Rhizobium albiziae APHNZY-24-4 with functions of promoting growth and inhibiting aspergillus flavus and application of rhizobium albiziae APHNZY-24-4

Abstract

The invention discloses a rhizobium mesonii strain APHNZY-24-4 which has the functions of promoting peanut growth and inhibiting aspergillus flavus, and the preservation number of the rhizobium mesonii strain APHNZY-24-4 is CCTCC (China Center for Type Culture Collection) M 2026228. According to the invention, integration of two functions of generating a specific bacteriostatic volatile matter, namely 3-methylbutyric acid, and efficient symbiotic nitrogen fixation on a single strain is realized in the rhizobium japonicum for the first time. 3-methylbutyric acid released by the strain can accurately down-regulate expression of toxin synthesis key genes and spore development core genes in aspergillus flavus, so that efficient bacteriostasis is realized on the molecular level. Through one-time inoculation, the 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 interdisciplinary field of agricultural microbiology and plant protection, specifically involving a strain of Rhizobium mesosinicum APHNZY-24-4 that has both efficient growth-promoting and nitrogen-fixing functions and strong inhibition of Aspergillus flavus, and its application in the green and safe production of peanuts. Technical Background

[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 security and increasing farmers' income. However, sustainable peanut production faces two core challenges: continuous yield increase and the control of aflatoxin contamination. Aflatoxins (AFs) produced when peanuts are infected by Aspergillus flavus are potent carcinogens that seriously endanger human and animal health and have become a key quality and safety bottleneck affecting international trade and industrial development.

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

[0005] As a legume, peanuts' most crucial and efficient yield-increasing pathway lies in establishing a specific symbiotic relationship with rhizobia, forming root nodules, and converting atmospheric nitrogen into nitrogen that the plant can directly utilize through biological nitrogen fixation. *Rhizobium mesosinicum* is a type of rhizobium widely found in soil and capable of forming symbiotic relationships with various legumes, possessing significant potential in promoting plant growth and nitrogen fixation. However, traditional rhizobium agents have limited functions, primarily focusing on nitrogen fixation and growth promotion, and generally lack direct biological control capabilities against peanut aflatoxin.

[0006] III. Limitations of Existing Aflatoxin Control Technologies

[0007] Currently, the control of aflatoxin mainly includes three categories: agricultural management, physicochemical methods, and biological control. The first two suffer from passive effects, high costs, or chemical residues; while mainstream biological control strains (such as Bacillus spp. and non-toxin-producing Aspergillus flavus) mainly focus on inhibiting bacteria and do not directly contribute to the core yield-increasing mechanism of peanuts—symbiotic nitrogen fixation. It is particularly noteworthy that the unique growth habit of peanuts, which "flowers above ground and fruits underground," makes it difficult for traditional above-ground chemical or biological control methods to directly act on the fruits in the soil during the critical period of pod development, creating a huge control gap and physical barrier.

[0008] IV. Systemic Defects of Existing Technological Approaches

[0009] The existing technological paths of "increasing production" and "preventing poisoning" have been separated into two independent systems, leading to the following systemic defects:

[0010] (1) Functional singularity and lack of synergy: Growth-promoting agents (including traditional rhizobia) generally lack effective inhibition of Aspergillus flavus; while biocontrol agents contribute very little to promoting the symbiotic nitrogen fixation process of peanuts. Simple combination of the two often leads to unstable effects or even mutual weakening due to competition or antagonism between microorganisms.

[0011] (2) Complex operation and high cost: It is necessary to purchase, store and apply two types of products separately, which significantly increases production costs, labor input and management complexity.

[0012] (3) Weak source control: There is a lack of an integrated biotechnology solution that can simultaneously promote nitrogen fixation and inhibit Aspergillus flavus at the source, starting from the same ecological niche (rhizosphere) and using the same microbial carrier during the peanut growing season and within the same growth cycle.

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

[0014] Based on the aforementioned industry bottlenecks and technological status, a clear technological gap exists in this field: there are currently no publicly reported or commercially available examples demonstrating that *Rhizobium mesosinicum* possesses a multifunctional strain capable of simultaneously and effectively inhibiting *Aspergillus flavus* by producing specific antibacterial volatiles (such as 3-methylbutyric acid) while maintaining highly efficient symbiotic nitrogen fixation. This invention aims to fill this gap by screening and developing a novel *Rhizobium mesosinicum* strain, APHNZY-24-4, which possesses both of these dual functions. This allows for the simultaneous regulation of the core yield-increasing mechanism (symbiotic nitrogen fixation) and the key toxin pollution source (*Aspergillus flavus* infection) of leguminous crops on a single microbial carrier. With a single application, this strain can establish a composite function integrating "nutrient supply" and "biological protection" 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 significant value in promoting green and safe peanut production. Summary of the Invention

[0015] To address the shortcomings of existing technologies, this invention provides a strain of Albizia julibrissin rhizobium APHNZY-24-4 that combines growth promotion and Aspergillus 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 *Rhizobium mesosinicum* strain APHNZY-24-4, which possesses both growth-promoting and aflatoxin-inhibiting functions. Its classification name is *Rhizobium mesosinicum*, its accession number is CCTCC M 2026228, its accession date is January 23, 2026, and it is deposited at the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University.

[0018] A second objective of this invention is to provide a microbial agent or preparation comprising at least one of Albizia julibrissin APHNZY-24-4, its fermentation product, or a culture thereof; wherein the fermentation product comprises fermentation broth, sterile supernatant, or an active substance extracted from the fermentation broth or sterile supernatant; and wherein 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 Albizia julibrissin APHNZY-24-4, or a microbial agent or preparation containing Albizia julibrissin APHNZY-24-4, in promoting peanut growth and nodulation nitrogen fixation.

[0020] The fourth objective of this invention is to provide the application of *Amygdalinia albinoides* APHNZY-24-4, or a microbial agent or preparation containing *Amygdalinia albinoides* APHNZY-24-4, 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 Albizia rhizobium APHNZY-24-4.

[0022] The fifth objective of this invention is to provide a method for inhibiting Aspergillus flavus, comprising the steps of: applying the aforementioned Albizia julibrissin APHNZY-24-4, or a microbial agent or preparation containing Albizia julibrissin APHNZY-24-4, 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: APHNZY-24-4 rhizobium of Albizia julibrissin can comprehensively promote the growth and nitrogen fixation of peanut plants. After inoculation, the peanut plant height and biomass increased by 36.1% and 30.4%, respectively, and the number of root nodules and the fresh weight of root nodules increased significantly by 107.2% and 22.5%, 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 77.4%, 97.5%, 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, 3-methylbutyric acid was identified as its key volatile antibacterial active substance. This compound was specifically enriched in the strain's metabolites (relative abundance was 2049.13 times that of the control group), and the pure product had a strong dose-dependent inhibitory effect on Aspergillus flavus (inhibition rate of 97.8% 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 its 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 "3-methylbutyric acid-mediated potent biocontrol" in the same strain of Albizia julibrissin. 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 *Rhizobium mesosinicum* strain APHNZY-24-4, which possesses both growth-promoting and aflatoxin-inhibiting functions. The strain is classified as *Rhizobium mesosinicum*. This *Rhizobium mesosinicum* APHNZY-24-4 was obtained by the inventors through screening. Its accession number is CCTCC M 2026228, and its accession date is January 23, 2026. 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 an APHNZY-24-4 phylogenetic tree based on multiple genes (16S, atpD, recA, dnaK, glnII, 23S).

[0031] Figure 3 A represents the field growth of peanut plants in the blank control (CK). Figure 3 B represents the field growth of peanut plants in the APHNZY-24-4 treatment group (T). Figure 3 C represents the root system of plants in the blank control (CK) / APHNZY-24-4 treatment group (T); Figure 3 D represents the blank control (CK) / APHNZY-24-4 treatment group (T) root nodules during the basal stage; Figure 3 E represents the overall plant height of the blank control (CK) / APHNZY-24-4 treatment group (T).

[0032] Figure 4 This invention relates to the effects of inoculation with *Albizia julibrissin* rhizobium APHNZY-24-4 on key phenotypic indicators of peanut seedling growth and symbiotic nodulation; among which, Figure 4 A represents the effect on plant height; Figure 4 B represents the effect on the fresh weight of the plant; Figure 4 C represents the effect on the number of root nodules; Figure 4 D represents the effect on the fresh weight of root nodules; in the figure, CK represents the blank control group, and T represents the APHNZY-24-4 treatment group; the column height represents the average value of each index, and the error bar represents the standard deviation (n ≥ 3). and The numbers represent highly significant (p < 0.01) and extremely significant (p < 0.001) differences between groups, 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 Albizia julibrissin APHNZY-24-4; wherein, Figure 4 A represents the expression level of NSP1, a core transcription factor gene in the nodule signaling pathway; Figure 4 B represents the expression level of NSP2, a co-regulatory factor gene of the nodule signaling pathway; Figure 4 C represents the expression level of the auxin early response factor gene SAUR; Figure 4 D represents the expression level of the auxin early response factor gene AUXIN. In the figure, CK represents the blank control group, and T represents the APFJPT-23-4 treatment group; the column height represents the gene expression level (based on standardized counts); the error bar represents the standard deviation (n=3). , 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 Albizia julibrissin rhizobium APHNZY-24-4 on the growth, sporulation, and aflatoxin synthesis of Aspergillus flavus using a double-plate method; wherein... Figure 6 A represents the diameter of Aspergillus flavus colonies; Figure 6 B represents conidia production; Figure 6 C represents aflatoxin production. In the figure, CK represents the blank control group, and T represents the APFJPT-23-4 treatment group; quantitative data are expressed as mean ± standard deviation (n ≥ 3); indicates highly significant differences between groups (independent samples t-test). ).

[0035] Figure 7 This is a bar chart showing the quantitative statistical results of Aspergillus flavus colony diameter in the blank control group (CK), blank control group (CK) + activated carbon (Car), Albizia julibrissin APHNZY-24-4 treatment group (T), and Albizia julibrissin APHNZY-24-4 treatment group (T) + activated carbon (Car); In the figure, The differences between groups were statistically significant (p < 0.001).

[0036] Figure 8 This invention relates to the effect of APHNZY-24-4 treatment of Albizia julibrissin 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, and T represents the APFJPT-23-4 treatment group; the boxes represent the quartile range, the midline represents the median, the whisker lines represent the range, and the 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 9This invention employs headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS) to detect and compare the relative abundance of the volatile component 3-methylbutyric acid in the blank control group (CK) and the APHNZY-24-4 treatment group (T) of *Albizia julibrissin* rhizobia. 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 < 0.01). 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 Albizia julibrissin rhizobium APHNZY-24-4.

[0040] The Albizia rhizobium APHNZY-24-4 of the present invention was isolated from peanut rhizosphere soil in the field treated with ARC microbial agent (the main components of which are: Bacillus amyloliquefaciens, Brevibacillus laterosporus, Bacillus mucilaginosus and Enterobacter ludwigii compound agent).

[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 *Rhizobium* spp., which is closely related to peanut symbiotic nitrogen fixation. For example... Figure 1 As shown, the average relative abundance of Rhizobium in the CK group was 0.0397%, while the average relative abundance in the ARC treatment group (T group) increased to 0.0573%, a significant increase of 44.5% (p < 0.01). This result provides direct ecological evidence for the subsequent targeted isolation and screening of functionally enhanced rhizobium strains (such as APHNZY-24-4) from the rhizosphere soil of the ARC treatment group, indicating that ARC inoculant can reshape the rhizosphere microbiota and specifically promote the colonization and enrichment of beneficial symbiotic bacteria—rhizobia.

[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 rhizobia 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 APHNZY-24-4.

[0045] Liquid culture: A single colony of APHNZY-24-4 was inoculated into 5 mL of YMB liquid medium and cultured at 28°C with shaking at 180 rpm for 72 hours until the late logarithmic growth phase (OD200). 600 (Values ​​are between 0.8 and 1.0). Short-term storage: Streak the above bacterial suspension on fresh YMA plates and store at 4°C, subculturing monthly. Long-term storage: Take the logarithmic growth phase bacterial suspension (OD... 600 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 APHNZY-24-4 pure culture 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 1342 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 APHNZY-24-4 had a 16S rRNA gene similarity of >99.5% with type strains such as Rhizobium mesosinicum CCBAU 25010.

[0051] Housekeeping gene identification: To accurately identify its species classification, partial sequences of the atpD, recA, and glnII housekeeping genes were further amplified and determined.

[0052] atpD gene: Amplified using primers atpD 255F and atpD 782R, yielding a sequence of approximately 489 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 508 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 579 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] Multigene phylogenetic analysis: The gene sequences of APHNZY-24-4 were aligned with the corresponding sequences of related rhizobium type strains in GenBank. A phylogenetic tree was constructed using MEGA 11.0 software using the neighbor-joining method (bootstrap = 1000). The results are clearly shown (see...). Figure 2 APHNZY-24-4 clustered with the type strain Rhizobium mesosinicum CCBAU 25010, confirming its taxonomic position as Rhizobium mesosinicum.

[0062] Example 2: Verification of the function of Albizia julibrissin APHNZY-24-4 in promoting peanut growth and nodulation nitrogen fixation.

[0063] Test materials: The tested peanut variety was Zhonghua 28. The soil was sandy loam, sterilized twice by autoclaving at 121℃ for 0.5 hours each time to eliminate the influence of indigenous rhizobia. Inoculum preparation: APHNZY-24-4 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.

[0064] Experimental treatments: APHNZY-24-4 treatment group (T): Plump and uniform peanut seeds were selected and uniformly mixed with the APHNZY-24-4 bacterial suspension at a ratio of 10% (v / w) of seed weight. After slightly drying, the seeds were sown in pots. Blank control group (CK): Seeds were mixed with an equal volume of sterile 0.85% NaCl solution, and the remaining operations were the same as the treatment group. Experimental setup: A completely randomized block design was adopted, with 12 replicates per group (i.e., 12 pots). 8 seeds were sown in each pot, and 3 strong seedlings were retained after thinning. Culture conditions: Cultured in an artificial climate chamber with a photoperiod of 16 hours light / 8 hours dark, day / night temperature set at 28℃ / 22℃, and relative humidity of 60%-70%. Routine freshwater management was used, and no nitrogen fertilizer was applied.

[0065] First, growth indicators were measured (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).

[0066] Plant height: The average plant height of the APHNZY-24-4 treatment group (T) was 36.63 ± 1.77 cm, which was significantly increased by 36.1% compared with the blank control group (CK, 26.92 ± 2.39 cm) (p = 1.17 × 10⁻⁶). -6 ).

[0067] Fresh weight: The average fresh weight of group T was 79.72 ± 2.8 g, which was significantly higher than that of group CK (61.15 ± 3.48 g) by 30.4% (p = 6.69 × 10⁻⁶). -8 ).

[0068] Nodule count: The average number of root nodules in group T was 170.83 ± 14.27 per plant, which was significantly increased by 107.2% compared with group CK (82.44 ± 7.89 per plant) (p = 9.03 × 10⁻⁶). -10 ).

[0069] Nodule weight: The average fresh weight of root nodules in the T group was 0.218 ± 0.023 g / plant, which was significantly increased by 22.5% compared with the CK group (0.178 ± 0.025 g / plant) (p = 0.0016).

[0070] Conclusion: Inoculation with APHNZY-24-4 significantly and comprehensively promoted the growth (plant height, fresh weight) and symbiotic nodulation ability (number of root nodules, root nodule weight) of peanut plants, indicating that this strain is a highly efficient peanut growth-promoting and symbiotic nitrogen-fixing strain.

[0071] Table 1: The promoting effect of APHNZY-24-4 inoculation treatment on peanut seedling growth and nodulation phenotype.

[0072]

[0073] Percentage improvement = [(Mean of group T - Mean of group CK) / Mean of group CK] × 100%. All data are expressed as mean ± standard deviation.

[0074] Inoculation with *Albizia julibrissin* rhizobium APHNZY-24-4 significantly promoted the growth and symbiotic nodulation of peanut seedlings, with the following direct phenotypic differences: Figure 3 As shown. In terms of overall shape, Figure 3The results showed that, compared with the blank control (CK), inoculation with the APHHNZY-24-4 strain significantly promoted the field growth of peanut plants. The treatment group (T) had taller plants, darker greener leaves, and more vigorous growth. Figure 3 B, E); its root system is more developed, and the number of nodules is significantly increased ( Figure 3 C). Especially during the needle-feeding period, the treatment group showed earlier and more abundant root nodule formation ( Figure 3 D). The results show that this strain effectively promotes peanut plant growth and enhances its nodulation ability under field conditions. This strong visual evidence, along with the aforementioned highly significant quantitative data (Table 1) showing a 36.1% increase in plant height, a 30.4% increase in plant fresh weight, and a 107.2% and 22.5% increase in root nodule number and fresh weight, respectively, constitutes a complete chain of evidence demonstrating that APHNZY-24-4 can efficiently promote peanut growth and significantly enhance its symbiotic nitrogen fixation ability.

[0075] Second, transcriptomics validation (molecular mechanism analysis).

[0076] Sample collection and processing: 30 days after sowing, root samples were randomly collected from three biological replicates of the APHNZY-24-4 treatment group (T) and the blank control group (CK) (three plants were mixed for each replicate), and were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0077] RNA sequencing and analysis: Total RNA was extracted using the TRIzol® Reagent method. After quality control, paired-end 150 bp sequencing was performed using the Illumina NovaSeq 6000 platform. After quality control, the raw data were aligned to the peanut reference genome using HISAT2 software, and differentially expressed genes were analyzed using the DESeq2 R package (screening threshold: p < 0.05).

[0078] Results: To elucidate the molecular basis of APHNZY-24-4's promotion of peanut growth and nodulation, transcriptome sequencing analysis was performed on peanut roots 30 days after inoculation. Differential expression analysis showed that APHNZY-24-4 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):

[0079] Symbiotic nodulation pathway: The expression levels of NSP1 and NSP2, the core transcription factors of the nodulation signaling pathway, were significantly upregulated in the T group, by 2.350-fold and 3.992-fold, respectively. These genes are the core regulatory switches that initiate the root nodulation process.

[0080] Plant hormone signaling: The expression level of SAUR, an early auxin response factor, was significantly upregulated by 12.395-fold; simultaneously, the expression level of AUXIN, a gene in the auxin biosynthesis / signaling pathway, was also significantly upregulated by 18.039-fold. This indicates that early growth programs such as cell division and elongation mediated by auxin are strongly and extensively activated.

[0081] Conclusion: The above molecular evidence indicates that APHNZY-24-4 inoculation systematically induces the co-regulation of key genes in the symbiotic signaling (NSP1, NSP2) and auxin signaling (SAUR, AUXIN) pathways in peanut roots. This reveals the intrinsic mechanism by which APHNZY-24-4 promotes nodulation and growth at the transcriptional level: by mimicking or enhancing natural symbiotic dialogue signals, it initiates and accelerates the host's symbiotic developmental program and growth response.

[0082] Table 2: Effects of APHNZY-24-4 treatment on the expression of key symbiotic and growth-related genes in peanut roots.

[0083]

[0084] Example 3: Functional verification of APHNZY-24-4 rhizobium in inhibiting Aspergillus flavus growth and toxin production

[0085] First, in vitro antagonism experiment (double-plate method).

[0086] To evaluate the antagonistic effect of APHNZY-24-4 against Aspergillus flavus, this study used the double-plate method to conduct an in vitro antagonistic experiment.

[0087] Pathogen: Aspergillus flavus LNZW-1, a standard strain of Aspergillus flavus that produces toxins.

[0088] 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.

[0089] Lower plate (90 mm in diameter): APHNZY-24-4 treatment group (T) uniformly coated with 100 µL of 1×10⁻⁶ solution. 8 Fresh bacterial suspension of APHNZY-24-4 (CFU / mL); blank control group (CK) spread on an equal volume of sterile YMB medium. After the bacterial suspension was absorbed, the upper plate was inverted.

[0090] Upper plate: Inoculate 2 µL of 1×10⁻⁶ solution at the center of the plate. 5 A suspension of Aspergillus flavus spores / mL.

[0091] Measurement indicators:

[0092] 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%.

[0093] 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.

[0094] 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).

[0095] The results show (see) Figure 6 (Table 3) APHNZY-24-4 treatment significantly and comprehensively inhibited the mycelial growth, spore reproduction, and toxin synthesis of Aspergillus flavus. The volatile substances produced by APHNZY-24-4 can effectively block the growth, reproduction, and toxin production of Aspergillus flavus simultaneously, achieving highly efficient source control.

[0096] Inhibition of mycelial growth: The colony diameter in the APHNZY-24-4 treatment group (T) was 1.62 ± 0.11 cm, significantly lower than that in the blank control group (CK, 7.18 ± 0.08 cm), with an inhibition rate of 77.4% (p = 2.55 × 10⁻⁶). -13 ).

[0097] Inhibition of spore reproduction: The sporulation rate in the APHNZY-24-4 treatment group was 1.78 ± 0.19 × 10⁻⁶. 6 The number of samples per dish was significantly lower than that of the blank control group (CK, 70.4 ± 6.8 × 10⁻⁶). 6 The inhibition rate reached 97.5% (p = 1.59 × 10⁻⁶ cells / plate). -8 ).

[0098] Inhibition of toxin synthesis: The aflatoxin B1 (AFB1) yield in the APHNZY-24-4 treatment group was 21.25 ± 7.6 ng / plate, which was significantly lower than that in the blank control group (CK, 587.92 ± 145.89 ng / plate), with an inhibition rate of 96.4% (p = 3.21 × 10⁻⁶). -9 ).

[0099] Table 3: Inhibitory effect of APHNZY-24-4 treatment on key life activities of Aspergillus flavus.

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

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

[0102] 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.

[0103] CK group (blank control): The lower plate was not inoculated with APHNZY-24-4 and no activated carbon was used.

[0104] CK + Car group (activated carbon control): The lower plate was not inoculated with APHNZY-24-4, but a layer of about 5 g of high-temperature sterilized granular activated carbon was spread on it.

[0105] Group T (with APHNZY-24-4, without activated carbon): The lower plate was uniformly coated with APHNZY-24-4 bacterial suspension (concentration: 1×10⁻⁶). 8 (CFU / mL), no activated carbon.

[0106] T + Car group (with APHNZY-24-4 and activated carbon): Half of the lower plate was coated with an equal amount of APHNZY-24-4 bacterial suspension, and the other half was covered with 5 g of sterilized activated carbon.

[0107] Results and analysis (see Figure 7 (Table 4) The adsorption effect of activated carbon significantly weakened the antibacterial effect of APHNZY-24-4.

[0108] Activated carbon itself had no effect: There was no significant difference in colony diameter between the CK group (7.18 ± 0.08 cm) and the CK + Car group (7.02 ± 0.08 cm), indicating that activated carbon itself had no effect on the growth of Aspergillus flavus.

[0109] APHNZY-24-4 releases potent antibacterial VOCs: the colony diameter in group T (1.62 ± 0.11 cm) was significantly smaller than that in group CK, with an antibacterial rate of 77.4%.

[0110] Activated carbon partially adsorbs key antibacterial substances: When activated carbon was added (T + Car group), the antibacterial effect was significantly weakened, the colony diameter (3.5 ± 0.1 cm) was significantly larger than that of the T group, and the antibacterial rate dropped to 51.3%, but was still significantly lower than that of the CK group.

[0111] Conclusion: This experiment demonstrates that the inhibitory effect of APHNZY-24-4 on Aspergillus flavus mainly depends on the volatile substances it releases that can be adsorbed by activated carbon.

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

[0113]

[0114] The key role of volatile organic compounds (VOCs) in inhibiting Aspergillus flavus mycelial growth was evaluated using activated carbon adsorption blocking experiments. Data are expressed as mean ± standard deviation. Inhibition rate (%) = [(average diameter of CK group - average diameter of treatment group) / average diameter of CK group] × 100%.

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

[0116] To elucidate the mechanism by which APHNZY-24-4 inhibits toxin production and sporulation in Aspergillus flavus at the molecular level, we performed transcriptome sequencing analysis on the treated Aspergillus flavus hyphae. Differential expression analysis showed that APHNZY-24-4 treatment specifically and significantly inhibited the expression of key genes in the core regulatory network of Aspergillus flavus development and toxin synthesis.

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

[0118] The expression of key genes regulating aflatoxin synthesis was suppressed: the expression levels of the core transcription factor gene aflR and its co-activator gene aflS in the APHNZY-24-4 treatment group (T) decreased to 26.47% and 15.77% of the control group, respectively, with both downregulation reaching a significant level (p < 0.05). The aflR / aflS complex is the master switch for activating the entire aflatoxin synthesis gene cluster, and its significant inhibition directly leads to the shutdown of the aflatoxin synthesis pathway.

[0119] The expression of key genes regulating conidial development pathways was strongly suppressed: the expression of abaA and wetA, core transcription factors regulating conidial development, was extremely suppressed, with expression levels in the treatment group only 1.07% and 8.23% 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.

[0120] Conclusion: The transcriptomic evidence above demonstrates that the volatile substances produced by APHNZY-24-4 can precisely interfere with the core life activity programs of Aspergillus flavus, namely, by strongly inhibiting the core regulatory hubs of toxin production (aflR / aflS) and sporulation (abaA / wetA), thereby achieving the dual effects of antibacterial and toxicity reduction.

[0121] Table 5: Inhibitory effect of APHNZY-24-4 treatment on the expression of key toxin-producing and sporulation genes in Aspergillus flavus.

[0122]

[0123] Example 4: Identification and verification of antibacterial active substances of Albizia julibrissin APHNZY-24-4.

[0124] To identify the characteristic volatile compounds produced by APHNZY-24-4, we performed untargeted metabolomics analysis of the volatile components of the strain culture using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS). By comparing the APHNZY-24-4 treatment group (T) with the control group (CK), a compound with highly significant differences was identified. This compound had a retention time of 23.823 min and a retention index of 1677.05 in the chromatogram. Its mass spectrum showed a similarity of 720 to 3-methylbutyric acid (3-methyl-) in the NIST standard library, with a molecular ion peak at m / z 43, thus confirming its identification.

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

[0126] 1) Extremely significant enrichment: The relative abundance of this compound in the APHNZY-24-4 treatment group was 2049.13 times that of the blank control group (Fold Change), and the difference between the groups was extremely significant (p = 0.0057).

[0127] 2) High model importance: In the orthogonal partial least squares discriminant analysis (OPLS-DA) model, its variable importance projection (VIP) value is as high as 1.50, indicating that it is one of the core volatile markers that distinguishes APHNZY-24-4 from the blank control.

[0128] Conclusion: Through comprehensive chromatographic-mass spectrometry identification and omics statistical analysis, we conclusively demonstrate that 3-methylbutyric acid is a characteristic volatile compound specifically and abundantly produced by APHNZY-24-4. This discovery provides a clear target for further investigation into the microbiological function of this compound.

[0129] Table 6: Identification and omics analysis results of 3-methylbutyric acid, a characteristic volatile substance of APHNZY-24-4.

[0130]

[0131] The retention index and high matching degree provide double verification for qualitative identification. The VIP value is much greater than 1.0, indicating that this compound is the most critical marker to distinguish the two groups of samples.

[0132] Second, purity verification experiment.

[0133] To directly verify the antibacterial activity of the key volatile substance 3-methylbutyric acid, we used the fumigation method to determine the effect of its pure graded dilutions on the mycelial growth of Aspergillus flavus (Table 7). The results showed that 3-methylbutyric acid had a strong and dose-dependent inhibitory effect on the mycelial growth of Aspergillus flavus.

[0134] High efficiency and complete inhibition: When added at the undiluted level, the inhibition rate on mycelial growth is as high as 97.8%, approaching complete inhibition. Even when diluted 1.6 times, its inhibition rate remains at an extremely high level of 81.9%.

[0135] A clear dose-response gradient: the antibacterial effect decreases sequentially with decreasing compound concentration. From an 8-fold dilution (inhibition rate 54.4%) to a 40-fold dilution (inhibition rate 20.7%), the antibacterial activity shows a regular decrease, indicating that its effective concentration range is relatively narrow.

[0136] Determining the activity threshold: When the dilution factor reaches 40 times or more, there is no substantial difference in the colony diameter between each treatment group and the control group (inhibition rate < 10%), indicating that 3-methylbutyric acid has basically lost its observable antibacterial activity at this concentration.

[0137] Conclusion: This pure product verification experiment conclusively demonstrates that 3-methylbutyric acid (3-methylbutyric acid) is itself a potent inhibitor of Aspergillus flavus mycelial growth. It exerts a significant antibacterial effect even at low concentrations, a characteristic consistent with the highly specific and significant enrichment (2502.44-fold) of this compound in the volatile substances of APHNZY-24-4. This strongly confirms that 3-methylbutyric acid is the key effector molecule mediating the volatile antibacterial effect of APHNZY-24-4.

[0138] Table 7: Effects of different dosages of 3-methylbutyric acid on the growth of Aspergillus flavus mycelia.

[0139]

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

[0141] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A *Amygdalinia albinoides* APHNZY-24-4 strain possessing both growth-promoting and aflatoxin-inhibiting functions, characterized in that... Its classification name is Rhizobium mesosinicum, accession number is CCTCC M 2026228, 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: Albizia julibrissin rhizobium APHNZY-24-4 as described in claim 1, 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.

3. The application of the Albizia julibrissin rhizobium APHNZY-24-4 as described in claim 1 or the microbial agent or preparation as described in claim 2 in promoting peanut growth and nodulation nitrogen fixation.

4. The use of the Albizia julibrissin Rhizobium APHNZY-24-4 as described in claim 1 or the microbial agent or preparation as described in claim 2 in inhibiting the growth, sporulation, and aflatoxin synthesis of Aspergillus flavus.

5. The application according to claim 4, characterized in that, The specific application involves achieving the inhibitory function through the volatile organic compounds released by the Albizia julibrissin rhizobium APHNZY-24-4.

6. A method for inhibiting Aspergillus flavus, characterized in that, The steps include: applying the Albizia rhizobium APHNZY-24-4 as described in claim 1 or the microbial agent or preparation as described in claim 2 to the peanut growing environment or peanut plants.

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