Bacillus mojavensis HF4 and application thereof in antagonism of plant pathogenic fungi

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus mojavensis HF4 and application of the bacillus mojavensis HF4 in antagonism of plant pathogenic fungi. The invention provides bacillus mojavensis HF4, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the bacillus mojavensis HF4 is CGMCC No.33569. The bacillus mojavensis HF4 is named as bacillus mojavensis HF4. The bacillus mojavensis HF4 has the effect of efficiently antagonizing plant pathogenic fungi, and has a remarkable antibacterial effect on various plant fungi; the strain can generate lipopeptides such as surfactin and fengycin which are rich in variety and homologues of the surfactin and the fengycin, has the potential of becoming efficient biocontrol endophytes, and has a better application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Bacillus mohaievii HF4 and application thereof in antagonizing plant pathogenic fungi. Background Art

[0002] Fungal pathogens are among the most important pathogens affecting crop production and food safety. Plant fungal diseases are primarily controlled by synthetic fungicides. However, excessive and inappropriate use of fungicides has caused environmental damage and health problems, and has increased fungicide resistance in plant pathogens.

[0003] As an alternative to traditional fungal control methods, the use of biocontrol agents is considered a safe and environmentally friendly strategy that can reduce the use of chemical fungicides. The antifungal potential of Bacillus as a biocontrol agent has been widely studied. Bacillus strains are commonly found in the soil and have a close relationship with plants, establishing a beneficial relationship with each other. At the same time, Bacillus strains can produce a variety of secondary metabolites that replace antibiotics, including lipopeptides. These bioactive metabolites have been considered as biocontrol agents that replace chemical pesticides. Among them, Bacillus mohaiwei ( Bacillus mojavensis ) is an antagonistic endophytic bacterium that can live within the intercellular spaces of plants, interacting with the host to influence plant growth and combat plant diseases. It shows great potential for application in crop disease control. Currently, there is still a need to develop Bacillus mohaiwei with high inhibitory activity against a variety of plant pathogens. Summary of the Invention

[0004] The invention provides a Bacillus mohaievii HF4 and application thereof in antagonizing plant pathogenic fungi.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides Bacillus mohaiwei ( Bacillus mojavensis )HF4, which was deposited on February 19, 2025, at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was named Bacillus mohaiwei Bacillus mojavensis , the deposit number is CGMCC No.33569.

[0007] The present invention isolates a strain of Bacillus mohaiwei ( Bacillus mojavensis ), named it Bacillus mohaiwei ( Bacillus mojavensis )HF4. Experimental verification has shown that this strain can inhibit a variety of plant pathogenic fungi, with high inhibition rates against 19 species. It can also produce a variety of antimicrobial lipopeptides, including surfactin A and surfactin B and their homologues, as well as fengycin A, fengycin B, fengycin C, fengycin S and their homologues, and can be used as a biocontrol agent to prevent and control plant fungal diseases.

[0008] In a second aspect, the present invention provides a microbial preparation comprising the above-mentioned Bacillus mohaiwei ( Bacillus mojavensis )HF4.

[0009] Among the microbial preparations described above, Bacillus mohaiwei ( Bacillus mojavensis )HF4 is preferably present in the form of live bacteria.

[0010] Wherein, the microbial preparation can be a liquid preparation or a solid preparation. The active ingredient of the microbial preparation comprises Bacillus mohaiwei ( Bacillus mojavensis The microbial preparation may further comprise auxiliary materials or carriers permitted in the field of microbial preparations.

[0011] In a third aspect, the present invention provides a method for preparing the above-mentioned microbial preparation, the method comprising: treating the above-mentioned Bacillus mohaiwei ( Bacillus mojavensis )HF4 was cultured to obtain a culture.

[0012] Preferably, the culture temperature is 30-37° C. Optional culture media include LB medium, Endo-KNO 3 medium, and the like.

[0013] In some embodiments of the present invention, the preparation method comprises: (1) Activate Bacillus mohaiwei on the plate ( Bacillus mojavensis )HF4, pick a single clone of the strain, inoculate it into LB medium, and culture it on a shaker at 30-37°C and 180-220 rpm for 8-14 hours to obtain the seed solution; (2) Bacillus mohaiwei ( Bacillus mojavensis )HF4 seed liquid was transferred to fermentation medium (Endo-KNO3 medium) at an inoculum rate of 3-5%, cultured on a shaker at 30-37°C and 180-220 rpm for 48-72 hours, and the fermentation broth was collected.

[0014] Preferably, in the microbial preparation, Bacillus mohaiwei ( Bacillus mojavensis )HF4 content is 1×10 9 ~1×10 10 cfu / mL.

[0015] In a fourth aspect, the present invention provides a biocontrol agent comprising a Bacillus mohaiwei ( Bacillus mojavensis )HF4, the Bacillus mohaiwei ( Bacillus mojavensis )HF4 fermentation culture, the Bacillus mohaiwei ( Bacillus mojavensis )HF4 fermentation supernatant or its dilution, one or more of the above-mentioned microbial preparations.

[0016] The method for preparing the fermentation culture comprises: Bacillus mojavensis )HF4 for fermentation and collecting the fermentation broth. The fermentation broth refers to all products obtained from the fermentation, including the bacterial cells and the fermentation broth supernatant.

[0017] The method for preparing the fermentation supernatant comprises: Bacillus mojavensis )HF4 was fermented to obtain a fermentation broth, the bacteria in the fermentation broth were removed, and the sterile supernatant was collected.

[0018] The above-mentioned removal of bacteria from the fermentation broth can be carried out by centrifugation and / or filtration.

[0019] In some embodiments of the present invention, the fermentation broth is centrifuged, the supernatant is collected, and then filtered to remove residual bacteria.

[0020] In some embodiments of the present invention, the method for preparing the biocontrol agent comprises: (1) Activate Bacillus mohaiwei on the plate ( Bacillus mojavensis )HF4, pick a single clone of the strain, inoculate it into LB medium, and culture it on a shaker at 30-37°C and 180-220 rpm for 8-14 hours to obtain the seed solution; (2) Bacillus mohaiwei ( Bacillus mojavensis )HF4 seed liquid was transferred to fermentation medium (Endo-KNO3 medium) at an inoculum rate of 3-5%, cultured on a shaker at 30-37°C and 180-220 rpm for 48-72 hours, and the fermentation broth was collected.

[0021] (3) The fermentation broth was centrifuged at 8,000-12,000 g, and the supernatant was collected. The supernatant was sterilized by filtering using a 0.22 μm filter membrane to obtain a sterile supernatant.

[0022] In some embodiments of the present invention, the method for preparing the biocontrol agent comprises: (1) Activation of Bacillus mohaiwei on plates (Bacillus mojavensis )HF4, pick a single clone of the strain, inoculate it into LB medium, and culture it on a shaker at 30-37°C and 180-220 rpm for 8-14 hours to obtain the seed solution; (2) Bacillus mohaiwei ( Bacillus mojavensis )HF4 seed liquid was transferred to fermentation medium (Endo-KNO3 medium) at an inoculum rate of 3-5%, cultured on a shaker at 30-37°C and 180-220 rpm for 48-72 hours, and the fermentation broth was collected.

[0023] In a fifth aspect, the present invention provides the above-mentioned Bacillus mohaiwei ( Bacillus mojavensis ) Use of HF4 or the microbial preparation or the biocontrol preparation in inhibiting plant pathogens.

[0024] In a sixth aspect, the present invention provides the above-mentioned Bacillus mohaiwei ( Bacillus mojavensis ) Use of HF4 or the microbial preparation or the biocontrol preparation in preventing and controlling plant pathogen infection or preventing and controlling plant diseases caused by plant pathogen infection.

[0025] In a seventh aspect, the present invention provides the above-mentioned Bacillus mohaiwei ( Bacillus mojavensis ) Use of HF4 or the microbial preparation or the biocontrol preparation in the preparation of products for inhibiting plant pathogens, preventing and controlling plant pathogen infections, or preventing and controlling plant diseases caused by plant pathogen infections.

[0026] In the present invention, the plant pathogen is preferably a plant pathogenic fungus, preferably a filamentous fungus.

[0027] Preferably, the plant pathogenic fungi include those selected from Gibberella ( FusaHum graminearum ), Curvularia volvacea ( Curvularia trifolii ), Rhizoctonia solani ( Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum ), Cysticercus spp. ( Physalospora piricola ), Fusarium equisetum ( Fusarium equiseti ), Apple Anthracnose ( Glomerella cingulat ), Phytophthora nicotianae ( Phytophthora parasitica ), Alternaria tabacum ( Alternaria alternata (Fr) Keissler), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Alternaria apples ( Alternaria alternata f.sp.mali), wilt-specialized Fusarium oxysporum ( Fusarium oxysporum f.sp.vasinfectum), cotton alternaria ( Alternaria gossypina ), Cladosporium cucurbitaceum ( Corynespora cassiicola ), cabbage ring spot pathogen ( Phyllosticta brassicea ), Diplodia theobrominum ( Lasiodiplodia theobromae ), Fusarium verticillium ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Olivaceous microsporum Pseudodiscotrichum ( Pestalotiopsis microspore ), Rice blast fungus ( Magnaporthe grisea )、Trichoderma viride ( Trichoderma viride )

[0028] The present invention provides Bacillus mohaiwei ( Bacillus mojavensis )HF4 has shown excellent antibacterial effect on plant pathogenic fungi such as tobacco phytophthora, Rhizoctonia solani, Fusarium oxysporum, Aspergillus niger, Alternaria nicotianae, Sclerotinia sclerotiorum, Alternaria sclerotiorum, Alternaria sclerotiorum, Botrytis cinerea, Rice blast fungus, and Trichoderma viride. The antibacterial effect can be achieved by Bacillus mohairi ( Bacillus mojavensis )HF4 viable bacteria, secondary metabolites produced by fermentation thereof, or dilutions obtained by diluting the secondary metabolites at different times take effect.

[0029] In an eighth aspect, the present invention provides the above-mentioned Bacillus mohaiwei ( Bacillus mojavensis ) Use of HF4 or the microbial preparation or the biocontrol preparation in the preparation of antimicrobial lipopeptides.

[0030] Preferably, the antimicrobial lipopeptides include surfactins and / or fengycins; more preferably, they include one or more selected from surfactin A or its homologues, surfactin B or its homologues, fengycin A or its homologues, fengycin B or its homologues, fengycin C or its homologues, and fengycin S or its homologues.

[0031] Among them, surfactin A or its homologues including C 12 -C 17 surfactin A. surfactin B or its homologues including C 14 、C 15 and C 18 surfactin B. fengycin A or its homologues including C 14 -C 17 Fengycin A. Fengycin B or its homologues including C 15 -C 17 Fengycin B, C with an unsaturated double bond in the carbon chain 14 -C16 fengycin B. fengycin C or homologues thereof include C 15 -C 17 fengycin C. fengycin S or homologues thereof include C 13 -C 14 fengycin S.

[0032] The bacillus mojavensis (Bacillus mojavensis) HF4 of the present application can produce at least 9 kinds of lipopeptides including surfactin A and surfactin B and homologues thereof, and 15 kinds of fengycin A, fengycin B, fengycin C and fengycin S and homologues thereof. Bacillus mojavensis The above-mentioned lipopeptides have the effect of antagonizing plant pathogenic fungi or plant diseases caused by the plant pathogenic fungi.

[0033] The bacillus mojavensis (Bacillus mojavensis) HF4 of the present application can produce at least 9 kinds of lipopeptides including surfactin A and surfactin B and homologues thereof, and 15 kinds of fengycin A, fengycin B, fengycin C and fengycin S and homologues thereof.

[0034] The bacillus mojavensis (Bacillus mojavensis) HF4 of the present application can produce at least 9 kinds of lipopeptides including surfactin A and surfactin B and homologues thereof, and 15 kinds of fengycin A, fengycin B, fengycin C and fengycin S and homologues thereof. 18 Surfactin B is a lipopeptide homologue that has not been reported yet. 11 Surfactin B is a lipopeptide homologue that has not been reported yet. 17 Surfactin B is a lipopeptide homologue that has not been reported yet. 15Surfactin is the most biologically active isomer, but a strain producing such a variety of fengycins has never been reported before; therefore, Bacillus mohaiwei HF4 can be used as a biocontrol endophyte for high-yield lipopeptides to control plant fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] 图1 The representative photos and inhibition rates of the sterile supernatant of Bacillus mohaiwei HF4 in Example 2 of the present invention against 19 species (21 strains) of fungi are shown; the letters in the upper left corner represent Gibberella ( FusaHum graminearum ), Curvularia volvacea ( Curvularia trifolii ), Rhizoctonia solani ( Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum ), Cysticercus spp. ( Physalospora piricola ), Fusarium equisetum ( Fusarium horsetail ), Apple Anthracnose ( Glomerella cingulate ), Phytophthora nicotianae ( Phytophthora parasitic ), Alternaria tabacum ( Alternaria alternata (Fr) Keissler), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Alternaria apple ( Alternaria alternata f.sp.mali), wilt-specialized Fusarium oxysporum ( Fusarium oxysporum f.sp.vasinfectum), cotton alternaria ( Alternaria gossypium ), Cladosporium cucurbitae ( Corynespora cassicola ), cabbage ring spot pathogen ( Phyllosticta brassica ), Diplodia theobrominum ( Lasiodiplodia theobromae ), Fusarium verticillium ( Fusarium whorl-shaped ), Botrytis cinerea ( Botrytis cinerea ), Olivaceous microsporum Pseudodiscotrichum ( Pestalotiopsis microspore ), Rice blast fungus ( Magnaporthe grisea )、Trichoderma viride ( Trichoderma green ); In the antibacterial effect picture of each fungal strain, the left side is the control group and the right side is the experimental group. The experiment was repeated three times.

[0037] Figure 2This is a representative photo of the inhibition of 16 species (18 strains) of fungi by the live bacteria of Bacillus mohaiwei HF4 in Example 2 of the present invention; the letters in the upper left corner represent the following in order: Lasiodiplodia theobromae ), Cysticercus spp. ( Physalospora piricola ), Gibberellic acid ( Grassland ), Botrytis cinerea ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Curvularia volvacea ( Cloverleaf clover ), Fusarium oxysporum ( Fusarium oxysporum ), Rice blast fungus ( Magnaporth gray ), Fusarium equisetum ( Fusarium horsetail ), Apple Anthracnose ( Glomerella cingulate ), Alternaria tabacum ( Alternaria alternata (Fr) Keissler), Fusarium verticillium ( Fusarium whorl-shaped ), Olivaceous microsporum Pseudodiscotrichum ( Pestalotiopsis microspore ), cabbage ring spot pathogen ( Phyllosticta brassicae ), Alternaria apple ( Alternaria alternata f.sp.mali), Alternaria paniculata ( Alternaria gossypina ), Phytophthora nicotianae ( Phytophthora parasitica ), wilt-specialized Fusarium oxysporum ( Fusarium oxysporum f.sp.vasinfectum), in the antibacterial effect picture of each fungal strain, the left side is the control group, the right side is the experimental group, and the experiment was repeated three times.

[0038] Figure 3 The results of the sterile supernatant of Bacillus mohairi HF4 in Example 3 of the present invention showing inhibition of Phytophthora nicotianae. (A) The results of the sterile supernatant of Bacillus mohairi HF4 at different dilutions on Phytophthora nicotianae. (B) The inhibition rate of the sterile supernatant of Bacillus mohairi HF4 on Phytophthora nicotianae at different dilutions.

[0039] Figure 4 The biocontrol effect of Bacillus mohaiwei HF4 on Phytophthora nicotianae in Example 3 of the present invention is shown. (A) Cryo-SEM image of HF4 sterile supernatant inhibiting Phytophthora nicotianae hyphae; scale bars: 200 μm and 40 μm. (B) 72-hour control effect of HF4 on detached tobacco leaves. The bacterial suspension of HF4 (1×10 8 cfu / mL) were treated with tobacco leaves and immediately inoculated with tobacco phytophthora. Data analysis was performed using t -Inspection, ** represents p <0.01.

[0040] Figure 5This is the ESI-MS / MS spectrum for molecular identification of the Surfactin compound in Example 4 of the present invention; (A) ESI-MS / MS spectrum of the crude methanol extract of the sterile HF4 supernatant, with ×10 above the vertical axis. 4 (B) Mass-to-charge ratio of 1,022.6756 secondary mass spectrum, containing red-marked characteristic fragment ion peaks 685 and 441 belonging to surfactin A, and the vertical axis is marked with ×10 3 (C) Mass-to-charge ratio of 1,022.6770. The characteristic fragment ion peaks 671 and 441 marked in red belong to surfactin B. The vertical axis is marked with ×10 2 .

[0041] Figure 6 ESI-MS / MS spectra for molecular identification of Fengycin compounds in Example 4 of the present invention; (A) The secondary mass spectrum with a mass-to-charge ratio of 1,464.8084, containing red-marked characteristic fragment ion peaks 1,080 and 966, belongs to fengycin A; (B) The secondary mass spectrum with a mass-to-charge ratio of 1,478.8234, containing red-marked characteristic fragment ion peaks 1,108 and 994, belongs to fengycin B; (C) The secondary mass spectrum with a mass-to-charge ratio of 1,478.8231, containing red-marked characteristic fragment ion peaks 1,094 and 980, belongs to fengycin C. (D) The secondary mass spectrum with a mass-to-charge ratio of 1,450.7926, containing red-marked characteristic fragment ion peaks 1,066 and 952, belongs to fengycin S. The vertical axis is marked with ×10 3 . DETAILED DESCRIPTION

[0042] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0043] The calculation formula of the fungal inhibition rate in the following examples is: Fungal inhibition rate (%) = (fungal diameter of the control group - fungal diameter of the experimental group) / fungal diameter of the control group × 100%.

[0044] The culture medium formula used in the following examples is as follows: LB medium: 5 g / L yeast extract, 10 g / L sodium chloride, 10 g / L tryptone, pH = 7.0. Sterilize at 121°C for 20 min.

[0045] Fermentation medium (Endo-KNO3 medium): glucose 28 g / L, KNO3 6 g / L, KH2PO4 1.2 g / L, MgSO4·7H2O 1.2 g / L, Na3C6H5O7·2H2O 0.2 g / L, CaCl2·2H2O 210 mg / L, FeSO4·7H2O 16 mg / L, EDTA-Na2 2.1 mg / L, trace elements 1 mL / L (H3BO4 2.86 g / L, MnCl2·4H2O 1.81 g / L, ZnSO4·7H2O 0.222 g / L, Na2MoO4·2H2O 0.021 g / L, CuSO4·5H2O 0.07 g / L), pH = 7.0. Sterilize at 121°C for 20 min.

[0046] Example 1 Isolation and identification of Bacillus mohaiwei HF4 The present invention isolated a Bacillus strain from the soil of a Beijing farm and identified it as Bacillus mohaiwei, which was named Bacillus mohaiwei ( Bacillus mojaveensis )HF4.

[0047] Bacillus mohaiwei ( Bacillus mojaveensis HF4 was deposited on February 19, 2025, at the General Microbiology Center of the China Culture Collection Administration (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), and was classified as Bacillus mohaiwei. Bacillus Mojave , the deposit number is CGMCC No.33569.

[0048] Example 2 Application of Bacillus mohaiwei HF4 in inhibiting 19 plant fungal diseases 1. Materials and Methods (1) Activate Bacillus mohaiwei HF4 on a plate, pick a single clone, inoculate it into 100 mL LB medium, and culture it overnight on a shaker at 37°C and 200 rpm to obtain seed liquid.

[0049] (2) The seed liquid of Bacillus mohaiwei HF4 was transferred to the fermentation medium at a 4% inoculation rate, and cultured on a shaker at 30°C and 200 rpm for 60 hours. The fermentation liquid (the bacterial count was 2.2×10 9 ~2.5×10 9 cfu / mL) and set aside.

[0050] (3) HF4 fermentation broth (bacterial count 2.2×10 9 ~2.5×109 cfu / mL) was centrifuged at 12,000 g for ultra-high-speed filtration, and sterilized by filtration using a 0.22 μm imported filter membrane three times, and the sterile supernatant was used for later use.

[0051] (4) The sterile supernatant was subjected to a punching experiment to inhibit fungi: a 6 mm diameter plant pathogenic fungus cake was inoculated at the center of the PDA solid culture medium; three holes were punched 3 cm from the center of the inoculated cake, and 200 μL of the sterile supernatant prepared in step (3) was added to each hole. Three replicates were performed in each group, and the cells were cultured at 28°C to detect the inhibitory effect of the sterile supernatant of Bacillus mohaiwei HF4 on plant pathogenic fungi.

[0052] (5) A 6 mm diameter cake of the tested plant pathogenic fungi was inoculated at the center of the PDA solid culture medium; 1 μL of HF4 fermentation liquid was inoculated 3 cm from the center of the inoculated cake, with 3 replicates per group. The culture was incubated at 28°C to detect the inhibitory effect of live bacteria of Bacillus mohaiwei HF4 on plant pathogenic fungi.

[0053] 2. Experimental results The antibacterial effect of the sterile supernatant of Bacillus mohaiwei HF4 on 19 fungi Figure 1 The results showed that the sterile supernatant of HF4 had an inhibitory effect on 19 filamentous fungi tested, such as Gibberella (45.2%), Curvularia volvacea (49.52%), Rhizoctonia solani (56.4%), Fusarium oxysporum (58.11%), Aspergillus niger (56.31%), Fusarium equisetifolia (38.93%), Colletotrichum apples (40.97%), Phytophthora nicotianae (59.6%), Alternaria nicotianae (58.35%), Sclerotinia sclerotiorum (54.6%), and Alternaria oleracea (54.25%). The results showed varying degrees of inhibition against Fusarium oxysporum (34.37%), Alternaria sinensis (54.88%), Coryneformis cucurbitae (42.76%), Brassica rapa (48.88%), Diplodia theobroma cacao (46.46%), Fusarium verticillioides (37.53%), Botrytis cinerea (53.88%), Pseudomonas aeruginosa (41.75%), Magnaporthe oryzae (56.45%), and Trichoderma viride (79.98%), with the highest inhibition rate reaching 79.98%. The numbers in parentheses indicate the inhibition rate of Bacillus mohaievii HF4 against fungi. This indicates that Bacillus mohaievii HF4 is a potential biocontrol agent for controlling plant fungal diseases.

[0054] The antibacterial effect of live Bacillus mohaiwei HF4 against 16 fungi Figure 2 The results showed that the live bacteria of Bacillus mohaiwei HF4 were effective against the cocoa hair color spores ( Lasiodiplodia theobromae ), Cysticercus spp. ( Physalospora piricola), Gibberellic acid ( Grassland ), Botrytis cinerea ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Curvularia volvacea ( Cloverleaf clover ), Fusarium oxysporum ( Fusarium oxysporum ), Rice blast fungus ( Magnaporthe grisea ), Fusarium equisetum ( Fusarium horsetail ), Apple Anthracnose ( Glomerella cingulat ), Alternaria tabacum ( Alternaria alternata (Fr) Keissler), Fusarium verticillium ( Fusarium verticillioides ), Olivaceous microsporum Pseudodiscotrichum ( Pestalotiopsis microspore ), cabbage ring spot pathogen ( Phyllosticta brassicea ), Alternaria apple ( Alternaria alternata f.sp.mali), Alternaria paniculata ( Alternaria gossypina ), Phytophthora nicotianae ( Phytophthora parasitica ), wilt-specialized Fusarium oxysporum ( Fusarium oxysporum It had inhibitory effects on 16 fungi (18 strains) including f.sp.vasinfectum.

[0055] Example 3: Excellent antibacterial effect of Bacillus mohaiwei HF4 on tobacco phytophthora 1. Materials and Methods (1) Activate Bacillus mohaiwei HF4 on a plate, pick a single clone, inoculate it into 100 mL LB medium, and culture it overnight on a shaker at 37°C and 200 rpm to obtain seed liquid.

[0056] (2) Transfer the seed liquid of Bacillus mohaiwei HF4 to the fermentation medium at a 4% inoculum volume, incubate on a shaker at 30°C and 200 rpm for 60 hours, and collect the fermentation liquid. Centrifuge a portion of the fermentation liquid for ultra-high-speed filtration and sterilize it by filtration through a 0.22 μm imported filter membrane. Take the sterile supernatant for later use.

[0057] (3) Sterile supernatant plate inhibition experiment: 600 μL of HF4 sterile supernatant was added to 60 mL of PDA solid culture medium (55°C), which was equivalent to diluting the sterile supernatant 100 times, and a gradient dilution inhibition experiment was set up; the sterile supernatant and PDA culture medium were mixed and poured into a culture dish, blown dry and set aside; a test bacterial cake with a diameter of 6 mm was inoculated at the center point of the PDA solid culture medium added with HF4 sterile supernatant, with 3 replicates per group, and cultured at 28°C.

[0058] 2. Experimental results The results showed that the sterile supernatant of HF4 had a good inhibitory effect on the pathogenic fungus of tobacco black shank disease, Phytophthora nicotianae. The inhibition rate of the sterile supernatant diluted 50 times was as high as 74.75%. It still had excellent antifungal activity after being diluted 500 times, with an inhibition rate of up to 51.71% ( Figure 3 Tobacco black shank is an important soil-borne fungal disease of tobacco caused by infection with Phytophthora nicotianae. It mainly infects the roots and stem base of tobacco, harming plants in the field. To determine the efficacy of Bacillus mohaiwei HF4 against tobacco black shank, the efficacy of HF4 against tobacco black shank pathogenic fungi on detached tobacco leaves was tested. A suspension of HF4 (1×10 8 cfu / mL) were treated with tobacco leaves and immediately inoculated with tobacco phytophthora. The results showed that Bacillus mohaiwei HF4 could cause abnormal swelling of the hyphae tips of tobacco phytophthora, affecting the normal growth of hyphae and significantly inhibiting the expansion of tobacco black shank lesions on detached tobacco leaves ( Figure 4 ).

[0059] Example 4: Identification of the Types of Lipopeptides Surfactins and Fengycins Produced by Bacillus mohaievii HF4 and Their Homologs 1. Materials and Methods (1) Activate Bacillus mohaiwei HF4 on a plate, pick a single clone, inoculate it into 100 mL LB medium, and culture it overnight on a shaker at 37°C and 200 rpm to obtain seed liquid.

[0060] (2) The seed liquid of Bacillus mohaiwei HF4 was transferred to the fermentation medium at a 4% inoculation rate and cultured on a shaker at 30°C and 200 rpm for 60 hours. The fermentation liquid was centrifuged for ultra-high-speed filtration and sterilized by filtration with a 0.22 μm imported filter membrane. The sterile supernatant was collected for later use.

[0061] (3) The supernatant was adjusted to pH 2.0 with 6 M HCl and placed in a 4°C refrigerator overnight. g After centrifugation for 15 minutes, discard the supernatant and retain the precipitate. Add 5 mL of methanol to dissolve the precipitate, 10,000 g Centrifuge for 10 min. At this time, the lipopeptide compounds exist in the methanol solution. Take the methanol solution and filter it with a 0.22 µm filter membrane for later use.

[0062] (4) Matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF / TOF) was used in reflectron mode to identify crude lipopeptide samples in the fermentation broth and determine the molecular weight of the lipopeptides. The structures of the lipopeptides in the fermentation broth were identified by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The liquid phase parameters were as follows: mobile phase A was 0.1% HPLC-grade formic acid in water, and mobile phase B was HPLC-grade methanol. The elution gradient was as follows: 0.1 min, 70% B; 0.1 min-2.0 min, 70% B; 2.0 min-8.0 min, 70%-100% B; 8.0 min-10 min, 100% B; 10.1 min, 70% B; 10.1 min-13 min 70% B. The flow rate was 0.3 mL / min. The chromatographic column was a Waters BEH C18 (50 mm × 2.1 mm, 1.7 µm particle). The analysis was performed in positive ion mode with the following MS tuning parameters: degassing rate 500 L / h, degassing temperature 500°C, cone gas flow 50 L / h, the source temperature was 120°C, the capillary voltage was 3.0 kV, and the cone voltage was 30 V. The data collection time was 0.5 sec, the mass-to-charge ratio range was 100 m / z–1,200 m / z, and the collision voltage was set to 25 V.

[0063] 2. Experimental results and analysis According to the total ion map of the sample ( Figure 5 , Figure 6 ), confirming that HF4 fermentation broth contains surfactin and fengycin. The ESI-MS / MS spectrum of the surfactin compound molecular identification is shown in the figure below. Figure 5 Combining MS / MS mass spectrometry information and literature reports, it was deduced that HF4 can secrete surfactin and its homologues, including C 12 -C 17 surfactin A and C 14 、C 15 and C 18 surfactin B, a total of 9 types (Table 1).

[0064] ESI-MS / MS spectra of fengycin compound molecular identification Figure 6 Combining MS / MS mass spectrometry information and literature reports, it was deduced that HF4 can secrete fengycin and its homologues, including C 14 -C 17 fengycin A,C 15 -C 17Fengycin B, C with an unsaturated double bond in the carbon chain 14 -C 16 fengycin B,C 15 -C 17 fengycin C and C 13 -C 14 Fengycin S, a total of 15 species (Table 2). Among the identified lipopeptides, C 18 Surfactin B is a previously unreported lipopeptide homolog. Furthermore, HF4 produced a continuous unsaturated olefin mixture of fengycin B, a species that has never before produced such a diverse range of fengycins. Both surfactin and fengycin are important antifungal compounds, and the use of HF4 in the development of antifungal biopharmaceuticals could have applications in medicine and the control of fungal diseases in crops.

[0065] Table 1 Surfactin molecular identification results

[0066] Note: B. mojavensis Identification of surfactin species and homologues in HF4 methanol crude extract.

[0067] Table 2 Fengycin molecular identification results

[0068] Note: B. mojavensis Identification of fengycin species and homologues in the crude HF4 methanol extract. *Indicates a double bond in the fatty acid chain.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Bacillus mohaiwei ( Bacillus mojavensis )HF4, characterized in that It is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.33569.

2. A microbial preparation, characterized in that: The microbial preparation comprises the Bacillus mohaievii ( Bacillus mojavensis )HF4.

3. The method for preparing the microbial preparation according to claim 2, characterized in that: The method comprises: treating the Bacillus mohaiwei ( Bacillus mojavensis )HF4 was cultured to obtain a culture.

4. A biocontrol agent, characterized in that It contains the Bacillus mohaiwei ( Bacillus mojavensis )HF4, Bacillus mohaiwei described in claim 1 ( Bacillus mojavensis )HF4 fermentation culture, the Bacillus mohaiwei of claim 1 ( Bacillus mojavensis )HF4 fermentation supernatant or its dilution, one or more of the microbial preparation according to claim 2.

5. The biocontrol agent according to claim 4, characterized in that The method for preparing the fermentation culture comprises: Bacillus mojavensis ) HF4 for fermentation and collecting the fermentation broth; And / or, the method for preparing the fermentation supernatant comprises: treating the Bacillus mohaiwei ( Bacillus mojavensis )HF4 was fermented to obtain a fermentation broth, the bacteria in the fermentation broth were removed, and the sterile supernatant was collected.

6. The Bacillus mohaiwei of claim 1 ( Bacillus mojavensis ) Use of the microbial preparation according to claim 2 or the biocontrol preparation according to claim 4 or 5 in inhibiting plant pathogens.

7. The Bacillus mohaiwei of claim 1 ( Bacillus mojavensis ) Use of HF4 or the microbial preparation according to claim 2 or the biocontrol preparation according to claim 4 or 5 in preventing and controlling plant pathogen infection or preventing and controlling plant diseases caused by plant pathogen infection.

8. The Bacillus mohaiwei of claim 1 ( Bacillus mojavensis ) Use of HF4 or the microbial preparation according to claim 2 or the biocontrol preparation according to claim 4 or 5 in the preparation of a product for inhibiting plant pathogens, preventing and controlling plant pathogen infections, or preventing and controlling plant diseases caused by plant pathogen infections.

9. The use according to any one of claims 6 to 8, characterized in that The plant pathogen is a plant pathogenic fungus; preferably a filamentous fungus; More preferably, the plant pathogenic fungi include those selected from Gibberella ( FusaHum graminearum ), Curvularia volvacea ( Curvularia trifolii ), Rhizoctonia solani ( Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum ), Cysticercus spp. ( Physalospora piricola ), Fusarium equisetum ( Fusarium equiseti ), Apple Anthracnose ( Glomerella cingulat ), Phytophthora nicotianae ( Phytophthora parasitica ), Alternaria tabacum ( Alternaria alternata (Fr) Keissler), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Alternaria apples ( Alternaria alternata f.sp.mali), wilt-specialized Fusarium oxysporum ( Fusarium oxysporum f.sp.vasinfectum), cotton alternaria ( Alternaria gossypina ), Cladosporium cucurbitaceum ( Corynespora cassiicola ), cabbage ring spot pathogen ( Phyllosticta brassicea ), Diplodia theobrominum ( Lasiodiplodia theobromae ), Fusarium verticillium ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Olivaceous microsporum Pseudodiscus hirsutus ( Pestalotiopsis microspore ), Rice blast fungus ( Magnaporthe grisea )、Trichoderma viride ( Trichoderma viride ) 10. The Bacillus mohaiwei of claim 1 ( Bacillus mojavensis ) Use of HF4 or the microbial preparation according to claim 2 or the biocontrol preparation according to claim 4 or 5 in the preparation of antimicrobial lipopeptides; Preferably, the antimicrobial lipopeptides include surfactins and / or fengycins; more preferably, they include one or more selected from surfactin A or its homologues, surfactin B or its homologues, fengycin A or its homologues, fengycin B or its homologues, fengycin C or its homologues, and fengycin S or its homologues.

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