound with extremely significant differences was identified. This compound had a retention time of 7.218 min and a retention index of 1066.171 in the chromatogram. Its mass spectrum showed a similarity of 879 to dimethyl disulfide (dimethyl) in the NIST standard library, with a molecular ion peak at m / z 94, thus confirming its identification.

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

[0130] 1) Extremely significant enrichment: The relative abundance of this compound in the APXJHT24-1 treatment group was 80.9641 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 0.000562).

[0131] 2) High model importance: In the orthogonal partial least squares discriminant analysis (OPLS-DA) model, its variable importance projection (VIP) value is as high as 1.352, indicating that it is one of the core volatile markers that distinguishes APXJHT24-1 from the blank control.

[0132] Conclusion: Combined chromatographic-mass spectrometry identification and omics statistical analysis conclusively demonstrate that dimethyl disulfide is a characteristic volatile compound specifically and abundantly produced by APXJHT24-1. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0133] Table 6: Identification and omics analysis results of dimethyl disulfide, a characteristic volatile substance in APXJHT24-1

[0134]

[0135] The retention index and high matching degree provide double verification for qualitative identification. The VIP value is much greater than 1.0, indicating that this compound is the most critical marker to distinguish the two groups of samples.

[0136] Second, purity verification experiment.

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

[0138] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.7%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 78.7%.

[0139] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 53.7%) to a 40-fold dilution (inhibition rate 18.6%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0140] Determining the activity threshold: When the dilution factor reaches 40 times or more, there is no substantial difference in the colony diameter between each treatment group and the control group (inhibition rate < 10%), indicating that dimethyl disulfide has basically lost its observable antibacterial activity at this concentration.

[0141] Conclusion: This pure product verification experiment conclusively demonstrates that dimethyl disulfide 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 APXJHT24-1, strongly confirming that dimethyl disulfide is a key effector molecule mediating the volatile antibacterial effect of APXJHT24-1.

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

[0143]

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

[0145] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A *Rhizobium sinense* strain APXJHT24-1 from Xinjiang, which possesses both growth-promoting and aflatoxin-inhibiting functions, characterized in that... Its classification name is Sinorhizobium xinjiangense, accession number is CCTCC M 2026229, accession date is January 23, 2026, and it is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent or preparation, characterized in that, It comprises at least one of the following: *Rhizobium sinense* APXJHT24-1 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 Xinjiang rhizobium APXJHT24-1 as described in claim 1 or the microbial agent or preparation as described in claim 2 in promoting peanut growth and nodulation nitrogen fixation.

4. The application of the Xinjiang Rhizobium APXJHT24-1 as described in claim 1 or the microbial agent or preparation as described in claim 2 in inhibiting the growth, sporulation, and aflatoxin synthesis of Aspergillus flavus.

5. The application according to claim 4, characterized in that, The specific application involves achieving the inhibitory function through the volatile organic compounds released by the Xinjiang rhizobium APXJHT24-1.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the Xinjiang rhizobium APXJHT24-1 as described in claim 1 or the microbial agent or preparation as described in claim 2 to the peanut planting environment or peanut plants.

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