Bacillus velezensis BJ-1141 and application thereof in preventing and treating plant diseases

Abstract

The invention provides a bacillus velezensis strain and application thereof. The name of the bacillus velezensis is BJ-1141, and the bacillus velezensis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32094. The strain has broad-spectrum antibacterial activity, and has a good inhibition effect on 24 plant pathogenic fungi such as rice pathogenic bacteria, wheat pathogenic bacteria, corn pathogenic bacteria, grape pathogenic bacteria, cherry pathogenic bacteria, solanaceous crop pathogenic bacteria and the like. Wherein the growth inhibition effect on sclerotinia sclerotiorum hyphae causing eggplant sclerotinia rot is optimal, and the inhibition rate can reach 95.57%. The BJ-1141 bacterial liquid has an obvious growth promoting effect on the growth of plants such as tomatoes and lettuce. After the BJ-1141 bacterial liquid is used for irrigating roots of potted tomatoes, the tomato fusarium wilt caused by tomato fusarium oxysporum can be effectively controlled, the BJ-1141 bacterial liquid has a good prevention and treatment effect, the prevention and treatment effect can reach 85.93%, and the prevention and treatment effect is obviously different from that of a control group.

Description

Technical Field

[0001] The present invention belongs to the field of microbial applications, and particularly relates to a Bacillus velezensis strain with broad-spectrum antibacterial activity and its applications, especially in the application of using this strain to prepare a microbial agent for controlling soil-borne diseases of protected vegetables. Background Art

[0002] Protected vegetables have become an important part of the development of the vegetable industry in China. However, with the increase in the development years of protected vegetables, unreasonable fertilization and continuous cropping of crops have led to increasingly serious soil obstacles such as deterioration of soil physical and chemical properties and imbalance of biological systems, and also exacerbated the occurrence of soil-borne diseases of protected vegetables. Among them, fusarium wilt caused by Fusarium oxysporum as the main pathogenic bacterium is the most common and serious fungal disease among soil-borne diseases. Since this type of disease generally occurs in the root and stem parts of plants, it has strong concealment, and when symptoms are found, it has often developed relatively seriously, missing the best control period, and ultimately resulting in serious damage to yield and quality. At present, for the control of soil-borne diseases of protected vegetables, treatment methods such as soil disinfection and high-temperature greenhouse steaming are mostly used. Although the effects are obvious, they will seriously damage the soil ecological environment and do not conform to the concept of sustainable development of green agriculture in China. Therefore, biological control methods with the advantages of being environmentally friendly, safe for humans and animals, and having a long-lasting control effect are gradually becoming an important means for controlling crop soil-borne diseases. Currently, the microbial pesticides commonly used for controlling soil-borne fungal diseases of vegetables mainly include Bacillus subtilis, Paenibacillus polymyxa, Trichoderma, etc. Compared with chemical pesticides, the types are significantly scarce. Therefore, screening new strains that can effectively control soil-borne fungal diseases of protected vegetables is of great significance for enriching the biological control resources of soil-borne diseases and creating new microbial pesticides.

[0003] Biocontrol bacteria of the genus Bacillus spp. are one of the most studied biocontrol bacteria at present, with characteristics such as stable physical and chemical properties, strong stress resistance, and a wide antibacterial spectrum, and some strains have been commercially applied. Among them, Bacillus velezensis, as a beneficial plant growth-promoting rhizobacterium, has obvious protective effects on various plant diseases such as mango anthracnose, konjac soft rot, cucumber leaf spot, cucumber downy mildew, tomato gray mold, rice bacterial leaf streak, wheat sheath blight, rapeseed sclerotinia rot, and black shank (Research status and application prospects of Bacillus velezensis. Chinese Journal of Microecology, 2024, 36(3): 351-356), and it is a biocontrol bacterium with excellent properties. At present, many strains of Bacillus velezensis have been widely studied, but the characteristics of each strain are different, and the number of strains that have been registered and applied in production is extremely small, and there is an urgent need to develop new strains. Summary of the Invention

[0004] The object of the present invention is to provide a strain of Bacillus velezensis BJ-1141 and its application in controlling plant diseases. This bacterium has a broad antibacterial spectrum and can effectively inhibit common plant pathogenic fungi, and has inhibitory effects on rice pathogens, wheat pathogens, corn pathogens, grape pathogens, cherry pathogens, and solanaceous crop pathogens, and has an obvious control effect on tomato wilt disease.

[0005] The Bacillus velezensis of the present invention was collected and isolated from the rhizosphere soil of rice in a severely rice blast-infected field in Hongshan District, Wuhan City, Hubei Province. Its taxonomic name is Bacillus velezensis, and the name is BJ-1141. It was deposited at the China General Microbiological Culture Collection Center (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on September 27, 2024, and the deposit number is CGMCC No. 32094.

[0006] When the Bacillus velezensis BJ-1141 strain of the present invention is cultured on an LB plate medium, the colony is milky white, round or nearly round, with a wrinkled and raised edge, and the surface is dry and granular. Based on the molecular identification of the 16S rRNA gene and the gyrA gene, this strain was identified as Bacillus velezensis. This strain can produce amylase, protease, cellulase, pectinase, and siderophore, and has the ability to form biofilms.

[0007] The present invention also provides the application of the Bacillus velezensis BJ-1141 in preventing and controlling common plant pathogenic fungal diseases, which has relatively broad-spectrum antibacterial activity. The pathogenic fungi are Magnaporthe oryzae, Fusarium graminearum, Bipolaris sorokiniana, Exserohilum turcicum, Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Dactylonectria macrodidyma, Paraeutypella citricola, Phaeoacremonium iranianum, Rosellinia necatrix, Phoma quercina, Botrytis cinerea, Passalora fulva, Fusarium oxysporum f.sp. Lycopersici, Fusarium oxysporum f.sp. radicis-lycopersici, Fusarium oxysporum f.sp. cucumerinum, Fusarium chlamydosporum, Plectosphaerella cucumerina, Corynespora cassiicola, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium solani, Phytophthora capsici, etc., one or more of them.

[0008] The present invention also provides the application of the Bacillus velezensis BJ-1141 strain or bacterial liquid in preventing and controlling tomato wilt and promoting tomato growth, and can be used to prepare biological pesticides or microbial bacterial fertilizers.

[0009] The application of the Bacillus velezensis or its antibacterial active substance in preparing biological pesticides and / or biological bacterial fertilizers having antagonistic activity against plant pathogenic fungi and / or growth-promoting effects on plants also belongs to the protection scope of the present invention.

[0010] The application of the Bacillus velezensis in promoting the growth of plant seedlings also belongs to the protection scope of the present invention. In the said application, preferably, the method for promoting the growth of plant seedlings is to dilute the bacterial liquid of Bacillus velezensis BJ-1141 to a concentration of 1×10 9 CFU / mL and perform root irrigation treatment on plants; the plants are preferably tomatoes and / or lettuce.

[0011] Compared with the prior art, the beneficial effects of the present invention include:

[0012] (1) The Bacillus velezensis BJ-1141 of the present invention has broad-spectrum antibacterial activity and has good inhibitory effects on 24 plant pathogenic fungi such as rice pathogenic bacteria, wheat pathogenic bacteria, corn pathogenic bacteria, grape pathogenic bacteria, cherry pathogenic bacteria and solanaceous crop pathogenic bacteria. Among them, the Bacillus velezensis BJ-1141 has the best inhibitory effect on the mycelial growth of Sclerotinia sclerotiorum causing eggplant sclerotinia, and the inhibition rate can reach 95.57%.

[0013] (2) The bacterial liquid of Bacillus velezensis BJ-1141 of the present invention has an obvious growth-promoting effect on the growth of plants such as tomatoes and lettuce.

[0014] (3) After the bacterial liquid of Bacillus velezensis BJ-1141 of the present invention is used for root irrigation of potted tomatoes, it can effectively control tomato wilt caused by Fusarium oxysporum f. sp. lycopersici, and has a good control effect. The control effect can reach 85.93%, and there is a significant difference compared with the control effect. Description of the Drawings

[0015] Figure 1 Colony characteristics of strain BJ-1141, wherein, A, colony morphology on LB plate; B, colony morphology under stereomicroscope;

[0016] Figure 2 Phylogenetic tree based on 16S rRNA gene and gyrA gene;

[0017] Figure 3 Detection of antibacterial and growth-promoting characteristics of strain BJ-1141, wherein, A, amylase; B, protease; C, cellulase; D, pectinase; E, siderophore;

[0018] Figure 4 Detection of biofilm formation ability of strain BJ-1141;

[0019] Figure 5 Plate antibacterial effect diagram of strain BJ-1141 against different plant pathogenic fungi;

[0020] Figure 6Control effect of strain BJ-1141 on tomato Fusarium wilt in pot experiments.

[0021] Biological material preservation

[0022] Name: Bacillus velezensis BJ-1141;

[0023] Taxonomic naming: Bacillus velezensis;

[0024] Date of deposit: September 27, 2024;

[0025] Depositary institution: China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing);

[0026] Deposit number: CGMCC No. 32094. Detailed implementation methods

[0027] Example 1: Isolation and identification of Bacillus velezensis BJ-1141

[0028] 1. Isolation and purification of the strain

[0029] Isolation of the strain: The strain isolation material was taken from the rhizosphere soil of rice in a severely rice blast-infected field in Hongshan District, Wuhan City, Hubei Province. Samples were taken at three points, with a distance of 10 meters between every two sampling points. 3 - 4 plants were taken from each point, and the samples were taken to a depth of 10 - 15 cm underground. 1 g of the rhizosphere soil of each collected plant was weighed and mixed evenly, and sterile water was added for gradient dilution to obtain soil dilutions with concentrations of 1×10 -5 g / ml, 1×10 -6 g / ml, and 1×10 -7 g / ml. The limited dilution method was used to isolate bacteria in a 96-well cell culture plate. First, 200 μl of 1 / 10 TSB liquid medium was added to each well, and then 10 μl of the soil dilution was added. After preliminary experiments, the soil dilution concentration (1×10 -6 g / ml) at which the number of turbid wells accounted for about 2 / 3 of the total number of wells of the dilution was selected for the formal experiment, and 10×96 wells were replicated. The 96-well plate was incubated at 25°C for 20 days.

[0030] Purification of the strain: The streak plate method was used. A sterilized toothpick was used to pick up the bacterial liquid in the turbid well and streaked on the LB solid medium for incubation at 25°C for 1 - 3 days, and single colonies were picked and preserved.

[0031] 2. Screening of the strain

[0032] The plate confrontation culture method was adopted. Magnaporthe oryzae was inoculated in the center of the PDA medium plate respectively. The isolated and purified bacterial strains were inoculated at four points 3 cm away from the fungal cake. The control group was only inoculated with the pathogen. It was cultured in the dark at 25 °C for 7 d, the antibacterial situation was observed, and the width of the antibacterial zone was measured. Each treatment was repeated 3 times. According to the inhibition rate, the strain with the most obvious antibacterial effect was selected, that is, the Bacillus velezensis BJ-1141 described in the present invention was obtained.

[0033] 3. Identification of the strain

[0034] The colonies of Bacillus velezensis BJ-1141 on the LB solid medium were milky white, round or nearly round, with the edges wrinkled and raised. Under the stereomicroscope, the surface of the colonies was observed to be dry and granular ( Figure 1 ). The genomic DNA of strain BJ-1141 was extracted using a bacterial genomic DNA extraction kit (Tiangen, Beijing). Using the genomic DNA of strain BJ-1141 as a template, the universal primers 27F and 1492R for the bacterial 16S rRNA gene and the universal primers gyrA-F and gyrA-R for the gyrA gene were selected for PCR amplification. The primer sequences were as follows:

[0035] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0036] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0037] gyrA-F: 5′-CAGTCAGGAAATGCGTACGTCCTT-3';

[0038] gyrA-R: 5′-CAAGGTAATGCTCCAGGCATTGCT-3'.

[0039] The PCR reaction system was (50 μL): 44 μL of 1.1×S4 Fidelity PCR Mix, 2 μL of DNA template, 2 μL of 27F / gyrA-F, and 2 μL of 1492R / gyrA-R.

[0040] The PCR amplification reaction procedure was: pre-denaturation at 95 °C for 5 min, denaturation at 98 °C for 10 s, annealing for 30 s, extension at 72 °C for 30 s, 35 cycles, and finally final extension at 72 °C for 5 min, and termination of the reaction at 4 °C (the annealing temperature for the 16S rRNA gene was 52 °C, and the annealing temperature for the gyrA gene was 60 °C).

[0041] The PCR amplification products were detected by 1% agarose gel electrophoresis and then sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for sequencing. The 16S rRNA gene sequence (Sequence 1 in the sequence listing) and gyrA gene sequence (Sequence 2 in the sequence listing) of the obtained strain BJ-1141 were compared with the sequences in the GenBank database of the NCBI website to obtain the strain species with the highest similarity to it.

[0042] The comparison results showed that the similarity of the 16S rRNA gene sequence of strain BJ-1141 to the 16S rRNA gene sequence of Bacillus velezensis D4 (GenBank accession number: MT271916.1) was 99.90%, and the similarity of the gyrA gene sequence (GenBank accession number: MT329071.1) was 99.78%. It was preliminarily identified as a strain of the Bacillus velezensis-related population.

[0043] The gene sequences of related strains with high homology were downloaded, and the 16S rRNA gene and gyrA gene sequences were compared and spliced using MEGA 11 software. A phylogenetic tree of strain BJ-1141 was constructed by the neighbor-joining method, and the bootstrap value was set to 1000. The results showed ( Figure 2 ), strain BJ-1141 was clustered together with the standard strain of Bacillus velezensis, Bacillus velezensis strain D4. Based on this, strain BJ-1141 was identified as Bacillus velezensis. Strain BJ-1141 was deposited in the China General Microbiological Culture Collection Center (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date was September 27, 2024, and the deposit number was CGMCC No. 32094.

[0044] 4. Detection of the antibacterial and growth-promoting characteristics of strain BJ-1141

[0045] 4.1 Amylase detection

[0046] Detection medium: 10 g of soluble starch, 5 g of yeast powder, 10 g of peptone, 10 g of NaCl, 17 g of agar, 1000 mL of water.

[0047] Detection method: Take 5 μL of the bacterial liquid of strain BJ-1141 with OD600 = 1 cultured for 24 h and spot it on the amylase detection medium, and culture it at 28 °C for 3 d. Flood the amylase detection medium with Lugol's iodine solution, and a clear transparent hydrolysis zone can be seen around the colonies producing amylase. Observe and measure the diameter (D) of the transparent zone and the diameter (d) of the colonies, and judge the amylase-producing ability of the strain according to the value of D / d.

[0048] 4.2 Protease Detection

[0049] Detection medium: A: 5 g of yeast extract, 10 g of peptone, 10 g of NaCl, 17 g of agar, 900 mL of water; B: 7 g of skim milk powder, 100 mL of water; A and B are sterilized separately and then mixed.

[0050] Detection method: Take 5 μL of the bacterial solution of strain BJ-1141 with OD600 = 1 cultured for 24 h and spot-inoculate it on the protease detection medium, culture at 28 °C for 3 d, observe whether a clear transparent hydrolysis zone appears around the colony. If it appears, it indicates the production of protease. Measure the diameter (D) of the transparent zone and the diameter (d) of the colony, and judge the protease-producing ability of the strain according to the value of D / d.

[0051] 4.3 Cellulase Detection

[0052] Detection medium: 7.5 g of sodium carboxymethyl cellulose, 1 g of KH 2 PO 4 1, 1 g of peptone, 0.5 g of yeast extract, 0.5 g of MgSO 4 0.5 g, 1.5 g of NaCl, 15 g of agar.

[0053] Detection method: Take 5 μL of the bacterial solution of strain BJ-1141 with OD600 = 1 cultured for 24 h and spot-inoculate it on the cellulase detection medium, culture at 28 °C for 3 d, stain with 1 g / L congo red for 1 h, and then soak and wash with 1 mol / L NaCl solution twice for decolorization, soak for 30 min each time. Observe whether a transparent zone appears around the colony, measure the diameter (D) of the transparent zone and the diameter (d) of the colony, and judge the cellulase-producing ability of the strain according to the value of D / d.

[0054] 4.4 Pectinase Detection

[0055] Detection medium: 8 g of pectin, 1.4 g of NH 4 Cl, 2 g of KH 2 PO 4 2, 6 g of K 2 HPO 4 ·3H 2 O, 0.1 g of MgSO 4 ·7H 2 O, 15 g of agar, 1 L of distilled water, natural pH.

[0056] Detection method: Take 5 μL of the bacterial liquid of strain BJ-1141 with an OD600 of 1 after 24 hours of cultivation and spot inoculate it on the pectinase detection medium. Incubate at 28 °C for 3 days, stain with 1 g / L congo red for 30 minutes, and then decolorize by soaking and washing twice with 1 mol / L NaCl solution, soaking for 30 minutes each time. Measure the diameter (D) of the transparent circle and the diameter (d) of the colony, and judge the pectinase-producing ability of the strain according to the value of D / d.

[0057] 4.5 Siderophore activity detection

[0058] The ability to produce siderophores was detected by the chrome azurol S agar colorimetric method, and the detection medium used was the improved solid siderophore detection medium (Beijing Coolaber Technology Co., Ltd.).

[0059] Detection method: Take 5 μL of the bacterial liquid of strain BJ-1141 with an OD600 of 1 after 24 hours of cultivation and spot inoculate it on the siderophore activity detection medium. Incubate at 28 °C for 3 days, observe whether there is a yellow halo around the colony, measure the diameter (D) of the yellow halo and the diameter (d) of the colony, and judge the siderophore-producing ability of the strain according to the value of D / d.

[0060] 4.6 Phosphorus-solubilizing ability detection

[0061] Detection medium: Glucose 10 g, calcium phosphate 5.0 g, magnesium chloride 5.0 g, magnesium sulfate heptahydrate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, agar 17 g, PH 7.0 ± 0.2 (25 °C).

[0062] Detection method: Take 5 μL of the bacterial liquid of strain BJ-1141 with an OD600 of 1 after 24 hours of cultivation and spot inoculate it on the NBRIP phosphorus-solubilizing medium. Incubate at a constant temperature of 28 °C for 3 days, observe whether there is a hydrolysis circle around the colony. Measure the diameter (D) of the hydrolysis circle and the diameter (d) of the colony, and determine the phosphorus-solubilizing ability of the strain according to the value of D / d. The stronger the phosphorus-solubilizing ability, the larger the hydrolysis circle.

[0063] 4.7 Potassium-solubilizing ability detection

[0064] Detection medium: Sucrose 10 g, magnesium sulfate heptahydrate 0.5 g, ammonium sulfate 0.2 g, sodium chloride 0.1 g, calcium carbonate 0.1 g, potassium feldspar powder 5 g, agar 20 g, water 1 L, pH 7.2.

[0065] Detection method: Take 5 μL of the bacterial liquid of strain BJ-1141 with an OD600 of 1 after 24 hours of cultivation and spot inoculate it on the potassium-solubilizing medium. Incubate at a constant temperature of 28 °C for 3 days, observe whether there is a hydrolysis circle around the colony. Measure the diameter (D) of the hydrolysis circle and the diameter (d) of the colony, and determine the potassium-solubilizing ability of the strain according to the value of D / d. The stronger the potassium-solubilizing ability, the larger the hydrolysis circle.

[0066] 4.8 Detection of nitrogen fixation ability

[0067] Modified Ashby's medium: K 2 HPO 4 0.2 g, MgSO 4 ·7H 2 O 0.2 g, NaCl 0.2 g, CaCO 3 5 g, K 2 SO 4 0.1 g, glucose 10 g, agar 15 g, pH 7.4 ± 0.2.

[0068] Detection method: Streak inoculate the strain BJ-1141 on the modified Ashby's medium and incubate at 28 °C for 5 - 7 d. Observe the colony size and morphology. Bacteria with nitrogen fixation ability will grow into viscous, translucent, white or brown colonies.

[0069] 4.9 Detection of biofilm formation ability

[0070] Inoculate a single colony of the strain into LB liquid medium and incubate at 28 °C and 200 r / min for 24 h. Then adjust the bacterial concentration with a UV spectrophotometer to three concentrations of OD600 = 0.4, 0.6, and 1.0. Take 4 mL of bacterial solutions with different concentrations and put them into sterilized test tubes respectively. Use sterile water as a control, seal the tubes, and incubate at 28 °C statically for 48 h. Pour out the bacterial solution in the test tubes, wash the test tubes with sterilized water, add 5 mL of 0.1% crystal violet staining solution, and place at 28 °C for 20 min. Pour out the crystal violet staining solution and wash the test tubes with sterilized water. Observe whether there is blue-violet on the test tube wall. If so, it indicates that the strain can produce biofilm. Then add 4 mL of 95% ethanol solution to the test tubes to dissolve the biofilm completely in the ethanol, and observe and evaluate the intensity of biofilm formation. The experiment is repeated 3 times.

[0071] The above research results show that the strain BJ-1141 can produce amylase, protease, cellulase, pectinase, and siderophore ( Figure 3 ), has the ability to form biofilm ( Figure 4 ), and does not have the ability to dissolve phosphorus and potassium and nitrogen fixation. The measurement results (Table 1) show that the strain BJ-1141 has strong protease and cellulase activities.

[0072] Table 1 Determination results of antibacterial and growth-promoting activities of Bacillus velezensis BJ-1141

[0073]

[0074]

[0075] Example 2: Determination of antibacterial activity of Bacillus velezensis BJ-1141 against common plant pathogens

[0076] Growth inhibition tests of Bacillus velezensis BJ-1141 obtained by separation were carried out on rice pathogens, wheat pathogens, corn pathogens, grape pathogens, cherry pathogens, and solanaceous crop pathogens. Using the plate confrontation culture method, a puncher (5 mm) was used to cut out the mycelial plugs from the colonies of Magnaporthe oryzae, Fusarium graminearum, Bipolaris sorokiniana, Exserohilum turcicum, Lasiodiplodia theobromae, Botryosphaeria dothide, Diaporthe eres, Dactylonectria macrodidyma, Paraeutypella citricola, Phaeoacremonium iranianum, Rosellinia necatrix, Nothophoma quercina, Botrytis cinerea, Passalora fulva, Fusarium oxysporum f.sp. Lycopersici, Fusarium oxysporum f.sp. radicis-lycopersici, Fusarium oxysporum f.sp. cucumerinum, Fusarium chlamydosporum, Plectosphaerella cucumerina, Corynespora cassiicola, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium solani, and Phytophthora capsici, which had been cultured on PDA medium at 25 °C for 3 - 7 d. The mycelial plugs were placed about 2 cm from the edge of the PDA plate, and a filter paper strip with a length × width of 4 × 0.5 cm was placed about 2 cm from the edge of the PDA plate on the other side. At the same time, 20 μL of the cultured bacterial liquid (OD 600(= 1.0) was dropped onto filter paper strips. Filter paper strips dropped with the same dose of clear water were used as controls. This was repeated 3 times. After culturing in an incubator at 25 °C until the radius of the control pathogenic bacteria colony in the direction of the filter paper strip exceeded the filter paper strip, the radius of the colony in the direction of the filter paper strip was measured, and the antibacterial rate was calculated.

[0077] Antibacterial rate (%) = (radius of control pathogenic bacteria colony - radius of treated pathogenic bacteria colony) / radius of control pathogenic bacteria colony × 100%

[0078] Table 2 Antibacterial activities of Bacillus velezensis BJ - 1141 against different phytopathogenic fungi

[0079]

[0080]

[0081] The results showed (Table 2, Figure 5 ). Strain BJ - 1141 had inhibitory effects on the tested phytopathogenic fungi to varying degrees. Among them, the inhibitory effect on the colony growth of Sclerotinia sclerotiorum was the strongest, with an average inhibitory rate reaching 95.57%. The antibacterial activity against Phytophthora capsici was the worst, with an average inhibitory rate of 34.25%. The inhibitory rates against the remaining 22 kinds of pathogenic fungi were all above 50%, indicating that the strain shown in the present invention had a broad antibacterial spectrum.

[0082] Example 3: Determination of the growth - promoting effect of Bacillus velezensis BJ - 1141 bacterial liquid

[0083] 1. Effect of Bacillus velezensis BJ - 1141 bacterial liquid on the growth of tomato seedlings

[0084] 1.1 Materials and methods

[0085] 1.1.1 Preparation of strain BJ - 1141 bacterial liquid

[0086] The strain BJ - 1141 was activated on LB solid medium and cultured in an incubator at 28 °C for 48 h. Single bacterial colonies were picked and inoculated into LB liquid medium. It was cultured at 28 °C and 180 r / min with constant shaking for 24 h as the seed liquid. The seed liquid was inoculated into a 500 mL conical flask containing 300 mL of LB liquid medium at 5% (volume fraction), and cultured at 28 °C and 180 r / min with constant shaking for 24 h, and then the bacterial liquid was collected and adjusted to 1×10 6 、1×10 7 、1×10 8 、1×10 9 for 4 concentrations for standby.

[0087] 1.1.2 Growth-promoting effect of Bacillus velezensis strain BJ-1141 bacterial solution on tomato seedlings

[0088] The growth-promoting effect of Bacillus velezensis strain BJ-1141 bacterial solution on tomato seedlings was determined by the root irrigation method. Tomato seeds were treated with 2% NaClO for 1 min, rinsed 3 times with sterile water, then treated with 75% ethanol for 1 min, and rinsed 3 times with sterile water. After disinfection, the seeds were evenly placed in a petri dish with 0.8% water agar, wrapped with a black bag, and placed in an incubator at 25 °C for germination. Tomato seeds with similar germination status were selected and sown in flower pots (diameter at the mouth 13.0 cm, diameter at the bottom 9.0 cm, height 12 cm) filled with nutrient substrate. After emergence, healthy plants with consistent growth vigor were retained. When the tomato seedlings had one true leaf, 50 mL of the prepared bacterial solution at different concentrations was used for root irrigation, and an equal amount of sterile water was used for root irrigation as a blank control. Root irrigation was carried out three times, with an interval of 7 d each time; there were 10 pots for each treatment, and the experiment was repeated 3 times. The flower pots were placed in a greenhouse at 25 °C for cultivation.

[0089] 1.1.3 Data statistics and analysis

[0090] On the 28th day after the first root irrigation, the growth status of the tomato seedlings was measured and photographed, including plant height, root length, fresh weight and dry weight of the above-ground part and roots, and the chlorophyll content was measured using a SPAD-502 chlorophyll meter at the same time. The data were statistically analyzed using SPSS 27 software.

[0091] 1.2 Determination results of related indexes of tomatoes after treatment with Bacillus velezensis BJ-1141 bacterial solution

[0092] The results showed (Table 3) that starting from the concentration of 1×10 8 CFU / mL, the plant height, fresh weight of the above-ground part, dry weight of the above-ground part, and chlorophyll content of the tomato seedlings treated with the bacterial solution were significantly higher than those treated with clear water; in addition, when the concentration was 1×10 9 CFU / mL, the stem diameter of the tomato seedlings was also significantly higher than that treated with clear water.

[0093] Table 3 Determination results of related indexes of tomatoes after treatment with Bacillus velezensis BJ-1141 bacterial solution

[0094]

[0095] 2. Effect of Bacillus velezensis BJ-1141 bacterial solution on the growth of lettuce seedlings

[0096] 2.1 Materials and methods

[0097] 2.1.1 Preparation of Bacillus velezensis strain BJ-1141 bacterial solution

[0098] The method was the same as 1.1.1 in Example 3.

[0099] 2.1.2 Growth promotion effect of Bacillus velezensis BJ-1141 bacterial liquid on lettuce seedlings

[0100] Sow lettuce seeds in flower pots (13.0 cm in diameter, 9.0 cm in bottom diameter, 12 cm in height) filled with nutrient substrate. After emergence, keep healthy plants with consistent growth. After lettuce seedlings grow one true leaf, pour 50 mL of each prepared bacterial liquid with different concentrations into the roots, and pour the same amount of sterile water into the roots as the blank control. Irrigate the roots three times in total, with a 7-day interval between each time; 10 pots for each treatment, repeated 3 times. Place the flower pots in a greenhouse at 25 °C for cultivation.

[0101] 2.1.3 Data statistics and analysis

[0102] Measure and record the growth status of lettuce seedlings and take pictures on the 28th day after the first root irrigation, measure plant height, plant width, leaf length, leaf width, single-plant weight, and measure the chlorophyll content using a SPAD-502 chlorophyll meter at the same time. Use SPSS 27 software to statistically analyze the data.

[0103] 2.2 Determination results of related indicators of lettuce after treatment with Bacillus velezensis BJ-1141 bacterial liquid

[0104] The results show (Table 4) that starting from the concentration of 1×10 6 CFU / mL, the plant height and plant width of lettuce seedlings treated with the bacterial liquid are significantly higher than those treated with clear water; starting from the concentration of 1×10 7 CFU / mL, the leaf length, leaf width, and single-plant weight of lettuce seedlings treated with the bacterial liquid are also significantly higher than those treated with clear water; when the concentration is 1×10 8 CFU / mL and 1×10 9 CFU / mL, the chlorophyll content of the plants treated with the bacterial liquid also begins to be significantly higher than that of the clear water control.

[0105] Table 4 Determination results of related indicators of lettuce after treatment with Bacillus velezensis BJ-1141 bacterial liquid

[0106]

[0107] Example 4: Determination of the control effect of Bacillus velezensis BJ-1141 bacterial liquid on tomato wilt in potted plants

[0108] 1.1 Materials and methods

[0109] 1.1.1 Preparation of Bacillus velezensis BJ-1141 bacterial liquid

[0110] The method is the same as 1.1.1 in Example 3, and the bacterial liquid concentration is adjusted to 1×10 7 、1×10 8 、5×10 8 、1×10 9 CFU / mL for standby.

[0111] 1.1.2 Preparation of pathogen spore suspension

[0112] Activate Fusarium oxysporum f.sp. lycopersici (Fol) in PDA solid medium and culture it in an incubator at 25 °C for 7 days. Cut out fungal cakes with a 7 mm punch, pick 5 fungal cakes and inoculate them into a 500 mL conical flask containing 300 mL of PDB liquid medium. After culturing at 25 °C and 180 r / min with constant shaking for 3 days, filter the bacterial liquid with magic gauze, centrifuge at 5000 r / min for 7 min, discard the supernatant, collect conidia, and prepare a conidia suspension with a concentration of 1×10 7 conidia / mL for standby use.

[0113] 1.1.3 Control effect of strain BJ-1141 on tomato wilt

[0114] The control effect of BJ-1141 bacterial liquid on tomato wilt was determined by the method of root injury irrigation. The cultivation method of tomato seedlings was the same as that in 1.1.2 of Example 3. Select healthy tomato seedlings at the two-leaf and one-heart stage with consistent growth, transplant them into flower pots (diameter 13.0 cm, bottom diameter 9.0 cm, height 12.0 cm), use about 1000 mL of soil per pot, and plant 1 plant per pot. After slightly injuring the roots of tomato seedlings, soak them in the pathogen conidia suspension for 30 min and then transplant them. Then, irrigate and inoculate 100 mL of the pathogen conidia suspension, and respectively conduct 50 mL of root irrigation treatment with 4 concentrations of BJ-1141 bacterial liquid 24 h after inoculating the pathogen. Use clear water root irrigation, root irrigation with only the pathogen, and root irrigation with only BJ-1141 bacterial liquid as controls, and use 700-fold liquid of 30% hymexazol aqueous solution (Jiangxi Heyi Chemical Co., Ltd., PD20110226) and 15-fold liquid of 500 million CFU / g Paenibacillus polymyxa suspension (Wuhan Kenuo Biotechnology Co., Ltd., PD20184026) as control agents. Each treatment has 10 plants and is repeated 3 times, and is cultured in a greenhouse at 25 - 30 °C.

[0115] Controls: ① Irrigate 50 mL of clear water; ② Irrigate 100 mL of the pathogen; ③ Irrigate 50 mL of BJ-1141 bacterial liquid at 1×10 9 CFU / mL.

[0116] 1.1.4 Data statistics and analysis

[0117] After inoculating the pathogen for 21 days, the disease incidence and disease index of each treatment were statistically analyzed, and the data were statistically analyzed using SPSS27 software.

[0118] Disease grading standard for tomato wilt:

[0119] 0: No symptoms;

[0120] 1: One or two leaves are significantly yellowed;

[0121] 2: Three or four true leaves are yellowed and the leaves wilt and droop;

[0122] 3: Five or six true leaves are yellowed or the true leaves wilt and droop;

[0123] 4: The whole plant severely wilts and dies.

[0124] The calculation formulas are as follows:

[0125] Incidence rate (%) = Number of diseased plants / Total number of surveyed plants × 100%

[0126] Disease index = ∑(Number of diseased plants at each level × Value of that disease level) / (Total number of plants × Highest disease level value) × 100%

[0127] Control effect (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100%

[0128] 1.2 Pot control effect of Bacillus velezensis BJ - 1141 bacterial liquid on tomato fusarium wilt

[0129] The results showed (Table 5, Figure 6 ), that the 4 treatment concentrations of Bacillus velezensis BJ - 1141 bacterial liquid all had good treatment effects on tomato fusarium wilt, and the control effects were all above 50%. With the increase of concentration, the treatment effect gradually improved. Among them, the bacterial liquids with concentrations of 5×10 8 CFU / mL and 1×10 9 CFU / mL had the best treatment effects, with incidence rates of 32.78% and 26.11% respectively, and the control effects could reach 81.13% and 85.93%. There was no significant difference in the incidence rate and control effect between the two. After drenching the roots of tomato seedlings with 700 - fold liquid of 30% hymexazol aqueous solution as the control agent, the incidence rate of tomato seedlings was 73.89% and the control effect was 48.95%; after drenching the roots of plants with 15 - fold liquid of 500 million CFU / g Paenibacillus polymyxa suspension agent, the incidence rate of the plants was 73.89% and the control effect was 50.82%. The treatment effects of the two control agents were significantly lower than that of strain BJ - 1141.

[0130] Table 5 Control effect of Bacillus velezensis BJ - 1141 bacterial liquid on tomato fusarium wilt

[0131]

[0132] The above-described embodiments only illustrate several implementation manners of the present invention. The description is relatively specific, but it is illustrative rather than restrictive for the present invention. For those of ordinary skill in the art, many modifications, variations, and improvements can be made without departing from the spirit and scope defined by the appended claims, and these all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus velezensis, named Bacillus velezensis BJ-1141, characterized in that: The Bacillus Velezii has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.32094.

2. Use of the Bacillus Velezii described in claim 1 in the preparation of a biocontrol agent, microbial fertilizer or biological pesticide for inhibiting plant pathogenic fungi.

3. The use according to claim 2, characterized in that: The plant pathogenic fungi are Magnaporthe oryzae, Fusarium graminearum, Bipolaris sorokiniana, Exserohilum turcicum, Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Dactylonectria macrodidyma, Paraeutypella citricola, Phaeoacremonium iranianum, Roselliniia necatrix, Nothophoma quercina, Bottytis cinerea, Passalora fulva, Fusarium oxysporum f.sp.Lycopersici), Fusarium oxysporum f.sp.radicis-lycopersici, Fusarium oxysporum f.sp.cucumerinum, Fusarium chlamydosporum, Plectosphaerella cucumerina, Corynespora cassiicola, Rhizoctonia solsni, Sclerotirnia sclerotiorum, Fusarium solani, and Phytophthora capsici.

4. A biocontrol agent for plant pathogenic fungi, characterized in that: The active ingredient of the biocontrol agent is the Bacillus Velezii described in claim 1.

5. The biocontrol agent according to claim 4, characterized in that The plant pathogenic fungi are Magnaporthe oryzae, Fusarium graminearum, Bipolaris sorokiana, Exserohilum turcicum, Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Dactylonectria macrodidyma, Paraeutypella citricola, Phaeoacremonium iranianum, Rosellini necatrix, Nothophoma quercina, Botrytis cinerea, Passalora fulva, Fusarium oxysporum f.sp.Lycopersici), Fusarium oxysporum f.sp.radicis-lycopersici, Fusarium oxysporum f.sp.cucumerinum, Fusarium chlamydosporum, Plectosphaerella cucumerina, Corynespora cassiicola, Rhizoctonia solani, Sclerotiniasclerotiorum, Fusarium solani, and Phytophthora capsici.

6. A microbial fertilizer for plant diseases, characterized in that: The active ingredient of the microbial fertilizer is the Bacillus Velezii described in claim 1.

7. The microbial fertilizer according to claim 6, characterized in that: The plant pathogens causing the plant diseases are Magnaporthe oryzae, Fusarium graminearum, Bipolaris sorokiniana, Exserohilum turcicum, Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Dactylonectria macrodidyma, Paraeutypella citricola, Phaeoacremonium iranianum, Rosellini necatrix, Nothophoma quercina, Botrytis cinerea, Passalorafulva, Fusarium oxysporum f.sp.Lycopersici), Fusarium oxysporum f.sp.radicis-lycopersici, Fusarium oxysporum fsp.cucumerinum, Fusarium chlamydosporum, Plectosphaerella cucumerina, Corynespora cassiicola, Rhizoctonia solani, Sclerotiniasclerotiorum, Fusarium solani, and Phytophthora capsici.

8. A biological pesticide for plant diseases, characterized in that: The active ingredient of the biological pesticide is the Bacillus Velezii described in claim 1.

9. The biopesticide according to claim 8, characterized in that: The biopesticide also includes adjuvants and / or additives acceptable to pesticide formulations.

10. Use of the Bacillus Velez subtilis according to claim 1 in promoting plant growth; the plants are preferably tomatoes and / or lettuce.

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