A microbial agent containing Bacillus velez XY40-1 and its preparation method and application

Abstract

The present invention discloses a microbial agent containing Bacillus velez XY40-1 and a preparation method and application thereof. The present invention creatively improves the fermentation medium formula of the fermentation links such as seed liquid and bacterial liquid, greatly shortens the fermentation cycle, significantly reduces the fermentation cost, enriches the content and types of microbial metabolites, and improves the functions of the series of products such as Bacillus velez agent in biological control and growth promotion. The biological pesticide suspension provided by the present invention can effectively prevent and control pepper blight; the microbial seed coating agent can significantly improve the germination rate of pepper, eggplant and cabbage seeds, and has a strong application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganism and plant disease prevention and control, and specifically relates to a microbial agent containing Bacillus Velezii XY40-1 and a preparation method and application thereof. Background Art

[0002] Bacillus velezensis, as a new species of Bacillus, is widely distributed in nature and has the characteristics of strong enzyme production ability, rich secondary metabolites, strong stress resistance, and high antibacterial activity. Studies have shown that Bacillus velezensis can effectively inhibit a variety of plant pathogens, such as Phytophthora capsici, Fusarium solani, Fusarium oxysporum, Rhizoctonia solani, and several pathogenic fungi on leafy vegetables, including brown spot fungus, stem spot mildew, Botrytis cinerea var. dianthus and Sclerotinia sclerotiorum, and effectively prevent and control a variety of plant diseases.

[0003] Microbial control inhibits agricultural pests and diseases by extracting and modifying bacteria, fungi, viruses and other organisms. Chemical control has problems such as widespread use and environmental risks. Long-term use of chemical agents can lead to drug resistance in pathogens and increase agricultural production costs. Using microorganisms as biological pesticides has the characteristics of being green, efficient, and able to continuously control pests and diseases.

[0004] In agriculture, seeds are an indispensable means of production and the foundation of agricultural production. At present, pests and diseases affect the germination rate and quality of seeds, and the traditional method of using pesticides to control pests and diseases has brought unbearable pressure to the ecological environment. The use of seed coating technology can not only improve the germination rate of seeds, but also improve the resistance of seeds to pests and diseases without affecting the ecological environment, and can also save costs and improve economic benefits. However, the negative impact of chemical seed coating agents on crop production is increasing, and there is an urgent need to develop green, low-toxic, green biological seed coating agents, among which biological seed coating agents based on microorganisms have become a development trend. At present, the only biological seed coating agent developed in China is Bacillus subtilis biological seed coating agent, and there are relatively few biological seed coating agents based on other microbial preparations, especially the research and development of Bacillus Velez biological seed coating agents is relatively lagging behind. Summary of the invention

[0005] The purpose of the present invention is to provide a microbial agent containing Bacillus Velez XY40-1 and a preparation method and application thereof, aiming to reduce the application of chemical pesticides and find a new direction for microbial control of Bacillus Velez.

[0006] The present invention is achieved in that:

[0007] In previous studies, the inventors screened out a strain of Bacillus velezensis XY40-1 that has good antagonism to citrus Geotrichum candidum, corn Curvularia and coconut gray spot pathogens. The strain was deposited in the China Center for Type Culture Collection on March 29, 2022, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: M 2022342.

[0008] The method for preparing the microbial agent containing Bacillus velezensis XY40-1 provided by the present invention comprises the following steps:

[0009] 1) Strain activation: inoculate Bacillus velezensis XY40-1 with a preservation number of CCTCC NO: M 2022342 on LB solid medium, and culture at 28-30° C. for 1-2 days to obtain an activated strain;

[0010] 2) Fermentation seed culture: The activated strain was inoculated into LB liquid culture medium, and cultured in a shaker at 160-200 rpm at 36-39° C. for 7-10 hours to obtain a seed solution;

[0011] 3) Fermentation: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 0.5% to 1.5%, and culture the culture medium at 36 to 39° C. and 160 to 200 rpm in a shaker for 10 to 18 hours to obtain the microbial agent.

[0012] Furthermore, in step 1), the formula of the LB solid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, and pH is 7.2-7.3.

[0013] Furthermore, in step 2), the formula of the LB liquid culture medium is: 20 g / L peptone, 10 g / L sodium chloride, 10 g / L yeast extract, 50 mmol / L zinc sulfate, 1000 nmol / L PQQ (pyrroloquinoline quinone), and the pH is 7.2-7.3.

[0014] Furthermore, in step 3), the formula of the fermentation medium is: 20 g / L soybean meal, 14 g / L glucose, 10 g / L magnesium sulfate, 50 mmol / L zinc sulfate, 20 g / L peptone, 10 g / L yeast extract powder, 10 g / L sodium chloride, 10 g / L potassium dihydrogen phosphate, 1000 nmol / L PQQ, and the pH of the fermentation tank is 7.2-7.3.

[0015] The microbial agent containing Bacillus velezensis XY40-1 is prepared according to the above preparation method.

[0016] The microbial agent is an original bacterial liquid that has not been concentrated.

[0017] Furthermore, in the microbial agent, the effective viable count of Bacillus velezensis XY40-1 is 8 billion to 8.5 billion CFU / ml.

[0018] Furthermore, the microbial agent also contains active secondary metabolites produced after fermentation of Bacillus velezensis XY40-1, including new cyclic lipopeptide antibiotics Locilomycin, Bacillaene, Butirosin, and Macrolactin H.

[0019] Furthermore, the biosynthetic gene cluster of the new cyclic lipopeptide antibiotic roxithromycin is a type III lanthipeptide gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The type III lanthipeptide gene cluster is derived from the 193714-215381 base sequence of Bacillus velezensis XY40-1.

[0020] Furthermore, the biosynthetic gene cluster of the butyryl bacteriocin is a PKS-like gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. The PKS-like gene cluster is derived from the 936194-972547 base sequence of Bacillus velezensis XY40-1.

[0021] Furthermore, the biosynthetic gene cluster of macrolide H is a trans-AT PKS gene cluster, whose nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, and the transAT-PKS gene cluster is derived from the 1423622-1511453 base sequence of Bacillus velezensis XY40-1.

[0022] Furthermore, the biosynthetic gene cluster of bacillusene is a NRPS-TransAT-PKS hybrid gene cluster, whose nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and the NRPS-TransAT-PKS hybrid gene cluster is derived from the 1730988-1831722 base sequence of Bacillus velezensis XY40-1.

[0023] Compared with the fermentation method disclosed in the international patent (patent number: 18 / 163,866) "Bacillus Venlezensis, Fermentation Method, Fermentation Product and its Application" previously applied by the inventor, the preparation method of the microbial agent containing Bacillus velezensis XY40-1 provided by the present invention creatively improves the fermentation medium formula of the fermentation links such as seed liquid and bacterial liquid, greatly shortens the fermentation cycle, significantly reduces the fermentation cost, enriches the content and types of microbial metabolites, improves the functions of a series of products such as Bacillus velezensis agent in biological control and growth promotion, and has more advantages in product application.

[0024] The invention discloses an application of a microbial agent containing Bacillus velezensis XY40-1 in inhibiting the growth of pathogenic bacteria or in preparing a pathogenic bacteria growth inhibitor.

[0025] Furthermore, the pathogen is at least one of Phytophthora pathogen, Sclerotium rolfsii pathogen, Anthracnose pathogen, Red star pathogen, Fusarium wilt pathogen, root rot pathogen and Ralstonia bacterial wilt pathogen.

[0026] The invention discloses an application of the microbial agent containing Bacillus Velezii XY40-1 in preparing an agent for preventing and controlling at least one of pepper phytophthora, pepper white rot, pepper anthracnose, tobacco brown spot pathogen, cucumber wilt pathogen, tomato root rot pathogen and pepper bacterial wilt pathogen.

[0027] The invention also provides a biological pesticide suspension, comprising the microbial agent containing Bacillus velezensis XY40-1.

[0028] Furthermore, the biopesticide suspension also contains the following components in mass concentration: wetting and dispersing agent 3.5% to 3.8%, thickener 6% to 8%, preservative 0.16% to 0.2%, antifreeze agent 4% to 5%, UV protectant 0.5% to 0.6%, penetrant 0.15 to 0.25%, synergist 6.6 to 7%, defoamer 0.1% to 0.5%, and the following components in molar concentration: PQQ 900 to 1100 nmol / L.

[0029] Further preferably, the biopesticide suspension further comprises the following ingredients in mass concentration: wetting and dispersing agent 3.8%, thickener 8%, preservative 0.16%, antifreeze agent 4%, UV protectant 0.5%, penetrant 0.2%, synergist 6.8%, defoamer 0.2%, and the following molar concentration of ingredients: PQQ 1000nmol / L.

[0030] It should be noted that in biological pesticide suspensions, the role of PQQ is mainly to promote the fermentation of microorganisms and maintain their activity.

[0031] Furthermore, the wetting and dispersing agent includes Tween + sodium lignin sulfonate, wherein the mass ratio of Tween to sodium lignin sulfonate is 1:(0.75-0.9); the thickener includes 2% xanthan gum mother liquor; the preservative includes Kathon; the antifreeze agent includes ethylene glycol; the UV protectant includes skimmed milk powder; the penetrant includes orange peel essential oil; the enhancer includes lactose peptide + butanol fat, wherein the mass ratio of lactose peptide to butanol fat is 5:95; the defoaming agent includes dimethyl silicone oil.

[0032] Furthermore, the main components of the lactopeptide are organic acids, free amino acids, potassium, phosphorus, etc., wherein the organic acid concentration is 200-250g / L, the free amino acid concentration is 100-150g / L, and the molecular weight is all <300, the potassium content is 50-80g / L, and the phosphorus content is 10-15g / L, and it is a natural mixture specifically extracted from corn fermentation.

[0033] Furthermore, the main components of organic acids in the lactopeptide are propionic acid, lactic acid, citric acid, succinic acid, formic acid, acetic acid, and malonic acid; the main components of free amino acids are tryptophan, cystine, methionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, leucine, isoleucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline.

[0034] It should be noted that the present invention found that lactopeptide has significant effects on plant growth promotion and disease inhibition, but is sensitive to concentration differences. Lower or higher concentrations are not conducive to plant growth. Only the concentration range of the present invention has better growth promotion, quality improvement and disease inhibition effects or significant differences on plants.

[0035] When the biological pesticide suspension of the present invention is used for preventing and controlling pepper phytophthora, it is diluted 100 to 200 times before spraying.

[0036] The present invention also provides a microbial seed coating agent, comprising the microbial agent of Bacillus velezensis XY40-1.

[0037] Furthermore, the microbial seed coating agent also contains the following components in mass concentration: wetting and dispersing agent 3.5% to 3.8%, thickener 6% to 8%, preservative 0.16% to 0.2%, antifreeze agent 4% to 5%, UV protectant 0.5% to 0.6%, defoaming agent 0.1% to 0.5%, film-forming agent 4% to 10%, warning color 0.2% to 1%, synergist 0.05 to 0.15%, and the following molar concentration components: PQQ 40 to 60 nmol / L.

[0038] Further preferably, the microbial seed coating agent also contains the following components in mass concentration: wetting and dispersing agent 3.8%, thickener 8%, preservative 0.16%, antifreeze agent 4%, UV protectant 0.5%, defoaming agent 0.2%, film-forming agent 6%, warning color 0.65%, synergist 0.1%, and the following molar concentration of components: PQQ 50nmol / L.

[0039] Furthermore, the wetting and dispersing agent includes Tween + sodium lignin sulfonate, wherein the mass ratio of Tween to sodium lignin sulfonate is 1:(0.75-0.9); the thickener includes 2% xanthan gum mother liquor; the preservative includes kasson; the antifreeze agent includes ethylene glycol; the UV protectant includes skimmed milk powder; the defoaming agent includes dimethyl silicone oil; the film-forming agent includes polyvinyl pyrrolidone; the warning color includes basic fuchsin; and the synergist includes gibberellin.

[0040] It should be noted that in microbial seed coating agents, the main function of PQQ is to promote seed germination.

[0041] The microbial seed coating agent is used in pepper, eggplant and cabbage seeds.

[0042] Preferably, the weight ratio of the microbial seed coating agent to seeds is 1:(25-30).

[0043] The application of the microbial agent containing Bacillus Velezii XY40-1 in the preparation of a preservative. Beneficial effects of the present invention:

[0044] The control effect of Bacillus Velezii XY40-1 on white rot fungus, anthracnose fungus and red star fungus can reach more than 80%, which are 85.78%, 81.70% and 80.72% respectively, and can be used in the technical field of microbial control; the biological pesticide suspension provided by the invention can effectively control pepper blight; the microbial seed coating agent can significantly improve the germination rate of pepper, eggplant and cabbage seeds, and has a strong application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The colony morphology of Bacillus velezensis XY40-1 on LB solid medium;

[0046] Figure 2 The antagonistic results of Bacillus velezensis XY40-1 against pepper anthracnose, tobacco brown spot disease, pepper white rot, tomato root rot, cucumber wilt, pepper phytophthora and pepper bacterial wilt;

[0047] Figure 3 The present invention provides pepper seeds coated with the microbial seed coating agent;

[0048] Figure 4 It is the result of pepper seeds germinating after being coated with the microbial seed coating agent of the present invention;

[0049] Figure 5 Eggplant seeds coated with the microbial seed coating agent of the present invention;

[0050] Figure 6 It is the result of germination of eggplant seeds after being coated with the microbial seed coating agent of the present invention;

[0051] Figure 7 The present invention is cabbage seeds coated with the microbial seed coating agent;

[0052] Figure 8 The invention discloses a germination result of cabbage seeds coated with the microbial seed coating agent. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] The Bacillus velezensis XY40-1 involved in the present invention was deposited in the China Center for Type Culture Collection on March 29, 2022, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2022342.

[0055] The bacterial culture medium used in the following examples is as follows:

[0056] LB solid medium (g / L): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, add water to dissolve, finally make up to 1000 mL, pH 7.2, and sterilize by high pressure (121°C, 30 min).

[0057] LB liquid culture medium (g / L): peptone 20 g / L, sodium chloride 10 g / L, yeast extract powder 10 g / L, zinc sulfate 50 mmol / L, PQQ 1000 nmol / L, finally fixed to 1000 mL, pH 7.2, and sterilized by high pressure (121°C, 30 min).

[0058] Fermentation medium (g / L): soybean meal 20 g / L, glucose 14 g / L, magnesium sulfate 10 g / L, zinc sulfate 50 mmol / L, peptone 20 g / L, yeast extract powder 10 g / L, sodium chloride 10 g / L, potassium dihydrogen phosphate 10 g / L, PQQ 1000 nmol / L, finally make up to 1000 mL, pH 7.2, and sterilize under high pressure (121°C, 30 min).

[0059] Example 1 Antagonism experiment of Bacillus velezensis XY40-1 against 7 plant pathogens

[0060] Plate enforcement: refers to inoculating Phytophthora, Sclerotium rolfsii, Anthracnose, Alternaria alternata, Fusarium wilt, root rot, and Ralstonia solanacearum in the center of the PDA plate, and then inoculating the strain Bacillus velezensis XY40-1 at a distance of 2 cm from the center of the culture medium by the cross method; the control group is inoculated with pathogens but not Bacillus velezensis XY40-1, and cultured at a constant temperature of 28°C. After the pathogens in the control group have grown all over the plate, the width of the inhibition zone is measured, and each group is treated 6 times. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter * 100%; see the results. Figure 2 And as shown in Table 1.

[0061] Table 1 Inhibitory effect of Bacillus velez XY40-1 on 7 plant pathogens

[0062]

[0063] from Figure 2 The experimental results shown in Table 1 show that Bacillus velezensis XY40-1 can effectively inhibit the growth of Phytophthora, Sclerotium rolfsii, Anthracnose, Red Star Pathogen, Fusarium wilt, root rot pathogens, and Ralstonia solanacearum, especially the control effect on Sclerotium rolfsii, Anthracnose, and Red Star Pathogen can reach more than 80%, which are 85.78%, 81.70%, and 80.72% respectively, and can be used in the field of microbial control technology.

[0064] Example 2 Preparation of a microbial agent containing Bacillus velezensis XY40-1

[0065] The specific preparation process is as follows:

[0066] 1) Strain activation: Bacillus velezensis XY40-1 with a preservation number of CCTCC NO: M 2022342 was inoculated on LB solid medium and cultured at 30° C. for 1 day to obtain an activated strain;

[0067] 2) Fermentation seed culture: The activated strain was inoculated into LB liquid culture medium, and cultured at 36° C. and 160 rpm in a shaker for 8 hours to obtain Bacillus velezensis XY40-1 seed liquid;

[0068] 3) Fermentation: Inoculate the seed liquid of Bacillus velezensis XY40-1 into the fermentation medium of the fermenter at an inoculum amount of 1%, and culture at 36° C. and 190 rpm in a shaker for 12 hours to prepare a microbial agent containing Bacillus velezensis XY40-1.

[0069] The microbial agent prepared in this example was tested, and it was found that the effective viable count of Bacillus velezensis XY40-1 in the original bacterial solution of the microbial agent that had not been concentrated was 8.2 billion CFU / ml.

[0070] Ultra high performance liquid chromatography-mass spectrometry was used for detection (chromatographic column: Acquity UPLC HSS T31.8um2.1*100mm), and it was found through detection and analysis that the microbial agent also contained fermentation metabolites such as new cyclic lipopeptide antibiotics Locilomycin, Butirosin, Macrolactin H, and Bacillaene.

[0071] Example 3 Silencing expression and verification of metabolite synthesis gene cluster

[0072] Based on the antiSMASH software, the biosynthetic gene clusters of the new cyclic lipopeptide antibiotics roxithromycin, butyryl sphingomyelin, macrolide H, and bacillusene from Bacillus velezensis XY40-1 were analyzed by bioinformatics, and it was found that there were synthetic gene clusters for the above metabolites, and there was one synthetic gene cluster for each metabolite. The biosynthetic gene cluster of the new cyclic lipopeptide antibiotic roxithromycin is a type III lanthipeptide gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The biosynthetic gene cluster of butyryl sphingomyelin is a PKS-like gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. The biosynthetic gene cluster of macrolide H is a trans-AT PKS gene cluster, and its nucleotide sequence is a full-length sequence sequentially connected by sequences such as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. The biosynthetic gene cluster of bacillusene is a NRPS-TransAT-PKS hybrid gene cluster, and its nucleotide sequence is a full-length sequence sequentially connected by sequences such as SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0073] By silencing the expression of the above-mentioned metabolite synthesis gene clusters respectively, their antagonistic confrontation with seven major pathogenic microorganisms was verified after fermentation.

[0074] Using Bacillus velez XY40-1 as a template, primers for the upstream and downstream flanking homology arm regions of the target genes required for the new cyclic lipopeptide antibiotics roxithromycin, butyryl glycosides, macrolide H, and bacillusene were designed (see Table 2), the gene fragments were amplified, and double enzyme digestion (EcoRⅠ / XhoⅠ) was performed with the resistance vector PTK, and the two enzyme digestion products were mixed with T4 ligase, and connected at 16℃ for 3h. The ligation products were transformed into Escherichia coli DH5α competent cells, and then spread on a 100mg / L ampicillin resistance plate at 36℃ for overnight culture, and the recombinants were screened and initially verified to be correct by enzyme digestion. Pick a single colony of Bacillus velez XY40-1 and inoculate it in 5mL LB liquid medium without antibiotics, and culture it at 36℃ and 160rpm / min for 8h. Take 1mL of Bacillus Velez XY40-1 bacterial solution and add it to 24mL of new antibiotic-free LB liquid medium, and shake and culture it on a shaker at 36℃ and 180rpm / min. Take 1mL of culture solution at five time periods of 2h, 2.5h, 3h, 3.5h, and 4h, add it to 1mL of antibiotic-free LB liquid medium, and shake and culture it at 36℃ and 200rpm / min for 1h. After culturing the bacterial solution for 1h in each time period, 50uL of the constructed plasmid was added, and after shaking and culturing at 36℃ and 180rpm / min for 0.5h, 20mL of bacterial solution was evenly coated on a double-resistance plate with erythromycin (1ug / mL) and kanamycin (5ug / mL), and placed in a 36℃ incubator for overnight culture. The above-mentioned Bacillus Velez XY40-1 after silencing the expression of the metabolite synthesis gene cluster was subjected to biocontrol confrontation (as shown in Table 3) to verify its biocontrol difference.

[0075] Table 2 Primers required for metabolite synthesis

[0076]

[0077] Table 3 The inhibitory effect of Bacillus velezensis XY40-1 on pathogens after silencing the expression of each metabolite synthesis gene cluster

[0078]

[0079]

[0080] Example 4 Preparation of biological pesticide suspension

[0081] Tween-80, sodium lignin sulfonate, 2% xanthan gum, ethylene glycol, kason, skim milk powder, orange peel essential oil, lactose peptide, butanol fat and dimethyl silicone oil were added to the microbial agent containing Bacillus velezensis XY40-1 prepared in Example 2, wherein the amount of Tween-80 was 2% of the mass of the microbial agent, the amount of sodium lignin sulfonate was 1.8% of the mass of the microbial agent, the amount of 2% xanthan gum was 8% of the mass of the microbial agent, and the amount of ethylene glycol was 4% of the mass of the microbial agent. %, the amount of kason is 0.16% of the mass of the microbial agent, the amount of skim milk powder is 0.5% of the mass of the microbial agent, the amount of orange peel essential oil is 0.2% of the mass of the microbial agent, the amount of lactose peptide is 0.34% of the mass of the microbial agent, the amount of butanol fat is 6.46% of the mass of the microbial agent, and the amount of dimethyl silicone oil is 0.2% of the mass of the microbial agent. Stir and mix thoroughly, and finally add PQQ, stir and mix to obtain a biological pesticide suspension. In the biological pesticide suspension, the concentration of PQQ is 1000nmol / L. Lactopeptide can be purchased through commercial channels (Zhucheng Haotian Pharmaceutical Co., Ltd.), and its main components are organic acids, free amino acids, potassium, phosphorus, etc., among which the organic acid concentration is 200-250g / L, the free amino acid concentration is 100-150g / L, and the molecular weight is <300, the potassium content is 50-80g / L, and the phosphorus content is 10-15g / L. It is a natural mixture extracted from corn fermentation.

[0082] Example 5: Control of pepper phytophthora by biological pesticide suspension

[0083] The field effect of the biological pesticide suspension prepared in Example 4 on the biocontrol of pepper phytophthora was verified. The biological pesticide suspension was diluted 150 times with water, and the pepper of the variety Xingshu 215 was irrigated with roots, 330 square meters per treatment, and the drug was used twice before the onset and at the early stage of the disease, respectively. Clean water and a chemical pesticide 20% pyraclostrobin suspension (diluted 2000 times, used twice before the onset and at the early stage of the disease, respectively) were used as a control group. The results are shown in Table 4.

[0084] Table 4 The results of the control of pepper phytophthora by Bacillus Velez biopesticide suspension

[0085]

[0086] Example 6 Preparation of microbial seed coating agent

[0087] Tween-80, sodium lignin sulfonate, 2% xanthan gum, ethylene glycol, kason, skim milk powder, dimethyl silicone oil, basic fuchsin, gibberellin, and polyvinyl pyrrolidone were added to the microbial agent containing Bacillus velezensis XY40-1 prepared in Example 2, wherein the amount of Tween-80 was 2% of the mass of the microbial agent, the amount of sodium lignin sulfonate was 1.8% of the mass of the microbial agent, the amount of 2% xanthan gum was 8% of the mass of the microbial agent, and the amount of ethylene glycol was 1.6% of the mass of the microbial agent. 4%, the amount of Kathon is 0.16% of the mass of the microbial agent, the amount of skim milk powder is 0.5% of the mass of the microbial agent, the amount of dimethyl silicone oil is 0.2% of the mass of the microbial agent, the amount of basic fuchsin is 0.65% of the mass of the microbial agent, the amount of gibberellin is 0.1% of the mass of the microbial agent (dissolved in 2 ml of anhydrous ethanol), the amount of polyvinyl pyrrolidone is 6% of the mass of the microbial agent, stir well to mix, and finally add 50nmol / L PQQ, stir well to mix, to obtain the microbial seed coating agent.

[0088] Comparative Example 1

[0089] Compared with Example 6, the difference of this comparative example is that PQQ is not added at the end, and other conditions remain unchanged.

[0090] Example 7 Determination of the effect of microbial seed coating agents on crop germination

[0091] 1. Effect of microbial seed coating agent on germination rate of pepper seeds

[0092] Take the microbial seed coating agent of Example 6, mix it with pepper seeds at a weight ratio of 1:25 for coating, dry it in the shade after coating, take it out and place it in a culture dish with moist filter paper for sowing, add 3mL of sterile water, add 1mL of sterile water every day, soak the seeds CK and the microbial seed coating agent prepared in Comparative Example 1 in clean water as a control, place it at 28°C for cultivation, count the seed germination potential after 4 days, count the number of germinations every day, and count the pepper seed germination rate after 7 days. The experimental results are shown in Table 5.

[0093] Germination potential (%) = (number of seeds germinated in 4 days / total number of seeds tested) × 100

[0094] Germination rate (%) = (number of seeds germinated in 7 days / total number of seeds tested) × 100

[0095] Table 5 Effect of microbial seed coating agent on pepper seed germination

[0096]

[0097] 2. Effect of microbial seed coating agent on germination rate of eggplant seeds

[0098] Take the microbial seed coating agent of Example 6, mix it with eggplant seeds at a weight ratio of 1:30 for coating, dry it in the shade after coating, take it out and sow it in a petri dish with moist filter paper, add 3mL of sterile water, add 1mL of sterile water every day, soak the seeds CK and the microbial seed coating agent prepared in Comparative Example 1 in clean water as a control, place it at 28°C for cultivation, count the seed germination potential after 4 days, count the number of germinations every day, and count the pepper seed germination rate after 7 days. The experimental results are shown in Table 6.

[0099] Germination potential (%) = (number of seeds germinated in 4 days / total number of seeds tested) × 100

[0100] Germination rate (%) = (number of seeds germinated in 7 days / total number of seeds tested) × 100

[0101] Table 6 Effects of microbial seed coating agents on germination of eggplant seeds

[0102]

[0103] 3. Effect of microbial seed coating agent on germination rate of cabbage seeds

[0104] Take the seed coating agent of Example 6, mix it with cabbage seeds at a weight ratio of 1:27 for coating, dry it in the shade after coating, take it out and sow it in a culture dish with moist filter paper, add 3mL of sterile water, add 1mL of sterile water every day, soak the seeds CK and the microbial seed coating agent prepared in Comparative Example 1 in clear water as a control, place it at 28°C for culture, and count the seed germination rate after 2 days. The experimental results are shown in Table 7.

[0105] Table 7 Effects of microbial seed coating agents on cabbage seed germination

[0106]

[0107] Figure 4 , Figure 6 , Figure 8 The experimental results in Tables 5 to 7 show that the microbial seed coating agent provided by the present invention can significantly improve the germination rate of pepper, eggplant and cabbage seeds.

Claims

1. A kind of containing Bacillus Velez Bacillus velezensis ) A method for preparing a microbial agent of XY40-1, characterized in that: The microbial agent is an unconcentrated original bacterial liquid, in which Bacillus Velezii ( Bacillus velezensis ) The effective viable count of XY40-1 is 8 billion to 8.5 billion CFU / ml; the microbial agent also contains Bacillus Velezii ( Bacillus velezensis ) Active secondary metabolites produced after XY40-1 fermentation, including new cyclic lipopeptide antibiotics Locilomycin, Bacillaene, Butirosin, and Macrolactin H; The preparation method of the microbial agent comprises the following steps: 1) Strain activation: Bacillus Velezii with the deposit number of CCTCC NO: M 2022342 ( Bacillus velezensis ) XY40-1 was inoculated on LB solid medium and cultured at 28-30°C for 1-2 days to obtain an activated strain; 2) Fermentation seed culture: The activated strain was inoculated into LB liquid culture medium, and cultured in a shaker at 160-200 rpm at 36-39°C for 7-10 hours to obtain seed solution; The formula of the LB liquid culture medium is: 20 g / L peptone, 10 g / L sodium chloride, 10 g / L yeast extract powder, 50 mmol / L zinc sulfate, 1000 nmol / L PQQ, pH 7.2-7.3; 3) Fermentation: inoculating the seed liquid into the fermentation medium at an inoculation rate of 0.5% to 1.5%, and culturing the culture medium at 160 to 200 rpm in a shaker at 36 to 39° C. for 10 to 18 hours to obtain the microbial agent; The formula of the fermentation medium is: 20g / L soybean meal, 14g / L glucose, 10g / L magnesium sulfate, 50mmol / L zinc sulfate, 20g / L peptone, 10g / L yeast extract powder, 10g / L sodium chloride, 10g / L potassium dihydrogen phosphate, 1000nmol / L PQQ, and the pH of the fermentation tank is 7.2-7.

3.

2. The method for preparing the microbial agent according to claim 1, characterized in that: The biosynthetic gene cluster of the novel cyclic lipopeptide antibiotic roxithromycin is a type III lanthipeptide gene cluster, and its nucleotide sequence is a full-length sequence sequentially connected by sequences such as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3. The type III lanthipeptide gene cluster is derived from Bacillus velezine ( Bacillus velezensis ) The base sequence of positions 193714-215381 of XY40-1; the biosynthetic gene cluster of butyryl bacteriocin is a PKS-like gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ IDNO:4, SEQ IDNO:5, SEQ IDNO:6, and SEQ IDNO:7, and the PKS-like gene cluster is derived from Bacillus velezine ( Bacillus velezensis ) The base sequence of positions 936194-972547 of XY40-1; the biosynthetic gene cluster of macrolide H is a trans-AT PKS gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ IDNO:8, SEQ IDNO:9, SEQ IDNO:10, SEQ IDNO:11, SEQ IDNO:12, SEQ IDNO:13, SEQ IDNO:14, SEQ IDNO:15, and SEQ IDNO:16, and the transAT-PKS gene cluster is derived from Bacillus velezinis ( Bacillus velezensis ) The 1423622-1511453 base sequence of XY40-1; the biosynthetic gene cluster of bacillusene is a NRPS-TransAT-PKS hybrid gene cluster, and its nucleotide sequence is a full-length sequence connected in sequence by sequences such as SEQ IDNO:17, SEQ IDNO:18, SEQ IDNO:19, SEQ IDNO:20, SEQ IDNO:21, SEQ IDNO:22, SEQ IDNO:23, SEQ IDNO:24, SEQ IDNO:25, SEQ IDNO:26, and SEQ IDNO:27, and the NRPS-TransAT-PKS hybrid gene cluster is derived from Bacillus velezine ( Bacillus velezensis ) The base sequence of XY40-1 is 1730988-1831722.

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