Bacillus velezensis SMB1 and application thereof in preventing and treating plant diseases

Abstract

The invention relates to bacillus velezensis SMB1 and application thereof in prevention and treatment of plant diseases, and belongs to the technical field of biological prevention and treatment of plant diseases. The strain is preserved in the China Center for Type Culture Collection on November 27, 2024, the preservation address is Luojia mountain, Luojia mountain, eight road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M20242647. The bacillus velezensis SMB1 provided by the invention has a remarkable bacteriostatic effect on plant pathogenic fungi, plant pathogenic oomycetes, plant pathogenic bacteria and animal pathogenic bacteria, has an extremely good broad-spectrum bacteriostatic function, has a great application potential in biological control of plant diseases, can reduce the use of chemical pesticides, and has a good application prospect. The pesticide residue in agricultural products is reduced, the environment is protected, and the urgent demand on pollution-free agricultural product production at present is met.

Description

Technical Field

[0001] The invention relates to a strain of Bacillus Velezii SMB1 and application thereof in preventing and controlling plant diseases, belonging to the technical field of biological prevention and control of plant diseases. Background Art

[0002] Agriculture is the cornerstone of human civilization and is closely related to global food security and economic development. Soil-borne crop diseases have a wide range of occurrence, spread quickly, and are difficult to prevent and control. They have become one of the important factors affecting crop growth and yield, and have seriously restricted the sustainable development of agriculture.

[0003] Plant soil-borne diseases refer to pathogenic microorganisms that live in the soil or in the remains of diseased plants in the soil. When conditions are right, they attack crops from the roots or stems and cause diseases. The most common and serious soil-borne plant diseases, such as wheat fusarium head blight, tomato gray mold, cotton wilt, tobacco bacterial wilt and Chinese cabbage soft rot, cause great economic losses to agricultural production. Plant soil-borne diseases are mainly caused by pathogenic microorganisms, including fungi, bacteria, viruses and nematodes. Common soil-borne pathogenic microorganisms include Fusarium oxysporum, Rhizoctonia solani, Verticillium dahliae, Pythium, Phytophthora, Ralstonia solanacearum and Erwinia.

[0004] Tobacco bacterial wilt disease caused by Ralstonia solanacearum is a major tobacco disease, and its severity is increasing year by year. The occurrence of this disease has seriously affected the yield and quality of tobacco, and restricted the sustainable development of the tobacco industry. Ralstonia solanacearum is a Gram-negative bacterium that mainly infects the host through root wounds, causing the leaves of the host plant to wilt and turn yellow, or even the entire plant to die.

[0005] In agricultural production, the most widely used methods for the prevention and control of soil-borne diseases, including tobacco wilt, are to cultivate resistant varieties and use chemical fungicides. However, disease-resistant varieties have a long growth cycle and low fertilizer utilization rate; while chemical fungicides are prone to environmental pollution, food safety, and killing beneficial microorganisms. In recent years, biological control has been used to prevent and control soil-borne diseases of crops due to its environmental friendliness and lack of heavy metal pollution. At present, the microorganisms successfully used in biological control of crop diseases mainly include fungi and bacteria, among which Bacillus has been widely studied.

[0006] Bacillus spp. is a Gram-positive bacteria, a very diverse microbial group, widely found in soil, water and humus, including Bacillus subtilis, Bacillus licheniformis, Bacillus polymyxa and Bacillus velez. Bacillus spp. effectively prevents and controls a variety of plant diseases mainly through antagonism, competition, inducing plant systemic resistance and promoting plant growth, and is an ideal biocontrol microorganism. At the same time, Bacillus spp. plays an irreplaceable role in the addition of organic fertilizers, biological pesticides, feed and soil activation.

[0007] At present, the Bacillus spores that have been published in my country for the prevention and control of plant diseases mainly include Bacillus subtilis for the prevention and control of red star disease of Nanguo Pear, Bacillus dystrophiae for the prevention and control of white rot disease of peanut, Bacillus pumilus for the prevention and control of gray leaf spot disease of tomato, and Bacillus amyloliquefaciens for the prevention and control of root knot nematode disease of tomato. However, it is still unclear whether these published Bacillus spores have a broad spectrum of antibacterial effects on plant pathogens, especially no reports on the prevention and control of tobacco bacterial wilt disease have been published. Therefore, actively exploring and screening new Bacillus spores with antibacterial potential from the soil is of great significance for the biological control of soil-borne plant diseases. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a strain of Bacillus Velezii SMB1 and application thereof in preventing and controlling plant diseases.

[0009] The technical solution of the present invention is as follows:

[0010] A strain of Bacillus velezensis SMB1, which was deposited in the China Center for Type Culture Collection on November 27, 2024, with the deposit address at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M20242647.

[0011] Preferably according to the present invention, the gene sequence of 16SrDNA of Bacillus velezensis SMB1 is as shown in SEQ ID NO.1.

[0012] The method for preparing the secondary metabolite of Bacillus Velez SMB1 comprises the following steps:

[0013] The Velez subtilis SMB1 is inoculated into LB liquid culture medium, and is shaken and cultured for 4 to 6 days under the conditions of 25 to 30° C. and 150 to 200 rpm to obtain a Velez subtilis SMB1 fermentation liquid; the Velez subtilis SMB1 fermentation liquid is centrifuged, the supernatant is collected, and then adsorption filtration is performed to obtain a secondary metabolite of the Velez subtilis SMB1.

[0014] Application of the above-mentioned Bacillus Velez SMB1, Bacillus Velez SMB1 fermentation broth or Bacillus Velez SMB1 secondary metabolites in the following (1) to (6):

[0015] (1) Prevent and control bacterial wilt diseases;

[0016] (2) Inhibit the growth of plant pathogenic fungi;

[0017] (3) Inhibit the growth of plant pathogenic oomycetes;

[0018] (4) Inhibit the growth of plant pathogenic bacteria;

[0019] (5) Inhibit the growth of animal pathogenic bacteria;

[0020] (6) Preparation of microbial agents for preventing and controlling plant diseases.

[0021] Preferably according to the present invention, the bacterial wilt disease is tobacco bacterial wilt disease or tomato bacterial wilt disease.

[0022] Preferably according to the present invention, the plant pathogenic fungi include one or more of Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium proliferatum, Valsa ambiens, Botryosphaeria berengeriana, Valsa mali, Colletotrichum gloeosporioides and Thanatephorus cucumeris.

[0023] Preferably according to the present invention, the plant pathogenic oomycetes include one or more of Phytophthora megasperma, Phytophthora capsici, Phytophthora cactorum and Pythium ultimum.

[0024] Preferably according to the present invention, the plant pathogenic bacteria include one or more of Ralstonia solanacearum CQPS-1, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Xanthomonas campestris pv. campestris, Pseudomonas sringae pv. tomato, and Erwinia carotovora subsp. carotovora.

[0025] Preferably according to the present invention, the animal pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0026] A bacterial agent for preventing and controlling plant diseases, comprising the above-mentioned Bacillus Velez SMB1, fermentation liquid of Bacillus Velez SMB1 or secondary metabolites of Bacillus Velez SMB1.

[0027] According to the preferred embodiment of the present invention, the concentration of Bacillus Velezii SMB1 in the bacterial agent is ≥ 1×10 7 CFU / mL, or the concentration of the secondary metabolites of Bacillus velez SMB1 was above 1 μg / mL.

[0028] A method for preventing and controlling plant diseases comprises diluting a fermentation liquid of Bacillus Velez SMB1 or a secondary metabolite of Bacillus Velez SMB1 by 100 to 500 times to obtain a microbial agent containing Bacillus Velez SMB1, and then using the agent in the form of seed soaking, flushing or furrow spraying.

[0029] Where the present invention is not exhaustive, prior art may be used.

[0030] Beneficial Effects

[0031] 1. The present invention screened a strain of Bacillus velezensis SMB1 capable of preventing and controlling plant diseases. The strain has significant antibacterial effects on 15 plant pathogenic fungi and oomycetes, including Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Phytophthora sojae, Phytophthora capsici, Phytophthora fragariae, Pyrotrichum oleraceus, Botrytis cinerea, Pythium ulminans, Colletotrichum melanocarpa, Pythium ultimum, and the inhibition rate can reach more than 30%. The strain has excellent broad-spectrum antibacterial function and has great application potential in the biological control of plant diseases.

[0032] 2. The fermentation liquid of Bacillus Velez subtilis SMB1 or the secondary metabolites of Bacillus Velez subtilis SMB1 of the present invention not only has a significant antibacterial effect on the above 15 kinds of plant pathogenic fungi, but also has a significant antibacterial effect on 9 kinds of pathogenic bacteria such as Ralstonia solanacearum, Xanthomonas oryzae, Pseudomonas syringae, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. By irrigating the roots with a microbial agent containing Bacillus Velez subtilis SMB1, tobacco wilt disease and tomato wilt disease caused by Ralstonia solanacearum can be effectively prevented and treated, and the incidence rate is reduced by more than 30%.

[0033] 3. The Bacillus Velez SMB1, the fermentation liquid of Bacillus Velez SMB1 or the secondary metabolite of Bacillus Velez SMB1 of the present invention have application prospects for preparing fungal agents for preventing and controlling plant diseases, and are expected to provide a new biocontrol agent for the prevention and control of plant fungal, bacterial and oomycete diseases, which can reduce the use of chemical pesticides, reduce pesticide residues in agricultural products, and protect the environment, meeting the current urgent demand for the production of pollution-free agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the complete genome map of Bacillus velez SMB1.

[0035] Figure 2 This is the evolutionary tree of Bacillus velez SMB1.

[0036] Figure 3 It is the synthetic protein and conserved domain of the secondary metabolite of Bacillus velez SMB1.

[0037] Figure 4 This is a graph showing the antibacterial effect of Bacillus Velez SMB1 on different plant pathogenic fungi and oomycetes.

[0038] Figure 5 This is a graph showing the antibacterial effects of the secondary metabolites of Bacillus Velez SMB1 on different plant pathogenic fungi and oomycetes.

[0039] Figure 6 This is a graph showing the antibacterial effects of the secondary metabolites of Bacillus Velez SMB1 on different pathogenic bacteria.

[0040] Figure 7 This is a graph showing the control effect of Bacillus Velez SMB1 on tomato bacterial wilt disease.

[0041] Figure 8 This is a graph showing the control effect of Bacillus Velez SMB1 on tobacco bacterial wilt disease.

[0042] Fig. 9 It is the rate of tobacco diseased plants with different disease indexes of tobacco bacterial wilt disease. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0044] The culture and formulation used in the embodiments of the present invention are as follows:

[0045] LB medium: 10 g sodium chloride, 10 g tryptone, 5 g yeast extract, 1000 mL distilled water, pH 7; 15 g agarose (solid medium).

[0046] PDA medium: 200 g peeled potatoes, 20 g glucose, 1000 mL distilled water; 15 g agarose (solid medium).

[0047] The plant culture conditions in the embodiment are: daytime temperature 25°C, 16h; nighttime temperature 22°C, 8h.

[0048] The Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium genus, Phytophthora sojae, Phytophthora capsici, Phytophthora fragariae, Pythium pyrifos, Botrytis cinerea, Pythium ultimum, Ralstonia solanacearum, Xanthomonas oryzae, Pseudomonas syringae, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli used in the examples are all conventional strains and can be purchased commercially.

[0049] Example 1. Isolation and identification of strain SMB1

[0050] 1. Sample source: Tobacco rhizosphere soil from Qiaogou Village, Shitou Town, Linqu County, Weifang City, Shandong Province, China.

[0051] 2. Isolation of strains: Weigh 5 g of tobacco rhizosphere soil into a conical flask containing 50 mL of sterile water, shake and mix at 28°C and 180 rpm, and -1 ~10 -6The soil suspension was diluted gradually, 50 μL of the soil suspension was pipetted onto LB solid medium and evenly spread with a spreading rod, and cultured at 28°C for 1 to 2 days; a single colony was picked and streaked on LB solid medium, and cultured at 28°C for 1 to 2 days to obtain the target Bacillus.

[0052] 3. Identification of strains: Pick the target Bacillus into a conical flask containing 10 mL LB liquid medium, shake and culture at 28°C and 180 rpm for 1 to 2 days, centrifuge at 8000 rpm for 10 min, and collect the bacteria; extract genomic DNA according to the instructions of the OMEGA bacterial DNA extraction kit; refer to the instructions of the 16s rDNA Bacterial Identification PCR Kit of TaKaRa Company, and perform PCR amplification using the extracted genomic DNA as a template;

[0053] Forward primer 27F: 5′-AGAGTTTGATCATGGCTCAG-3′;

[0054] Reverse primer 1492R: 5′-AAGTCGTAACAAGGTAAGCG-3′;

[0055] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, post-extension at 72°C for 5 min, and 30 cycles.

[0056] According to the instructions of the OMEGA gel recovery kit, the PCR product was recovered and purified; the PCR product was sent to Qingdao Qingke Biotechnology Co., Ltd. for sequencing analysis, and the 16s rDNA sequence of the target Bacillus (as shown in SEQ ID NO.1) was analyzed by using the BLAST tool of NCBI for similarity with the reported typical strain sequences. The results showed that the similarity value with the genus Bacillus Velezii reached up to 99.93%, and it was determined that it was likely to be a strain of Bacillus Velezii.

[0057] 4. Evolutionary tree analysis of strains: The genomic DNA was sent to BGI Biotech for whole genome sequencing. The published whole genome sequence of Bacillus was downloaded from the Genome database of NCBI, and the whole genome evolutionary tree analysis was performed using MEGA7 software.

[0058] The whole genome sequencing results of the target Bacillus are as follows Figure 1 As shown, the genome length is 4.01Mbp, the GC content is 46.4%, there are 3805 protein coding genes and 86 tRNA genes; the whole genome evolution tree result is as follows Figure 2 shown.

[0059] Depend on Figure 1 and Figure 2 It can be seen that the similarity between the target Bacillus and Bacillus velezensis PC2906, Bacillus velezensis G0524 and Bacillus amyloliquefaciens JRS8 is 98.7%, 98.6% and 98.1% respectively, and the number of differential proteins is 338, 309 and 369 respectively; the highest similarity with other Bacillus is only 81%. Accordingly, the inventors of the present application named the strain Bacillus velezensis SMB1, S handongUniversity, State Key Laboratory of M icrobial Technology, B acteria,No.1).

[0060] The Bacillus velez SMB1 strain was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20242647, and the deposit date: November 27, 2024; the deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0061] Example 2: Prediction of gene clusters for secondary metabolite synthesis in Bacillus velez SMB1

[0062] The whole genome of Bacillus Velez SMB1 was analyzed using antiSMASH 6.1.0 to predict the synthetic gene clusters of secondary metabolites. The synthetic gene clusters of secondary metabolites of Bacillus Velez SMB1 and their secondary metabolites are shown in Table 1. The synthetic proteins and conserved domains of secondary metabolites of Bacillus Velez SMB1 are shown in Figure 3 shown.

[0063] Table 1

[0064]

[0065] As shown in Table 1, there are 14 secondary metabolite synthesis gene clusters in the whole genome of Bacillus velez SMB1, including 2 transAT-PKS gene clusters, 2 NRPS gene clusters, 2 terpene gene clusters, 1 phosphonate gene cluster, 1 PKS-like gene cluster, 1 transAT-PKS or T3PKS or NRPS gene cluster, 1 transAT-PKS or T3PKS or betalactone gene cluster, 1 T3PKS gene cluster, 1 NRPS or RiPP-like gene cluster, 1 lanthipeptide-class-ii gene cluster and 1 other gene cluster.

[0066] Depend on Figure 3 It can be seen that the domain prediction results of each secondary metabolite synthesis gene cluster showed that proteins SMB1_00353, SMB1_00354 and SMB1_00355 are responsible for the biosynthesis of surfactin; proteins SMB1_01445, SMB1_01446, SMB1_01447, SMB1_01448, SMB1_01449, SMB1_01450 and SMB1_01451 are responsible for the biosynthesis of macrolactin H; protein SMB1_01708, SMB1_01709, SMB1_01710, SMB1_01711 and SMB1_01712 are responsible for the biosynthesis of Bacillaene; SMB1_01865, SMB1_01864, SMB1_01863, SMB1_01862, SMB1_01861, SMB1_01837, SMB1_01836 and SMB1_01835 are responsible for the biosynthesis of Fengycin. In addition, Bacillus velez SMB1 also has the potential to synthesize secondary metabolites Bacillibactin, Bacilysin, Mersacidin and Butirosin.

[0067] The above results indicate that the fermentation broth of Bacillus Velez SMB1 and the secondary metabolites of Bacillus Velez SMB1 theoretically contain a variety of antibacterial and antibacterial substances.

[0068] Example 3: Determination of the antibacterial effect of Bacillus Velezii SMB1 on plant pathogenic fungi

[0069] The antibacterial effect of Bacillus Velezii SMB1 on plant pathogenic fungi was determined as follows:

[0070] (1) Pathogenic fungi and oomycetes such as Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium laminarum, Phytophthora sojae, Phytophthora capsici, Phytophthora fragariae, Pyrotrichum oleraceus, Botrytis cinerea, Pythium ultimum, Colletotrichum cucumeris, and Pythium ultimum were inoculated on PDA solid culture medium, and cultured at 28°C for 2 to 3 days;

[0071] (2) The Bacillus Velezii SMB1 isolated in Example 1 was inoculated into a conical flask containing 50 mL of LB liquid medium and cultured at 28° C. and 180 rpm until the OD 600 =1.0, to obtain the fermentation liquid of Bacillus Velez SMB1; take 1 mL of the fermentation liquid of Bacillus Velez SMB1 into a sterile 2 mL centrifuge tube, centrifuge at 8000 rpm for 10 min, discard the supernatant to collect the bacteria, suspend the bacteria in 1 mL of sterile water to obtain a bacterial suspension of Bacillus Velez SMB1;

[0072] (3) Using a sterile cork borer, take a 0.5 cm diameter bacterial cake along the edge of each pathogenic fungus and oomycete hyphae in step (1), and place the bacterial cake upside down on one end of the PDA solid culture medium. Using a pipette, draw 3 μL of the SMB1 bacterial suspension in step (2) on the other end of the PDA solid culture medium plate, and incubate at 28° C. for 2 to 7 days; the pathogenic fungi and oomycetes without the addition of the SMB1 bacterial suspension are the control group, and the pathogenic fungi and oomycetes with the addition of the SMB1 bacterial suspension are the treatment group;

[0073] (4) When the pathogenic fungi and oomycetes in the control group have grown all over the PDA solid culture medium plate, the colony diameters of the pathogenic fungi and oomycetes in the control group are measured, and the inhibition rate of the growth of each plant pathogenic fungus and oomycete by Bacillus Velezii SMB1 is calculated. The results are as follows: Figure 4 shown.

[0074] Calculation formula: Inhibition rate (%) = {colony diameter of pathogenic fungi or oomycetes in the control group (cm) - colony diameter of pathogenic fungi or oomycetes in the treatment group (cm) / colony diameter of pathogenic fungi or oomycetes in the control group (cm)} × 100.

[0075] Depend on Figure 4 It can be seen that Bacillus Velezii SMB1 can significantly inhibit the growth of pathogenic fungi and oomycetes such as Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Phytophthora sojae, Phytophthora capsici, Phytophthora fragariae, Pyrotrichum fusellae, Botrytis cinereae, Pythium ulminans, Colletotrichum cucurbitae, and Pythium ultimum, and has excellent broad-spectrum antibacterial function.

[0076] Among them, the inhibition rate of the growth of Rhizoctonia solani, Sclerotinia sclerotiorum, Apple Anthracnose, Botrytis cinerea and Strawberry Phytophthora can reach more than 50%; the inhibition rate of Fusarium graminearum, Botrytis cinerea, Fusarium solani, Soybean Phytophthora, Strawberry Phytophthora, Pear Black Cortex, Botrytis cinerea, Apple Black Cortex, Apple Anthracnose and Melon Phytophthora can reach 40-50%; the inhibition rate of Fusarium oxysporum, Capsicum Phytophthora and Ultimate Pythium can reach 30-40%.

[0077] Example 4: Determination of the Antibacterial Effect of Secondary Metabolites of Bacillus Velezii SMB1 on Plant Pathogenic Fungi

[0078] The antibacterial effect of the secondary metabolites of Bacillus Velezii SMB1 on plant pathogenic fungi was determined as follows:

[0079] (1) Pathogenic fungi and oomycetes such as Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium laminarum, Phytophthora sojae, Phytophthora capsici, Phytophthora fragariae, Pyrotrichum oleraceus, Botrytis cinereae, Pyrotrichum malus, Colletotrichum malus and Pseudomonas aeruginosa were inoculated on PDA solid culture medium, and cultured at 28°C for 2 to 3 days;

[0080] (2) The Velez subsp. SMB1 isolated in Example 1 was inoculated into a conical flask containing 100 mL of LB liquid culture medium, and cultured at 28° C. and 180 rpm for 5 days to obtain a fermentation broth of the Velez subsp. SMB1; the fermentation broth of the Velez subsp. SMB1 was centrifuged at 8000 rpm for 10 min, the supernatant was collected, 40 mL of the supernatant was taken, filtered through a C18 SPE adsorption column, and then 2 mL of methanol was added to the C18 SPE adsorption column to obtain a secondary metabolite solution of the Velez subsp. SMB1;

[0081] (3) Using a sterile cork borer, take a 0.5 cm diameter bacterial cake along the edge of each pathogenic fungus and oomycete hyphae in step (1), place the bacterial cake upside down in the middle of the PDA solid culture medium, place an Oxford cup 2 cm away, use a pipette to draw 80 μL of the SMB1 secondary metabolite solution in step (2) into the Oxford cup, and culture it at 28°C for 2 to 7 days; at the same time, use 10 μg of hygromycin as a positive control and 80 μL of methanol as a negative control;

[0082] (4) The radius of the negative control group and the treatment group of pathogenic fungi and oomycetes colonies was measured, and the inhibition rate of the secondary metabolites of Bacillus Velezii SMB1 on the growth of various plant pathogenic fungi and oomycetes was calculated. The results are as follows: Figure 5 shown.

[0083] Calculation formula: Inhibition rate (%) = {colony radius of pathogenic fungi or oomycetes in the negative control group (cm) - colony radius of pathogenic fungi or oomycetes in the treatment group (cm) / semi-diameter of pathogenic fungi or oomycetes in the control group (cm)} × 100.

[0084] Depend on Figure 5 It can be seen that the secondary metabolites produced by Bacillus Velezii SMB1 have significant antibacterial effects on Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Phytophthora sojae, Phytophthora capsici, Phytophthora fragariae, Pyrotrichum oleraceus, Botrytis cinerea, Pyrotrichum malus, Colletotrichum apples and Pseudomonas aeruginosa, and have excellent broad-spectrum antibacterial function.

[0085] Among them, the inhibition rate against sclerotinia, pear black rot fungus and apple black rot fungus reached more than 98%; the inhibition rate against Rhizoctonia solani, soybean Phytophthora, Botrytis cinerea, Fusarium oxysporum, strawberry Phytophthora, Botrytis cinerea and apple Anthracnose reached more than 80%; the inhibition rate against Fusarium solani, pepper Phytophthora, Fusarium graminearum and cucurbitacillus reached more than 50%.

[0086] Example 5. Determination of the antibacterial effect of secondary metabolites of Bacillus velez SMB1 on pathogenic bacteria

[0087] The antibacterial effect of the secondary metabolites of Bacillus Velezii SMB1 on pathogenic bacteria was determined as follows:

[0088] (1) Ralstonia solanacearum, Xanthomonas oryzae, Pseudomonas syringae, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli were cultured in conical flasks containing 50 mL of LB liquid medium and shaken at 28°C and 180 rpm until the OD 600 =1.0, dilute with sterile LB liquid medium to OD 600 =0.01;

[0089] (2) The Velez subsp. SMB1 isolated in Example 1 was inoculated into a conical flask containing 100 mL of LB liquid culture medium, and cultured at 28° C. and 180 rpm for 5 days to obtain a fermentation broth of the Velez subsp. SMB1; the fermentation broth of the Velez subsp. SMB1 was centrifuged at 8000 rpm for 10 min, the supernatant was collected, 40 mL of the supernatant was taken, filtered through a C18 SPE adsorption column, and then 2 mL of methanol was added to the C18 SPE adsorption column to obtain a secondary metabolite solution of the Velez subsp. SMB1;

[0090] (3) Use a pipette to draw 100 μL of the diluted pathogenic bacterial suspension onto the LB solid culture medium, spread the pathogenic bacterial suspension evenly with a spreading rod, place an Oxford cup, use a pipette to draw 80 μL of the SMB1 secondary metabolite solution prepared in step (2) into the Oxford cup, and incubate at 28° C. for 2 to 3 days; at the same time, use 6 μg of kanamycin as a positive control and 80 μL of methanol as a negative control.

[0091] (4) The radius of the pathogenic bacterial colonies in the negative control group and the treatment group was measured, and the inhibition rate of the secondary metabolites of Bacillus Velezii SMB1 on the growth of each pathogenic bacteria was calculated. The results are as follows: Figure 6 shown.

[0092] Depend on Figure 6 It can be seen that the secondary metabolites produced by Bacillus Velezii SMB1 can significantly inhibit the growth of Ralstonia solanacearum, Xanthomonas oryzae, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli, and have excellent broad-spectrum antibacterial function.

[0093] Among them, the diameter of the antagonism zone against rice bacterial leaf blight pathogen is 4.42±0.1cm, the diameter of the antagonism zone against rice streak pathogen is 4.31±0.06cm, the diameter of the antagonism zone against Xanthomonas campestris is 2.83±0.06cm, the diameter of the antagonism zone against Ralstonia solanacearum is 2.54±0.06cm, the diameter of the antagonism zone against Erwinia carotovora is 1.56±0.1cm, the diameter of the antagonism zone against Escherichia coli is 1.91±0.1cm, the diameter of the antagonism zone against Pseudomonas syringae is 2.03±0.06cm, the diameter of the antagonism zone against Pseudomonas aeruginosa is 1.53±0.06cm, and the diameter of the antagonism zone against Staphylococcus aureus is 1.83±0.06cm.

[0094] Example 6: Evaluation of the effect of Bacillus Velez SMB1 on controlling tomato bacterial wilt disease

[0095] The efficacy of Bacillus Velez SMB1 in controlling tomato bacterial wilt disease was evaluated as follows:

[0096] (1) The Bacillus Velezii SMB1 isolated in Example 1 was inoculated into a conical flask containing 100 mL of LB liquid medium and cultured at 28° C. and 180 rpm until the OD 600 =1.0; centrifuge at 6000rpm for 10min, discard the supernatant and collect the cells, dilute with sterile water to OD 600 =0.04, SMB1 bacterial agent was obtained;

[0097] (2) Soak tomato seeds in 75% alcohol for 1 min, wash with sterile water for 30 s, transfer to a culture dish containing filter paper moistened with sterile water, and place in a 4°C incubator in the dark for 2 days;

[0098] (3) The culture dish was transferred to a light incubator until the seeds germinated, and then transplanted into a pot containing sterile soil and cultured for another 30 days;

[0099] (4) Ralstonia solanacearum was cultured in a conical flask containing 50 mL of LB liquid medium and shaken at 28°C and 180 rpm until the OD 600 =1.0, centrifuge at 6000rpm for 10min, discard the supernatant and collect the cells, dilute with sterile water to OD 600 =0.01;

[0100] (5) In the control group, sterilized bamboo sticks were randomly inserted into the soil along the roots of tomatoes to cause mechanical damage, and 10 mL OD 600 = 0.01 diluted suspension of Ralstonia solanacearum; sterilized bamboo sticks were randomly inserted into the soil along the roots of tomatoes in the treatment group to cause mechanical damage, and 10 mL OD 600 = 0.01 diluted suspension of Ralstonia solanacearum, and 10 mL OD 600 = 0.04 SMB1 agent; continue to culture in the light incubator for 7 to 10 days, and count the number of wilted tomato plants; each treatment has at least 50 plants, and each group of experiments is repeated at least three times. The effect of tomato wilt disease is evaluated as follows: Figure 7 shown.

[0101] Depend on Figure 7 It can be seen that all the leaves of tomato plants inoculated with Ralstonia solanacearum wilted, while the number of wilting leaves of tomato plants irrigated with SMB1 was significantly reduced, and even no wilting symptoms appeared. This shows that irrigating the roots with microbial agents containing Bacillus Velezii SMB1 can effectively prevent and control tomato bacterial wilt caused by Ralstonia solanacearum.

[0102] Example 7: Evaluation of the effect of Bacillus Velezii SMB1 on controlling tobacco bacterial wilt disease

[0103] The efficacy of Bacillus Velez SMB1 in controlling tobacco bacterial wilt disease was evaluated as follows:

[0104] (1) The Bacillus Velezii SMB1 isolated in Example 1 was inoculated into a conical flask containing 100 mL of LB liquid medium and cultured at 28° C. and 180 rpm until the OD 600 =1.0; centrifuge at 6000rpm for 10min, discard the supernatant and collect the cells, dilute with sterile water to OD 600= 0.01, 0.02 and 0.04, and three SMB1 inoculants were obtained;

[0105] (2) The Velez subsp. SMB1 isolated in Example 1 was inoculated into a conical flask containing 100 mL of LB liquid culture medium, and cultured at 28° C. and 180 rpm for 5 days to obtain a Velez subsp. SMB1 fermentation broth; the Velez subsp. SMB1 fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was collected, and filtered with a 0.22 μm bacterial filter to obtain a SMB1 fermentation broth supernatant;

[0106] (3) Soak tobacco seeds in 75% alcohol for 1 min, wash with sterile water for 30 s, transfer to a pot containing sterile soil, and place in a 4°C incubator in the dark for 2 days;

[0107] (4) The flower pots were transferred to a light incubator, and the seeds were germinated to the four-leaf stage, and then transplanted into pots containing sterile soil and cultured for another 30 days;

[0108] (5) Cultivate Ralstonia solanacearum in a conical flask containing 50 mL of LB liquid medium and shake at 28°C and 180 rpm until the OD 600 =1.0, centrifuge at 6000rpm for 10min, discard the supernatant and collect the cells, dilute with sterile water to OD 600 =0.01;

[0109] (6) In the control group, sterilized bamboo sticks were randomly inserted into the soil along the roots of tobacco to cause mechanical damage, and 10 mL OD 600 = 0.01 diluted bacterial suspension of Ralstonia solanacearum; sterilized bamboo sticks were randomly inserted into the soil along the roots of tobacco in treatment group 1 to cause mechanical damage, and 10 mL of SMB1 fermentation liquid supernatant was irrigated along the roots; sterilized bamboo sticks were randomly inserted into the soil along the roots of tobacco in treatment group 2 to cause mechanical damage, and 10 mL of OD 600 = 0.01 SMB1 inoculant; in treatment group 3, sterilized bamboo sticks were randomly inserted into the soil along the roots of tobacco to cause mechanical damage, and 10 mL of SMB1 fermentation liquid supernatant and OD were irrigated along the roots. 600 = 0.02 SMB1 agent; in treatment group 4, sterilized bamboo sticks were randomly inserted into the soil along the roots of tobacco to cause mechanical damage, and 10 mL OD 600 =0.04 of SMB1 inoculant; continue to culture in the light incubator for 7 to 10 days, and count the number of withered tobacco plants;

[0110] (7) Count the number of tobacco with different disease indexes and calculate the incidence rate. The disease index standards are as follows:

[0111] Level 0: The whole plant is disease-free;

[0112] Level 1: There are occasional chlorotic spots on the stem, or less than 1 / 2 of the leaves on the diseased side wilt;

[0113] Level 3: There are black streaks on the stem, but not exceeding 1 / 2 of the stem height, or 1 / 2 to 2 / 3 of the leaves on the diseased side wilt;

[0114] Level 5: The black streaks on the stem exceed 1 / 2 of the stem height but do not reach the top of the stem, or more than 2 / 3 of the leaves on the diseased side wilt;

[0115] Level 7: Black streaks on the stem reach the top of the stem, or all leaves of the diseased plant wilt;

[0116] Level 9: All diseased plants died.

[0117] Incidence rate (%) = (number of diseased plants / total number of tobacco plants surveyed) × 100.

[0118] Each treatment had at least 50 plants, and each group of experiments was repeated at least three times. The effect evaluation of tobacco bacterial wilt disease was as follows: Figure 8 As shown in Figure 2, the incidence of tobacco bacterial wilt disease is as follows: Fig. 9 shown.

[0119] Depend on Figure 8 It can be seen that all leaves of tobacco plants inoculated with Ralstonia solanacearum withered, while OD 600 The leaves of tobacco plants treated with SMB1 at a concentration of = 0.04 showed almost no wilt symptoms.

[0120] Depend on Fig. 9 It can be seen that the zero-level disease rate of tobacco plants inoculated with Ralstonia solanacearum was 42.9%, while the supernatant of SMB1 fermentation liquid and OD 600 =0.01, OD 600 =0.02 and OD 600 =0.04, the 0-level morbidity of tobacco plants was 66.7%, 60%, 60% and 93.7% respectively; the 1-level morbidity of tobacco plants inoculated with Ralstonia solanacearum was 0, while the supernatant of SMB1 fermentation liquid, OD 600 =0.01, OD 600 =0.02 and OD 600 =0.04, the first-level morbidity of tobacco plants was 20%, 6.7%, 13.3% and 6.3% respectively; the third-level morbidity of tobacco plants inoculated with Ralstonia solanacearum was 0, while the same time, SMB1 fermentation liquid, OD 600 =0.01, OD 600 =0.02 and OD 600=0.04 of SMB1 inoculant, the 1st level morbidity of tobacco plants was 0, 6.6%, 20% and 0 respectively; the 7th level morbidity of tobacco plants inoculated with Ralstonia solanacearum was 57.1%, while the 7th level morbidity of tobacco plants inoculated with SMB1 fermentation liquid, OD 600 =0.01, OD 600 =0.02 and OD 600 =0.04 SMB1 microbial agent, the first-level morbidity of tobacco plants were 13.3%, 26.7%, 6.7% and 0 respectively. This shows that the microbial agent containing Bacillus Velez SMB1 can effectively prevent and control tobacco bacterial wilt disease caused by Ralstonia solanacearum by irrigating the roots with the fermentation liquid of Bacillus Velez SMB1, and the morbidity is reduced by more than 30%.

[0121] The above-described embodiments are only preferred specific implementation schemes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus velezensis SMB1 was deposited in the China Center for Type Culture Collection on November 27, 2024. The deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCCNO: M20242647.

2. The method for preparing the secondary metabolites of Bacillus Velezii SMB1 according to claim 1, characterized in that: The steps include: The Velez subtilis SMB1 is inoculated into LB liquid culture medium, and is shaken and cultured for 4 to 6 days under the conditions of 25 to 30° C. and 150 to 200 rpm to obtain a Velez subtilis SMB1 fermentation liquid; the Velez subtilis SMB1 fermentation liquid is centrifuged, the supernatant is collected, and then adsorption filtration is performed to obtain a secondary metabolite of the Velez subtilis SMB1.

3. Use of the Bacillus Velez SMB1 according to claim 1, the fermentation liquid of the Bacillus Velez SMB1 according to claim 2, or the secondary metabolites of the Bacillus Velez SMB1 in the following (1) to (6): (1) Prevent and control bacterial wilt diseases; (2) Inhibit the growth of plant pathogenic fungi; (3) Inhibit the growth of plant pathogenic oomycetes; (4) Inhibit the growth of plant pathogenic bacteria; (5) Inhibit the growth of animal pathogenic bacteria; (6) Preparation of microbial agents for preventing and controlling plant diseases.

4. The use according to claim 3, characterized in that The bacterial wilt disease is tobacco bacterial wilt disease or tomato bacterial wilt disease.

5. The use according to claim 3, characterized in that The plant pathogenic fungi include one or more of Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Pyrotrichum oleraceus, Botrytis cinerea, Pyrotrichum oleraceus, Colletotrichum apples, and Colletotrichum cucumeris.

6. The use according to claim 3, characterized in that The plant pathogenic oomycetes include one or more of soybean phytophthora, pepper phytophthora, strawberry phytophthora, and ultimum pythium; the plant pathogenic bacteria include one or more of Ralstonia solanacearum, oryzae bacterial blight, rice leaf spot pathogen, Xanthomonas campestris, Pseudomonas syringae, and Erwinia carotovora.

7. The use according to claim 3, characterized in that The animal pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

8. A fungal agent for preventing and controlling plant diseases, characterized in that: The bacterial agent includes the Bacillus Velez SMB1 according to claim 1, the fermentation liquid of the Bacillus Velez SMB1 according to claim 2, or the secondary metabolite of the Bacillus Velez SMB1.

9. The microbial agent for preventing and controlling plant diseases according to claim 8, characterized in that: The concentration of Bacillus Velez SMB1 in the bacterial agent is ≥ 1×10 7 CFU / mL, or the concentration of the secondary metabolites of Bacillus velez SMB1 was above 1 μg / mL.

10. A method for preventing and controlling plant diseases, characterized in that: The microbial agent containing Bacillus Velez SMB1 is obtained by diluting the fermentation liquid of Bacillus Velez SMB1 or the secondary metabolite of Bacillus Velez SMB1 described in claim 2 by 100 to 500 times, and then used in the form of seed soaking, flushing or ditch spraying.

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