Bacillus amyloliquefaciens W1 and application of bacillus amyloliquefaciens W1 in prevention and treatment of tobacco mildew

Abstract

The invention discloses bacillus amyloliquefaciens W1 and application thereof in preventing and treating tobacco mildew, and belongs to the field of microorganisms. The bacillus amyloliquefaciens W1 is preserved in the China Center for Type Culture Collection on September 30, 2024, and the preservation number of the bacillus amyloliquefaciens W1 is CCTCC (China Center for Type Culture Collection) NO: M20242138. The bacillus amyloliquefaciens W1 provided by the invention has an ideal prevention and treatment effect on tobacco mildew caused by aspergillus tubingensis, talaromyces funosus, aspergillus niger and aspergillus flavus, and the mycelium form of the mildew is destroyed by generating various lipopeptide substances, so that the antibacterial function is realized. Microbial resources and reference basis are provided for tobacco storage mildew prevention and control, and a theoretical foundation is laid for further exploration of a biological prevention and control mechanism of bacillus.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Bacillus amyloliquefaciens W1 and application thereof in preventing and controlling tobacco leaf mildew. Background Art

[0002] Tobacco is an important cash crop, and my country is the world's largest tobacco producer and consumer. High-quality tobacco leaves are the core raw materials of cigarettes and are vital to cigarette companies, but their coordinated nutritional components create favorable conditions for the mildew of stored tobacco leaves. It is reported that about 12.9% of flue-cured tobacco in my country cannot be used for cigarette manufacturing every year due to fungal corruption in storage. Tobacco mildew is an infectious disease caused by fungi, which can affect the appearance and intrinsic quality of tobacco leaves and cause serious economic losses. Once tobacco leaves produce mold, the fungi will quickly absorb nutrients, destroy their tissue structure, and excrete blue, green and black pigments, releasing unpleasant odors. In addition, as fungi grow, they will secrete various mycotoxins and allergens into tobacco leaves, including aflatoxin, patulin and ochratoxin, sphingosine-like mycotoxins, sphingosine-like mycotoxins, posing a threat to human health. At present, Aspergillus, Mucor and Alternaria are isolated from moldy tobacco leaves with a high frequency. Zhu Guining found that Aspergillus microorganisms are the main microorganisms that cause tobacco leaves to mildew in Guangxi warehouses. Zhou et al. studied tobacco leaves with mildew in Jilin, Guizhou and Shanghai and found that the dominant fungal groups were Aspergillus, Xerostomyces and Basidiomyces. The above studies show that the dominant fungi of tobacco leaves mildew in different regions are different.

[0003] The prevention and control of tobacco leaf mildew is currently achieved through a variety of methods, such as chemical control, physical control and biological control. However, due to health and environmental safety issues, the use of chemical agents is limited; physical control is not popular due to its high cost and cumbersome operation. Therefore, the use of sustainable biological control strategies is an emerging research field and has become an important research direction for tobacco leaf mildew prevention. At present, the research on antagonistic bacteria for tobacco leaf mildew is still in its preliminary stage, and the antagonistic mechanism involved has not been fully understood. In addition, the dominant molds of tobacco leaf mildew in different regions are different. Therefore, it is of great significance to explore excellent strains with biological activity against different tobacco leaf mildew pathogens and clarify the biological control mechanism of the strains, so as to solve the problem of tobacco leaf mildew by biological control and improve the quality of tobacco leaves in different regions. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Bacillus amyloliquefaciens W1 and its application in preventing and controlling tobacco leaf mildew, so as to solve the problems existing in the above-mentioned prior art. The Bacillus amyloliquefaciens W1 provided by the present invention has an ideal prevention and control effect on tobacco leaf mildew caused by Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger and Aspergillus flavus, and destroys the mycelial morphology of the mold by producing a variety of lipopeptide substances, thereby achieving the function of antibacterial. The present invention provides microbial resources and reference basis for the prevention and control of stored tobacco mildew, and also lays a theoretical foundation for further exploring the biological control mechanism of Bacillus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Bacillus amyloliquefaciens W1, which was deposited in the China Center for Type Culture Collection on September 30, 2024, with a deposit number of CCTCC NO: M20242138.

[0007] The present invention also provides a microbial preparation, the active ingredients of which include the Bacillus amyloliquefaciens W1 and / or its metabolites.

[0008] Furthermore, the concentration of the Bacillus amyloliquefaciens W1 is 10 7 ~10 9 CFU / mL.

[0009] Furthermore, the metabolites of Bacillus amyloliquefaciens W1 are lipopeptide substances; the lipopeptide substances include Bacilysin, C16 FengycinA and C14 Iturin B.

[0010] The present invention also provides the use of the Bacillus amyloliquefaciens W1 or the microbial preparation in preventing and treating tobacco leaf mildew disease, wherein the pathogens of the tobacco leaf mildew disease include Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger and Aspergillus flavus.

[0011] Furthermore, the tobacco leaves are tobacco leaves in storage.

[0012] The present invention also provides a method for preventing and treating tobacco leaf mildew, comprising the steps of applying the microbial preparation to tobacco leaves;

[0013] The pathogenic bacteria of tobacco leaf mildew disease include Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger and Aspergillus flavus.

[0014] Furthermore, the tobacco leaves are tobacco leaves in storage.

[0015] The present invention discloses the following technical effects:

[0016] The present invention isolates and screens a strain of Bacillus amyloliquefaciens W1 from tobacco leaves in storage in Guiyang, Guizhou Province. The strain has a highly effective antagonistic effect on various molds such as Aspergillus tubingensis, Talaromyces fusca, Aspergillus niger and Aspergillus flavus, and can be used for the prevention and control of tobacco mildew during the storage stage. At the same time, the present invention has found through experiments that the strain destroys the mycelial morphology of the mold by producing a variety of lipopeptide substances, thereby achieving an antibacterial function. In vitro experimental results show that the Bacillus amyloliquefaciens W1 provided by the present invention has an ideal prevention and control effect on tobacco mildew caused by Aspergillus tubingensis, Talaromyces fusca, Aspergillus niger and Aspergillus flavus.

[0017] The present invention provides microbial resources and reference basis for the prevention and control of stored tobacco mildew, and also lays a theoretical foundation for further exploring the biological control mechanism of Bacillus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The morphological characteristics and phylogenetic relationships of dominant molds; (A) is the morphological characteristics of pathogen colonies on PDA medium; (B) is the phylogenetic tree of pathogens constructed based on ITS sequences using MEGA 11 software;

[0020] Figure 2 The antibacterial activity of antagonistic bacteria W1 against four fungi; A is the initial screening result of the plate confrontation method; B is the re-screening result of the Oxford cup method; in A and B, the control group is in the first row; the W1 treatment group is in the second row;

[0021] Figure 3 This is the phylogenetic tree of antagonistic bacteria W1 constructed based on ITS sequences using MEGA 11 software;

[0022] Figure 4 This is a graph showing the effect of the fermentation liquid of antagonistic bacteria W1 on the prevention and treatment of tobacco leaf mildew;

[0023] Figure 5 The antibacterial activity of crude lipopeptide of antagonistic bacteria W1 against mold; A is treated with clean water; B is treated with lipopeptide;

[0024] Figure 6Total ion chromatogram of lipopeptides generated by W1 for UPLC-MS / MS identification;

[0025] Figure 7 Mass spectrum of Bacilysin in crude lipopeptide extract produced by W1 for UPLC-MS / MS identification;

[0026] Figure 8 Mass spectrum of C16 FengycinA in crude lipopeptide extract produced by W1 for UPLC-MS / MS identification;

[0027] Fig. 9 Mass spectrum of C14 Iturin B in crude lipopeptide extract produced by W1 for UPLC-MS / MS identification;

[0028] Fig.10 The results are scanning electron microscopy analysis of the inhibitory effect of lipopeptides on the mycelium growth of pathogenic fungi; A is treated with clean water; B is treated with lipopeptides.

[0029] Biological Deposit

[0030] Bacillus amyloliquefaciens W1.

[0031] Classification and nomenclature: Bacillus amyloliquefaciens;

[0032] Deposit number: CCTCC NO: M20242138;

[0033] Preservation time: September 30, 2024;

[0034] Depository: China Center for Type Culture Collection;

[0035] Deposit address: Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example

[0042] 1. Materials and Methods

[0043] 1. Materials

[0044] Tobacco leaf samples: The moldy tobacco leaf samples came from the moldy tobacco samples found during the storage period in Tongren, Qiannan, Bijie and Guiyang, the main tobacco producing areas in Guizhou. After sampling, they were placed in sterile bags and then transported to the laboratory for subsequent testing.

[0045] Culture medium: Red Bengal medium was used for the isolation and purification of pathogens; potato dextrose agar (PDA) medium was used for the culture of molds; LB solid and liquid medium were used for the culture of antagonistic bacteria.

[0046] 2. Isolation, identification and pathogenicity testing of pathogenic bacteria

[0047] Weigh 15 g of moldy tobacco leaves and add them to 85 mL of sterile water. Incubate them in a shaking incubator at 180 rpm and 28°C for 1 h. Take 1 mL of the culture supernatant and perform gradient dilutions (10 -4 ~10 -6 ) and spread on Bengal rose medium, culture at 28℃ for 5-7 days, select strains with different morphology and vigorous growth for isolation, repeat 3-4 times. Then use PDA medium to culture dominant fungi.

[0048] Fungal genomic DNA was extracted using a rapid fungal genomic DNA extraction kit (Shenggong, catalog number: B518229), and PCR amplification was performed using ITS universal primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO.1) and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO.2). The reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 2 min, 30 cycles, and finally extension at 72°C for 5 min. The obtained gene sequences were compared with other fungal sequences in the National Center for Biotechnology Information (NCBI) database, and phylogenetic analysis was performed using the neighbor-joining method using the MEGA 11 software (MEGA 11, Temple University, Philadelphia, PA, USA) system.

[0049] 3. Isolation, screening and identification of antagonistic Bacillus strains

[0050] 15 g of healthy tobacco leaves were aseptically inoculated into 85 mL of sterile saline, shaken at 180 rpm for 20 min at 32 °C, and heated in a water bath at 80 °C for 15 min. Then 150 μL of the liquid was transferred into 5 mL of Luria-Bertani (LB) broth and cultured at 180 rpm for 12 h at 32 °C. The culture was diluted to 10 -3 , 10 -4 and 10 -5 , then inoculated on LB medium and cultured at 32℃ for 12h. Single colonies were selected from the plate and numbered individually for preliminary screening of antagonistic bacteria. The antifungal activity of antagonistic bacteria against each dominant fungus was evaluated by plate confrontation method. Antagonistic bacteria with better antibacterial effect were selected for Oxford cup rescreening and subsequent control tests, and named W1.

[0051] The genomic DNA of the selected strains was extracted by DNA extraction kit (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China) and used as template for PCR amplification using universal primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO.3) and 1492R: 5′-GGTTACCTTGTTAGACTT-3′ (SEQ ID NO.4). The PCR product was sent to Science Compass for sequencing. BLAST was used to analyze the sequence similarity of the 16S rDNA gene sequence, and the phylogenetic tree was constructed using the neighbor-joining method using MEGA11.0 software.

[0052] 4. Observation of the antagonistic effect of strain W1 on dominant fungi using scanning electron microscopy

[0053] Observe the effect of antagonistic bacteria on the mycelial morphology of mold. The antagonistic bacteria and mold were cultured on a plate for 5 days, and the control group was treated with an equal amount of sterile water instead. The samples were fixed in a 4% glutaraldehyde solution for 24 hours, washed three times with phosphate buffer, and dehydrated in an ethanol aqueous solution (ethanol volume fractions were 50%, 70%, 85%, 95% and 100%), and then the samples were dried in a critical point dryer under CO2 and sprayed with gold. Finally, the samples were examined using SEM (SU8100, Hitachi, Japan). Each group of treatments was repeated three times.

[0054] 5. Effect of antagonistic bacteria W1 fermentation liquid on control effect

[0055] Use the foliar spraying method. After sterilizing 50g of tobacco leaves, spray 10mL of mixed fungal spore suspension (spore concentration is 10 5 CFU / mL), and then sprayed with 10mL of antagonistic bacteria fermentation liquid as the treatment group, a total of 3 treatments: (a) spraying 10 7 CFU / mL antagonistic bacteria fermentation broth; (b) spray 10 8 CFU / mL antagonistic bacteria fermentation liquid; (c) spray 10 9 CFU / mL antagonistic bacteria fermentation liquid. First spray 10mL of mixed fungus spore suspension, and then spray 10mL of clean water as the control group. After culturing at 28℃ and 75% relative humidity for 20 days, observe the degree of tobacco leaf mildew, and repeat the experiment 3 times.

[0056] 6. Study on the control mechanism of antagonistic bacteria W1 on tobacco leaf mildew

[0057] 6.1. Extraction and antifungal activity of antagonistic bacterial W1 lipopeptide

[0058] The acid precipitation method was used to extract lipopeptides from the supernatant of the fermentation broth of strain W1. The specific method was as follows: the fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 min, the supernatant was adjusted to pH 2 with hydrochloric acid, and precipitated overnight. Then, the crude lipopeptide precipitate was harvested by centrifugation at 10,000 rpm for 20 min at 4°C. Subsequently, methanol was added to dissolve the precipitate, and the precipitate was extracted with a Buchner funnel, and finally rotary evaporated and freeze-dried to obtain a crude extract of antibacterial lipopeptides.

[0059] The Oxford cup antibacterial method was used to test the antifungal activity. The activated dominant fungal strain was inoculated into the center of the PDA medium. An Oxford cup was placed 2 cm away from the center of the medium. The crude lipopeptide extract (concentration 100 mg / L) was added into the medium. The culture was carried out at 28°C for 7 days. Each group of tests was repeated three times. The colony diameter was measured and the antibacterial rate was calculated.

[0060] 6.2. UPLC-MS Analysis of Antimicrobial Substances

[0061] The identification of lipopeptides was performed by ultra-high performance (UP) LC-QTOF mass spectrometry (MS). Chromatographic column: Waters HSST3 (100×2.1 mm, 1.8 μm); mobile phase: phase A is ultrapure water solution (containing 0.1% formic acid), phase B is acetonitrile solution (containing 0.1% formic acid); flow rate 0.3 mL / min; column temperature 40°C; injection volume 2 μL; elution gradient: 0 min phase A / phase B (100:0, v / v), 1 min phase A / phase B (100:0, v / v), 12 min phase A / phase B (5:95, v / v), 13 min phase A / phase B (5:95, v / v), 13.1 min phase A / phase B (100:0, v / v), 17 min phase A / phase B (100:0, v / v). An autosampler was used for injection of 1 μL. MS analysis was performed using a Q Exactive HFX high-resolution mass spectrometer with an electrospray ionization source. The analysis was performed in positive ion mode in the mass range of 100-1600 Da. The source temperature and capillary voltage were set to 150°C and 3.0 kV, respectively. Argon (Ar) was used as collision gas.

[0062] 7. Data Analysis

[0063] SPSS 27.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. LSD (least significant difference method) with P < 0.05 was used for analysis of variance (ANOVA). All experiments were repeated 3 times, and the results were expressed as mean ± standard deviation. Origin 2022 software was used for drawing.

[0064] 2. Results

[0065] 1. Identification and pathogenicity test of dominant fungi

[0066] Four fungi were isolated from moldy tobacco leaves and named M-1, M-2, M-3 and M-4. The fungi were isolated and purified, and the characteristics of colony culture were observed after purification, such as Figure 1As shown in (A). The colonies of strain M-1 were white at first, turned black after 5 days of culture, and had semi-hairy edges; strain M-2 grew faster, and began to appear white and fluffy, then gradually turned green, and the surface of the colonies was powdery; strain M-3 had a loose texture and spread rapidly, initially white, then turned yellow-green, and finally turned black; strain M-4 had a loose texture and grew relatively fast, initially white, and gradually turned yellow. According to the sequence alignment with the NCBI database, the isolated mold ITS sequences (Gene Bank accession numbers PQ135063-PQ135066) showed 100%, 99.46%, 100% and 100% homology with Aspergillus tubingensis (MH185806.1), Talaromyces funiculosus (MT940763.1), Aspergillus niger (ON365676.1) and Aspergillus flavus (OR534538.1), respectively. The phylogenetic tree was constructed using the neighbor-joining (NJ) method, and the results are shown in Figure 2. Figure 1 (B), strains M-1, M-2, M-3, and M-4 clustered with Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger, and Aspergillus flavus, respectively. Therefore, the morphological identification combined with the phylogenetic tree results identified the four dominant fungi as Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger, and Aspergillus flavus.

[0067] 2. Screening and identification of antagonistic bacteria W1

[0068] In the present invention, a total of 65 candidate strains were screened from the surface of healthy tobacco leaves, and the antagonistic activity of these strains against 4 dominant fungi was preliminarily detected by plate confrontation test. Among them, strain W1 had the highest antibacterial activity against the 4 dominant fungi at the same time, with inhibition rates of 64.91%, 61.75%, 51.14% and 76.19% respectively. Figure 2 A). The strain W1 was screened again using the Oxford cup method. The antifungal activity of strain W1 against four fungi in vitro was determined by measuring the diameter of the inhibition zone after strain W1 treated the fungi to verify its potency and stability in passage. The results are shown in Figure 2 The diameters of the inhibition zones of B against the four fungi were 24.1, 22.2, 23.4 and 22 mm respectively.

[0069] The BLAST analysis results of the 16S rRNA gene sequence (1451 bp, GenBank accession number: PQ135968) showed that the homology between strain W1 and Bacillus amyloliquefaciens (OM541330.1) was 100%. The phylogenetic tree based on the 16S rRNA gene sequence was clustered with Bacillus amyloliquefaciens (OM541330.1) using the neighbor-joining (NJ) method. Figure 3 ). Therefore, strain W1 was identified as Bacillus amyloliquefaciens W1.

[0070] 3. Effect of strain W1 concentration on control effect

[0071] The in vitro leaf protection test of strain W1 showed that 5 days after the leaves were inoculated with pathogens, different concentrations of W1 suspension had a significant inhibitory effect on the growth of fungi. The inhibitory effect increased significantly with the increase of concentration. Figure 4 As shown in the figure, after the mixed bacterial liquid was inoculated on the surface of CK and sprayed with clean water for 20 days, a large number of molds grew and covered the surface of the flue-cured tobacco, causing the tobacco leaves to rot. After the W1 bacterial suspension was used for prevention and treatment, the concentration of the bacterial suspension was 10 9 The treatment with CFU / mL had the best effect, with no mildew spots on the tobacco leaf surface and no odor; the bacterial suspension was diluted 10 times and 100 times (10 8 CFU / mL and 10 7 CFU / mL) also has a good preventive effect on mildew. The tobacco leaves are not rotten and almost no obvious mildew spots can be seen.

[0072] 4. Antifungal activity of crude lipopeptide from strain W1

[0073] The crude lipopeptide of strain W1 was extracted by acid precipitation method, and its antibacterial effect on 4 fungi was as follows: Figure 5 As shown, Figure 5 The results showed that the crude lipopeptide of strain W1 could significantly inhibit the growth of four fungi, with the inhibition rates of Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger and Aspergillus flavus being 46.7%, 59.26%, 66.37% and 45.56%, respectively.

[0074] 5. Lipopeptide identification

[0075] The present invention uses HPLC-MS to perform qualitative mass spectrometry analysis on the crude lipopeptide extract of strain W1, and combines the peaks ( Figure 6) showed that the crude extract contained three components, and each component was then analyzed by UPLC-Triple TOF-MS. Figures 7 to 9 Further analysis of the results in Table 1 revealed an ion peak at m / z 271.06, corresponding to the ion form [M+H] + The molecular weight is 270.06, which is consistent with the molecular weight of the dipeptide antibacterial substance Bacilysin; there is an ion peak at the m / z value of 732.4, and the corresponding ion form is [M+2H] 2+ The molecular weight is 1462.04, which is consistent with the molecular weight of the cyclic lipopeptide Fengycin A-C16; there is an ion peak at the m / z value of 1044.53, and the corresponding ion form is [M+H] + The molecular weight was 1043.53, which was consistent with the molecular weight of the cyclic lipopeptide Iturin B-C14. Therefore, it was inferred that strain W1 produced three antimicrobial peptides: Bacilysin, C16 FengycinA and C14 Iturin B, achieving the biocontrol effect on tobacco leaf mildew.

[0076] Table 1 Identification of lipopeptides of Bacillus amyloliquefaciens W1 by UPLC-QTOF-MS

[0077]

[0078] 6. Scanning electron microscopy (SEM) observation of the effect of lipopeptides on fungal hyphae

[0079] The morphology of mycelium of each fungus after lipopeptide treatment was observed by scanning electron microscopy. Fig.10 As shown in the figure, the mycelia of the four molds in the water control group were round and full, with complete morphological structure; the mycelia of the four molds treated with strain W1 showed serious twisting, wrinkling, collapse or deformation, and disintegration occurred. It can be seen that the lipopeptides produced by the fermentation of strain W1 can destroy the mycelial morphology of each mold, thereby achieving an antibacterial effect.

[0080] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens W1, characterized in that: The strain was deposited in the China Center for Type Culture Collection on September 30, 2024, with the deposit number CCTCC NO: M20242138.

2. A microbial preparation, characterized in that: The active ingredients include the Bacillus amyloliquefaciens W1 and / or its metabolites as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that The concentration of Bacillus amyloliquefaciens W1 is 10 7 ~10 9 CFU / mL.

4. The microbial preparation according to claim 2, characterized in that The metabolites of Bacillus amyloliquefaciens W1 are lipopeptide substances; the lipopeptide substances include Bacilysin, C16 FengycinA and C14 Iturin B.

5. Use of the Bacillus amyloliquefaciens W1 according to claim 1 or the microbial preparation according to any one of claims 2 to 4 in preventing and treating tobacco leaf mildew, characterized in that: The pathogenic bacteria of tobacco leaf mildew disease include Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger and Aspergillus flavus.

6. The use according to claim 5, characterized in that: The tobacco leaves are tobacco leaves in storage.

7. A method for preventing and treating tobacco leaf mildew, characterized in that: The method comprises the steps of applying the microbial preparation according to any one of claims 2 to 4 to tobacco leaves; The pathogenic bacteria of tobacco leaf mildew disease include Aspergillus tubingensis, Talaromyces funiculosus, Aspergillus niger and Aspergillus flavus.

8. The method according to claim 7, characterized in that The tobacco leaves are tobacco leaves in storage.

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