Stenotrophomonas sp. and its fermentation product and application

Abstract

The application provides a Stenotrophomonas geniculata and a fermentation product and application thereof, relates to the technical field of biology, and the Stenotrophomonas geniculata D963 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33555. The Stenotrophomonas geniculata D963 can directly inhibit the generation of aflatoxin without affecting the growth of Aspergillus flavus, is not prone to causing rapid spread of drug-resistant strains, has a good prevention and control effect on aflatoxin, can effectively inhibit the formation of aflatoxin pollution in crops, and can be used for preparing an aflatoxin pollution inhibitor.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a stenotrophomonas and a fermentation product and application thereof. BACKGROUND

[0002] Aspergillus flavus is a saprophytic fungus widely distributed in nature, which has strong adaptability and tolerance to the environment. The aflatoxins produced by Aspergillus flavus have widely polluted the world, becoming one of the important threats in agricultural production that needs to be highly vigilant. Aspergillus flavus mainly produces aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2), and some strains can also produce aflatoxin G, cyclopiazonic acid (CPA) and other aflatoxins. Among them, AFB1 is the main toxin, which is listed as a class I carcinogen by the International Agency for Research on Cancer (IARC), can induce liver cancer, and is considered as one of the strongest chemical carcinogens known. During the pre-production, production and post-production stages of crops, this toxin can contaminate peanuts, corn, rice and other major grain and oil crops, posing a major threat to food safety and human health.

[0003] Due to the strong chemical stability of aflatoxins, there is little decomposition during the agricultural production and processing process, and the conventional cooking method is difficult to effectively remove the toxin, so it is necessary to control the amount of aflatoxin produced in crops to reduce toxin pollution. At present, there is a lack of effective chemical pesticides to prevent and control Aspergillus flavus, and the commonly used fungicides in corn and peanut fields have limited effect on the prevention and control of Aspergillus flavus, and even stimulate the production of aflatoxins, while causing pesticide residue pollution problems. Degradation of toxins by chemical and physical techniques is also a way, but the existing chemical method degrades completely, but cannot reach the level of green safety; the physical method removes the toxin quickly, but the degradation is not complete. Compared with the above two methods, biological control can effectively remove the toxin and ensure the quality of food, and has strong specificity and biological safety.

[0004] The current common biological control includes aflatoxin-degrading bacteria, non-aflatoxin-producing Aspergillus strains, and Aspergillus antagonists. The use of aflatoxin-degrading bacteria can effectively degrade the aflatoxins secreted by Aspergillus flavus, but the degradation or conversion products are unknown in terms of human toxicity, which has potential risk; the introduction of non-aflatoxin-producing Aspergillus strains may bring other biological safety and health risks, including the stability problem caused by the variation in Aspergillus strains, and the problem of producing CPA and other toxins by non-toxin-producing strains. Therefore, it is necessary to find a safe and non-toxic, high-efficiency detoxification, and stable control effect alternative biocontrol strain and aflatoxin pollution inhibitor.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a Stenotrophomonas geniculata D963 to at least solve one of the technical problems existing in the prior art.

[0007] The second purpose of the present application is to provide a fermentation product.

[0008] The third purpose of the present application is to provide a microbial agent.

[0009] The fourth purpose of the present application is to provide the application of the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned microbial agent in preventing and controlling aflatoxin pollution.

[0010] The fifth purpose of the present application is to provide the application of the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned microbial agent in inhibiting aflatoxin or preparing a product for inhibiting aflatoxin.

[0011] The sixth purpose of the present application is to provide a preparation for inhibiting aflatoxin.

[0012] The seventh purpose of the present application is to provide a method for preventing and controlling aflatoxin pollution.

[0013] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0014] In a first aspect, the present application provides a Stenotrophomonas geniculata D963, which is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 33555.

[0015] Further, the Stenotrophomonas geniculata D963 is derived from rice panicles.

[0016] In a second aspect, the present application provides a fermentation product, and the preparation method comprises: liquid fermentation of the above-mentioned Stenotrophomonas geniculata D963, taking the fermentation broth after the fermentation is completed, and obtaining the fermentation product.

[0017] In a third aspect, the present application provides a microbial agent, which comprises the above-mentioned Stenotrophomonas geniculata D963 or the above-mentioned fermentation product.

[0018] In a fourth aspect, the present application provides an application of the Stenotrophomonas geniculata D963, the fermentation product or the microbial inoculum in preventing and controlling the aflatoxin pollution.

[0019] In a fifth aspect, the present application provides an application of the Stenotrophomonas geniculata D963, the fermentation product or the microbial inoculum in inhibiting the aflatoxin or in preparing a product for inhibiting the aflatoxin.

[0020] Further, the aflatoxin includes aflatoxin B1 or / and aflatoxin B2.

[0021] In a sixth aspect, the present application provides a preparation for inhibiting the aflatoxin, which includes the Stenotrophomonas geniculata D963, the fermentation product or the microbial inoculum.

[0022] Further, the preparation further includes an adjuvant;

[0023] Preferably, the adjuvant includes at least one of a solvent, a co-solvent, an emulsifier, a wetting agent, a dispersing agent, a sticking agent or a stabilizer.

[0024] Preferably, the dosage form of the preparation includes at least one of a powder, a granule, a suspension, an oil, a fumigant, a slow-release agent, a seed coating agent, a microcapsule, an effervescent tablet or a spray.

[0025] In a seventh aspect, the present application provides a method for preventing and controlling the aflatoxin pollution, which includes applying a solution containing the Stenotrophomonas geniculata D963, the fermentation product, the microbial inoculum or the preparation to a prevention and control object.

[0026] Preferably, the prevention and control object includes a crop or / and an agricultural product.

[0027] Preferably, the application to the crop includes seed soaking treatment of the crop.

[0028] Preferably, the seed soaking treatment is for at least 10 hours.

[0029] Preferably, the seed is a seed for pregermination.

[0030] Preferably, the crop is a grain and oil crop.

[0031] Preferably, the grain and oil crop includes at least one of peanut, corn, rice or wheat.

[0032] Preferably, applying to the agricultural product comprises spraying to the agricultural product;

[0033] Preferably, the agricultural product is a storage period agricultural product;

[0034] Preferably, the agricultural product comprises at least one of a grain, tea leaves or cotton seeds.

[0035] The present application provides Stenotrophomonas geniculata D963, which can directly inhibit the production of aflatoxin without affecting the growth of Aspergillus flavus, is not prone to cause rapid spread of drug-resistant strains, has good prevention and control effect on aflatoxin, can effectively inhibit the formation of aflatoxin pollution in crops, and can be used for preparing an aflatoxin pollution inhibitor. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0037] Figure 1 Colony morphology of Stenotrophomonas geniculata D963 provided for Example 1 of the present application;

[0038] Figure 2 Phylogenetic tree of 16S rDNA sequence of Stenotrophomonas geniculata D963 provided for Example 1 of the present application and related model strains;

[0039] Figure 3 GyrB gene sequence phylogenetic analysis of Stenotrophomonas geniculata D963 provided for Example 1 of the present application and related model strains;

[0040] Figure 4 Comparison of prevention and control effects of different biocontrol bacteria on AFB1 and AFB2 pollution provided for Example 2 of the present application;

[0041] Figure 5 Comparison of detoxification effects of Stenotrophomonas geniculata D963 and Aspergillus flavus spores with different relative concentrations provided for Example 2 of the present application;

[0042] Figure 6 Comparison of prevention and control effects of sterile supernatant of different biocontrol bacteria on aflatoxin pollution provided for Example 3 of the present application;

[0043] Figure 7Stability of the sterile supernatant of Stenotrophomonas geniculata D963 provided in Example 3 of the present application;

[0044] Figure 8 Comparison of the aflatoxin contamination prevention and control effects of the fermentation products of Stenotrophomonas geniculata D963 obtained by different extraction methods provided in Example 4 of the present application. DETAILED DESCRIPTION

[0045] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meanings of the terms will be clear in view of the description, given the benefit of the present disclosure. For any potentially ambiguous

[0046] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods in the art and such methods and techniques are explained fully in the literature in the field. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Techniques, 4th Ed., John Wiley & Sons, Inc., New York (1999); and the series Methods in Enzymology, Academic Press, Inc., San Diego, CA.

[0047] The technical solutions of the present application will be described clearly and completely in combination with examples below. Obviously, the described examples are part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all the other examples obtained by those of ordinary skill in the art without making creative efforts are within the protection scope of the present application.

[0048] In one aspect of the present application, a Stenotrophomonas geniculata D963 is provided, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33555. The Stenotrophomonas geniculata D963 CGMCC No. 33555 is referred to as Stenotrophomonas geniculata D963 hereinafter.

[0049] Deposit Description

[0050] Strain name: Stenotrophomonas geniculata D963;

[0051] Latin name: Stenotrophomonas geniculata D963;

[0052] Depositary: China General Microbiological Culture Collection Center;

[0053] Abbreviation of the depositary: CGMCC;

[0054] Address: No. 1, Beichen Road, Haidian District, Beijing;

[0055] Date of deposit: February 18, 2025;

[0056] Collection number of the depositary: CGMCC No. 33555.

[0057] The strain is isolated from rice panicles, and research shows that, compared with antagonistic bacteria inhibiting the growth of Aspergillus flavus, the strain can directly inhibit the production of aflatoxin without affecting the growth of Aspergillus flavus and is not easy to cause rapid spread of drug-resistant strains; has good prevention and control effect on aflatoxin, can effectively inhibit the formation of aflatoxin pollution in crops, and can be used for preparing an aflatoxin pollution inhibitor.

[0058] According to another aspect of the present application, a fermentation product is also provided, and the preparation method comprises: liquid fermentation of the above-mentioned oligotrophic bacteria D963, and obtaining the fermentation product after the fermentation is completed.

[0059] In some specific embodiments, the liquid fermentation comprises inoculating the pre-cultured oligotrophic bacteria D963 bacterial liquid into a liquid culture medium for liquid fermentation. In some specific embodiments, the pre-culturing comprises inoculating an oligotrophic bacteria D963 single colony into a liquid culture medium for at least 24 hours of culture. In some specific embodiments, the inoculation amount of the pre-cultured oligotrophic bacteria D963 bacterial liquid into the liquid culture medium is 0.05% to 0.3%, preferably 0.2%.

[0060] The culture medium for culturing the oligotrophic bacteria D963 can be selected from a Sabouraud liquid culture medium.

[0061] Specifically, a single colony of the oligotrophic bacteria D963 can be vertically picked up by using an inoculation needle, and then placed in 50 ml of a Sabouraud culture medium for pre-culturing. After 24 hours of culture, 10 mL of bacterial liquid is aspirated, centrifuged at 8000 rpm for 5 min, and then the supernatant is discarded. Then, an equal amount of clean water is added for washing, and after shaking and mixing, the bacterial liquid is centrifuged at 5000 rpm for 5 min. Again, an equal amount of clean water is added and shaken and mixed, and then 100 μL of the mixed bacterial liquid is added to 50 mL of a SD liquid culture medium for culture. After completion, the culture medium containing the oligotrophic bacteria D963 is placed in a constant-temperature shaking incubator at 28°C for 4 days, and the fermentation liquid of the oligotrophic bacteria D963 can be obtained.

[0062] In some specific embodiments, the taking of the fermentation liquor further comprises extracting the fermentation product. In some specific embodiments, the extraction is by extractant extraction or macroporous resin adsorption method (MR) extraction; preferably, the extractant is selected from at least one of methanol, ethyl acetate (EA), dichloromethane (DCM) or petroleum ether (PET). The three organic solvents increase in polarity in turn, and have a wide range of polarity, which can ensure that the active secondary metabolites in the Stenotrophomonas D963 fermentation liquor are extracted. At the same time, the macroporous resin adsorption method (MR) can also be used to extract the Stenotrophomonas D963 secondary metabolites in a high-throughput and rapid manner, and the Stenotrophomonas D963 fermentation product can be obtained based on the MR extraction method.

[0063] According to another aspect of the present application, a microbial agent is also provided, comprising the Stenotrophomonas D963 or the fermentation product described above.

[0064] Since the Stenotrophomonas D963 and its fermentation product have good inhibitory effect on aflatoxins, the Stenotrophomonas D963, or the fermentation product thereof, or the microbial agent containing the Stenotrophomonas D963 or the fermentation product thereof can be used as an effective component for preventing and controlling aflatoxin pollution, or inhibiting aflatoxins, or preparing products for inhibiting aflatoxins.

[0065] According to another aspect of the present application, the Stenotrophomonas D963, the fermentation product or the microbial agent described above are also provided for use in preventing and controlling aflatoxin pollution.

[0066] According to another aspect of the present application, the Stenotrophomonas D963, the fermentation product or the microbial agent described above are also provided for use in inhibiting aflatoxins or preparing products for inhibiting aflatoxins.

[0067] In some specific embodiments, the aflatoxins comprise aflatoxin B1 (AFB1 toxin) or / and aflatoxin B2 (AFB2 toxin).

[0068] According to another aspect of the present application, a preparation for inhibiting aflatoxins is also provided, comprising the Stenotrophomonas D963, the fermentation product or the microbial agent described above.

[0069] In some specific embodiments, the preparation further comprises an adjuvant;

[0070] In some specific embodiments, the adjuvant comprises at least one of a solvent, a cosolvent, an emulsifier, a wetting agent, a dispersant, a sticking agent or a stabilizer;

[0071] In some specific embodiments, the dosage form of the preparation comprises at least one of a powder, a granule, a suspension, an oil, a fumigant, a slow-release agent, a seed coating agent, a microcapsule, an effervescent tablet or a spray.

[0072] According to another aspect of the present application, there is also provided a method for preventing and controlling aflatoxin contamination, comprising applying a solution containing the above-mentioned oligotrophic bacteria D963, the above-mentioned fermentation product, the above-mentioned bacterial agent or the above-mentioned preparation to a prevention and control object; which can effectively inhibit the content of aflatoxin in crops.

[0073] In some specific embodiments, the prevention and control object comprises crops or / and agricultural products.

[0074] Preferably, the grain and oil crops comprise at least one of peanuts, corn, rice or wheat. The agricultural products are agricultural products in storage period; preferably, the agricultural products comprise at least one of grains, tea leaves or cotton seeds.

[0075] In some specific embodiments, applying to crops comprises seed soaking treatment of seeds of crops.

[0076] In some specific embodiments, the seed soaking treatment time is at least 10 hours.

[0077] Specifically, the seed soaking treatment time can be, but is not limited to, 10 h, 11 h, 12 h, 13 h, 14 h, 16 h or any other time of more than 10 hours. In order to achieve a better prevention and control effect, in some specific embodiments, the seeds are seeds that have been pregerminated.

[0078] In some specific embodiments, applying to agricultural products comprises spraying on agricultural products.

[0079] In order to achieve a better prevention and control effect of aflatoxin contamination, in some specific embodiments, the concentration of oligotrophic bacteria D963 in the spraying liquid for spraying on agricultural products is 10 3 ~ 10 7 CFU / mL; or, the concentration of the fermentation product of oligotrophic bacteria D963 in the spraying liquid for spraying on agricultural products is 200 ~ 400 μg / mL.

[0080] Specifically, the concentration of oligotrophic bacteria D963 in the spraying liquid can be, but is not limited to, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL or 10 7 CFU / mL, and can also be 10 3 ~ 10 7The concentration of the oligotrophomonas fermentation product in the sprayed liquid can be, but is not limited to, 200 μg / mL, 240 μg / mL, 280 μg / mL, 300 μg / mL, 340 μg / mL, 380 μg / mL, or 400 μg / mL, or any concentration between 200 and 400 μg / mL.

[0081] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0082] Sabouraud broth: 10.0g peptone, 40.0g glucose, dissolved in distilled water and brought to a final volume of 1L, sterilized at 121°C for 20 minutes. Add 2% agar when preparing solid Sabouraud broth.

[0083] Aspergillus flavus CGMCC No. 3.4408.

[0084] The biocontrol bacteria (Aspergillus cvjetkovicii, Trichoderma virens PS1-7, Bacillus subtilis, B. velezensis, B. licheniformis, Stenotrophomonas geniculate D963, Pseudomonas mosselii S3-F1, P. mosselii S3-F2, P. hibiscicola, Sphingomonasmelonis, and Streptomyces N201) were obtained from the biocontrol bacteria library constructed by the Laboratory of Plant-Microbial Interaction Molecular Ecology, Institute of Pesticides and Environmental Toxicology, Zhejiang University.

[0085] Example 1

[0086] 1. Isolation and purification of strain D963: Rice panicle samples were collected in August 2021. After rinsing with sterile water, the panicles were ground in a sterile mortar with sterile water. 2 ml of the grinding solution was diluted 2 times, 5 times and 10 times respectively. 100 μL of the diluted solution was spread on LB medium and cultured at 28℃ for 3 days. Multiple colonies grew on the plates. Based on different colors and morphologies, single colonies were obtained after three streak purifications.

[0087] 2. Identification of strain D963:

[0088] (a) Morphological characteristics: such as Figure 1 As shown, strain D963 forms pale yellow, round, smooth, raised, and opaque single colonies on Sabouraud dextrose agar.

[0089] (II) 16S rRNA gene analysis: The 16S rRNA gene sequence was used for species identification. The primers used for PCR amplification were 27F / 1492R, and a 1465 bp gene sequence was obtained (the nucleotide sequence is shown as SEQ ID NO: 1). Based on the standard strain sequence in the NCBI database, the homology was analyzed by sequence alignment, and the phylogenetic tree was constructed by MEGA11 software, to further determine the species attribution of each isolated strain. The phylogenetic analysis was calculated by maximum likelihood method (ML) and GTR model, and the reliability of the phylogenetic relationship was evaluated by Bootstrap support rate. The constructed phylogenetic tree is shown in FIG. 1. Figure 2

[0090] (III) gyrB gene analysis: The gyrB gene sequence was used for species identification. The primers used for PCR amplification were 5'-ATGAGCGACGAACAGAACACC-3' (SEQ ID NO: 3) and 5'-TCAGATATCCAGGTTTGCGACC-3' (SEQ ID NO: 4), and a 2366 bp gene sequence was obtained (the nucleotide sequence is shown as SEQ ID NO: 2). Based on the standard strain sequence in the NCBI database, the homology was analyzed by sequence alignment, and the phylogenetic tree was constructed by MEGA11 software, to further determine the species attribution of each isolated strain. The phylogenetic analysis was calculated by maximum likelihood method (ML) and GTR model, and the reliability of the phylogenetic relationship was evaluated by Bootstrap support rate. The constructed phylogenetic tree is shown in FIG. 2. Figure 3

[0091] Based on the morphological characteristics and gene sequence characteristics, strain D963 was identified as Stenotrophomonas geniculata.

[0092] Example 2: Effect of strain D963 on the prevention and control of aflatoxin production by Aspergillus flavus

[0093] 1. Constructing a co-culture system of biocontrol bacteria and Aspergillus flavus: The spore suspension of Aspergillus flavus 3.4408 strain was taken out from the refrigerator and activated. When the mycelium on the plate grew to 6 cm, a 5 mm fungus cake was punched out with a puncher and inoculated into 50 mL of SD medium, and at the same time, 50 microliters of activated biocontrol bacterial liquid was inoculated.

[0094] ​​Aspergillus cvjetkovicii, Trichoderma virens PS1-7, Bacillus subtilis, B. velezensis, B. licheniformis, Stenotrophomonasgeniculate D963, Pseudomonas mosselii S3-F1, P. mosselii S3-F2, P. hibiscicola, Sphingomonas melonis and Streptomyces N201, and mix of T. virens PS1-7, Stenotrophomonasgeniculate D963 and P. hibiscicola were used as biocontrol agents. Meanwhile, a control group was set up, using metabolites instead of biocontrol agents, and the metabolite selected was 100 ppm of 2,4-di-tert-butylphenol (DTBP). After inoculation, it was incubated at 28°C, 150 rpm for four days.

[0095] 2. TLC detection of aflatoxin production: The culture solution of the biocontrol agent and aflatoxin co-culture system constructed in step 1 was taken out and centrifuged at 12000 rpm for 10 min, and the supernatant was filtered with filter cloth to remove the mycelium. The supernatant was extracted with ethyl acetate, and a 3-micron pore size capillary was used to spot on the silica gel plate. At the same time, 25 μg / mL of aflatoxin AFB1, AFB2, AFG1 and 3 μg / mL of AFG2 standard solution were prepared. Then, the developing agent of chloroform: acetone = 9: 1 was run to the distance of 0.5 cm on the silica gel plate, and after the silica gel plate was dried, the aflatoxin production was observed under 365 nm ultraviolet light. The biocontrol agent with dim or no fluorescence of aflatoxin AFB1 was selected as the biocontrol agent with inhibitory effect on toxin production, and the results are shown in Figure 4

[0096] From the results of Figure 4 , after 48h, 72h and 96h of co-culture, compared with Aspergillus flavus (CK), it was found that T. virens PS1-7, P. hibiscicola and Stenotrophomonasgeniculate D963 and mix had relatively obvious inhibitory effect, among which Stenotrophomonasgeniculate D963 had the best inhibitory effect on toxin, and P. mosselii S3-F2 had certain inhibitory effect at 48h and 72h.

[0097] ​3. Effect of the relative concentrations of Aspergillus flavus spores (Af) and strain D963 (Sg) on ​​the inhibitory effect in the co-culture system: 100 μL of 5 × 10⁻⁶ strains were mixed with 100 μL of 5 × 10⁻⁶ strains. 6 Add 10 mL of Sabouraud dextrose agar to a spore suspension of spores / mL, then add 10 μL of a 5 × 10⁻⁶ mcg solution. 6 CFU / mL, 5×10 7 CFU / mL, 5×10 8 CFU / mL, 5×10 9 CFU / mL of D963 bacterial culture was co-cultured with Aspergillus flavus spores. Treatments with Af:Sg ratios of 10:1, 1:1, 1:10, and 1:100 were obtained. Results are shown below. Figure 5 As shown, A represents the growth of the co-culture system under different treatments, and B represents the detection by thin-layer chromatography.

[0098] from Figure 5 The results showed that when the ratio of Aspergillus flavus 3.4408 spores to the number of colonies of strain D963 was less than 1:1, the inhibitory effect on aflatoxin was significantly enhanced.

[0099] Example 3: Control effect of sterile supernatant on Aspergillus flavus

[0100] In this embodiment, Aspergillus flavus was cultured in the fermentation supernatant of a mixture of Trichoderma virens PS1-7, P. hibiscicola, Stenotrophomonas geniculate D963, and Trichoderma virens PS1-7, P. hibiscicola, Stenotrophomonas geniculate D963, and P. mosselii S3-F2, which showed good inhibitory effects on toxins, to compare the inhibitory effects of different fermentation products on Aspergillus flavus.

[0101] 1. Preparation of sterile supernatant: A single colony was vertically picked using an inoculation needle and pre-cultured in 50 ml of Sabouraud dextrose agar for 24 hours. 10 mL of the bacterial culture was then centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and an equal volume of water was added for washing. The mixture was then vortexed and centrifuged again at 5000 rpm for 5 min. An equal volume of water was added again and vortexed. 100 μL of the mixed bacterial culture was added to 50 mL of SD liquid medium for further incubation. After incubation, the culture medium containing *Oligotrophomonas* was incubated at 28°C with shaking for 4 days to obtain the fermentation broth. The supernatant was then centrifuged at 12000 rpm for 10 min and filtered through a 0.22 μm microporous membrane to obtain a sterile supernatant containing the fermentation products.

[0102] 2. Comparison of Aspergillus Inhibition Effects

[0103] Four fermentation supernatants prepared according to step 1 were used to culture Aspergillus flavus 3.4408 for four days to obtain co-culture solutions. The thin-layer chromatography method described in Example 2 was used to process and detect the production of aflatoxin in the co-culture solutions. The results are as follows: Figure 6 As shown, A: Aspergillus flavus 3.4408 bacterial culture, T: Trichoderma virens PS1-7 bacterial culture, Ph: P. hibiscicola bacterial culture, Sg: Stenotrophomonas geniculate D963 bacterial culture, M: co-culture of Trichoderma virens PS1-7, P. hibiscicola, and Stenotrophomonas geniculate D963, Tm: sterile supernatant from co-culture of A and T, Pm: sterile supernatant from co-culture of A and Ph, Sm: sterile supernatant from co-culture of A and Sg, and Mm: sterile supernatant from co-culture of A and M.

[0104] from Figure 6 The results showed that for *Trichoderma virens* PS1-7, A+T had an inhibitory effect on aflatoxin, but A+Tm showed no effect, indicating that the inhibitory effect of this bacterium on aflatoxin did not originate from Tm. For *P. hibiscicola*, different treatments showed poor inhibitory effects on aflatoxin. For *Stenotrophomonasgeniculate* D963, compared with A, the bands of A+Sg, Sm, and A+Sm were all weaker, indicating that the *Stenotrophomonasgeniculate* D963 bacterial culture and its sterile supernatant containing fermentation products could inhibit aflatoxin production. For co-cultured bacterial cultures, A+M, Mm, and A+Mm all achieved high inhibitory effects, indicating that the bacterial culture or sterile supernatant of strain D963, or the co-culture culture or sterile supernatant of mixed strains containing strain D963, could inhibit the production of aflatoxin by *Aspergillus flavus*.

[0105] 2. Stability testing

[0106] The fermentation supernatant of strain D963 obtained in step 1 was sterilized at 121°C and then cultured with Aspergillus flavus 3.4408 for four days to obtain a co-culture solution. The solution was then processed using the thin-layer chromatography method described in Example 2 to detect the production of aflatoxin. The results are as follows: Figure 7As shown, compared with Af, the band after sterilization still presents weakening, which indicates that the activity after sterilization is slightly reduced compared with before sterilization, but still has certain aflatoxin inhibition effect and has certain chemical stability. Therefore, the strain D963 and the fermentation product thereof have application prospect in preparation of aflatoxin pollution inhibitor.

[0107] Example 4: Prevention and control effect of the fermentation product of the strain D963 on Aspergillus flavus

[0108] In this example, the fermentation supernatant of the strain D963 prepared by the method in Example 3 was extracted with ethyl acetate (EA), dried, concentrated and dissolved in 200 μL of DMSO solution to obtain the fermentation product of the strain D963 (Sg metabolite), which was added to 50 mL of liquid SD medium to obtain the medium containing the Sg metabolite extracted by EA. At the same time, 50 mL of the fermentation supernatant of the strain D963 prepared by the method in Example 3 was taken as the medium containing the Sg metabolite without extraction. Aspergillus flavus 3.4408 was inoculated into the SD medium containing the Sg metabolite without extraction and the SD medium containing the Sg metabolite extracted by EA, respectively, and the co-cultured bacteria liquid on the 4th day and the 6th day was taken, which was treated by the thin layer chromatography method in Example 2 and the aflatoxin production in the co-cultured bacteria liquid was detected.

[0109] The results are shown in Table 2. Figure 8 As shown in Table 2, A: Aspergillus flavus 3.4408 and the medium without Sg metabolite, Sm: A and the medium containing Sg metabolite, from 1, 2, 3 three repeated groups, it can be seen that the fermentation product of the strain D963 extracted by ethyl acetate has certain inhibition effect on aflatoxin, but the sterile supernatant without extraction has the best inhibition effect on aflatoxin.

[0110] In summary, the stenotrophomonas and the fermentation product thereof can be used for prevention and control of crop aflatoxin pollution, and can be mainly used for preparation of aflatoxin pollution inhibitor of crops such as peanuts and corns. The preparation of the embodiments of the present application can be applied to grain storage to prevent and control aflatoxin pollution.

[0111] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A strain of Stenotrophomonas hollisii (S. hollisii) D963, characterized in that, Stenotrophomonas geniculata )D963, characterized in that, The oligella knee (Oligella knee) Stenotrophomonas geniculata D963 is preserved in China Microbial Culture Collection Center, and the preservation number is CGMCC No. 33555.

2. The Leptospirillum bacteria of claim 1, wherein the Leptospirillum bacteria is Leptospirillum genus bacteria. Stenotrophomonas geniculata ) D963, characterized in that, The Stenoloma oligotrophica (S. oligotrophica) Stenotrophomonas geniculata D963 is derived from a rice panicle.

3. A fermentation product, characterized in that, The preparation method comprises the following steps: subjecting the oligella kuehneri (O. Stenotrophomonas geniculata ) D963 in claim 1 or 2 to liquid fermentation, taking the fermentation liquor after the fermentation is completed, and obtaining a fermentation product.

4. A bacterial agent characterized in that, The use of a fermentation product according to claim 3 or 4 for the production of a pharmaceutical composition for the treatment of a disease or disorder caused by a bacterial infection. Stenotrophomonas geniculata ) D963 or a fermentation product according to claim 3.

5. The Leptospirillum genus as claimed in claim 1 or 2, wherein the Leptospirillum genus is Leptospirillum kihistum. Stenotrophomonas geniculata ) D963, the application of the fermentation product as claimed in claim 3 or the microbial agent as claimed in claim 4 in preventing and controlling aflatoxin pollution.

6. The S. celeris of claim 1 or 2, Stenotrophomonas geniculata ) D963, the use of the fermentation product of claim 3 or the microbial agent of claim 4 in the manufacture of a product for inhibiting aflatoxins.

7. Use according to claim 5 or 6, characterized in that, The aflatoxin includes aflatoxin B1 or / and aflatoxin B2.

8. An aflatoxin-inhibiting formulation, characterized by, The Stenotrophomonas geniculata D963 of claim 1 or 2, the fermentation product of claim 3, or the microbial inoculant of claim 4 are further included.

9. The formulation of claim 8, wherein, An adjuvant is further included.

10. The preparation according to claim 9, characterized in that The adjuvant includes at least one of a solvent, a co-solvent, an emulsifier, a wetting agent, a dispersing agent, a sticking agent, or a stabilizer.

11. The formulation of claim 8, wherein, The dosage form of the preparation includes at least one of a powder, a granule, a suspension, an oil, a fumigant, a slow-release agent, a seed coating agent, a microcapsule, an effervescent tablet, or a spray.

12. A method for preventing and controlling aflatoxin contamination, characterized by, including the administration of a solution of the oligella kioshii (Oligella kioshii) of claim 1 or 2, Stenotrophomonas geniculata ) D963, the fermentation product of claim 3, the microbial agent of claim 4, or the preparation of claims 8 to 11 to the prevention and control object.

13. The method of claim 12, wherein, The control object includes a crop or / and an agricultural product.

14. The method of claim 13, wherein, The application to the crop includes seed soaking treatment of the crop.

15. The method of claim 14, wherein, The seed soaking treatment is for at least 10 hours.

16. The method of claim 14, wherein, The seed is a pregerminated seed.

17. The method of claim 13, wherein, The crop is a grain and oil crop.

18. The method of claim 17, wherein, The grain and oil crop includes at least one of peanut, corn, rice, or wheat.

19. The method of claim 13, wherein, The application to the agricultural product includes spraying to the agricultural product.

20. The method of claim 19, wherein, The agricultural product is a storage period agricultural product.

21. The method of claim 19, wherein, The agricultural product includes at least one of a cereal, tea, or cottonseed.

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