Bacillus velezensis ZX101 and application thereof in prevention and treatment of tea tree leaf blight

Abstract

The invention discloses bacillus velezensis ZX101 and application thereof in prevention and treatment of tea tree leaf blight. According to the bacillus velezensis ZX101, the bacillus velezensis ZX101 with high inhibitory activity on Diapotheca mongkongensis is obtained by separating and screening tea tree leaves in a sunshine tea garden, and the preservation number of the bacillus velezensis ZX101 is CCTCC (China Center for Type Culture Collection) NO: M2024470; the strain biological agent has good biocontrol potential for the tea tree leaf blight caused by Diapotheca mongkongensis, and a new strain resource is provided for preventing and treating the tea tree leaf blight in production. Meanwhile, the foliar fertilizer with bacillus velezensis ZX101 as an effective component is prepared, synergistic interaction of all substance components of the foliar fertilizer is achieved, and the foliar fertilizer is suitable for biological prevention and control of the tea tree leaf blight and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biocontrol strains, and particularly relates to a strain of Bacillus velezensis ZX101 and application thereof in the prevention and treatment of tea leaf blight. Background Art

[0002] Tea leaf blight is the most common leaf disease in tea gardens. It is widely distributed and occurs in all tea-growing areas in China. Diseased leaves fall off early, branches and tips wither, weakening the tree and reducing yield. In severe cases, the tea garden appears brown and young tea trees may die. Pathogens of the genus Metarhizium are the main pathogens that cause tea leaf blight.

[0003] At present, the prevention and control of tea leaf blight is mainly based on agricultural prevention and control and chemical prevention. On the basis of using disease-free seeds and relatively resistant (tolerant) varieties and strengthening cultivation management to improve plant resistance, comprehensive prevention and control measures are adopted with a focus on spraying during the growth period. However, the long-term and large-scale use of chemical pesticides damages the ecological environment and harms human health. In recent years, with people's increasing attention to tea food safety issues and the rise of organic tea planting, how to safely and effectively prevent and control tea leaf blight in the tea production process has become an urgent problem to be solved.

[0004] The research of Bacillus velezii mainly focuses on the fields of feed, medicine, textile, aquatic products, sewage treatment, plant protection, etc. As a new type of biocontrol microbial factor, Bacillus velezii has attracted much attention in plant disease prevention and control, promoting plant growth, etc. Bacillus velezii can effectively inhibit a variety of plant pathogens, such as pepper phytophthora, fusarium solani, fusarium oxysporum, and Rhizoctonia solani. And several pathogenic fungi on leafy vegetables include brown spot fungus, stem spot mildew, gray Botrytis cinerea dianthus variant and rapeseed sclerotinia fungus, effectively preventing and controlling a variety of plant diseases. It has been reported that it can be used to prevent and control diseases such as wheat leaf blight, tea tree tea ring spot, pear gray mold and penicillium, rice blast and tomato early blight. CN117821339A discloses a strain of Bacillus velezii B2 and its application in preventing and controlling wheat leaf blight, effectively reducing the growth of micronodular mold hyphae, and having a good prevention and control effect on wheat leaf blight caused by micronodular mold, with a prevention effect of up to 95.4% in the greenhouse. CN110564646A discloses a strain of Bacillus Velez CY30 and its application in the prevention and treatment of tea ring spot disease. Bacillus Velez CY30 is used for foliar spraying to prevent and treat tea ring spot disease on tea trees, and root irrigation has the effect of promoting the growth and increasing the yield of tea trees.

[0005] The efficacy of biocontrol strains is affected not only by the biological characteristics of the strains themselves, but also by the soil environment, climate and geographical conditions. Currently, some biocontrol strains have problems such as short duration and unstable effects. Therefore, further screening of biocontrol strain resources with stronger adaptability and exploring the efficient mode of action of biocontrol strains are of great significance for the prevention and control of tea leaf blight. Summary of the invention

[0006] In view of the above problems, the present invention provides a strain of Bacillus velezensis ZX101 and an application thereof in the prevention and treatment of tea leaf blight. The strain is Bacillus velezensis ZX101, which can significantly inhibit the growth of Diaporthe hongkongensis. At the same time, Bacillus velezensis ZX101 is used as an effective ingredient, and pullulan, trehalose and potassium humate are used to improve the attachment rate and survival rate of Bacillus velezensis ZX101 on the leaf surface. The prepared foliar fertilizer realizes the synergistic enhancement of various material components, is suitable for the biological prevention and treatment of tea garden leaf blight, and shows broad application prospects.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention isolates and screens Bacillus velezensis ZX101 with high inhibitory activity against Diaporthe hongkongensis from tea leaves in Rizhao tea garden. The strain has been deposited in China Center for Type Culture Collection on March 14, 2024, with a deposit number of CCTCC NO: M2024470, and the deposit address is Wuhan University, Wuhan, China. The strain grows on nutrient agar medium, and the colony is milky white and opaque, with a sticky surface, rough and wrinkled skin, uneven edges, and a single colony is irregular in shape.

[0009] The invention provides a biological bacterial agent with Bacillus velezensis ZX101 as an effective component.

[0010] The preparation method of the above-mentioned Bacillus velezensis ZX101 biological agent is characterized in that, under sterile conditions, the activated Bacillus velezensis ZX101 is inoculated into LB liquid culture medium, and cultured at 35-40°C and 150-200rpm for 20-30h to prepare the Bacillus velezensis ZX101 biological agent with a concentration of 1×10 8 -1×10 9 cfu / mL.

[0011] The invention provides a foliar fertilizer with Bacillus velezensis ZX101 as an effective ingredient.

[0012] The preparation method of the above-mentioned foliar fertilizer is:

[0013] (1) Preparation of seed liquid of Bacillus velez ZX101 and Aureobasidium pullulans with a deposit number of CGMCC No. 33984;

[0014] (2) Preparation of fermentation medium;

[0015] (3) inoculating the Bacillus velezensis ZX101 seed solution obtained in step (1) into the fermentation medium obtained in step (2) for culturing;

[0016] (4) adding the budding Aureobasidium pullulans seed solution prepared in step (1) to the fermentation medium obtained in step (3) and continuing to culture to obtain a mixed fermentation solution;

[0017] (5) Adding trehalose and potassium humate to the mixed fermentation liquid obtained in step (4) to obtain foliar fertilizer.

[0018] Furthermore, the preparation method of the Bacillus Velez ZX101 seed liquid in step (1) is: inoculating Bacillus Velez ZX101 into LB culture medium, culturing at 35-40° C. for 20-30 hours, and preparing the Bacillus Velez ZX101 seed liquid.

[0019] Furthermore, the method for preparing the budding Aureobasidium pullulans seed solution in step (1) is: inoculating budding Aureobasidium pullulans into potato glucose culture medium, culturing at 25-30°C for 45-50h, and preparing the budding Aureobasidium pullulans seed solution.

[0020] Furthermore, the preparation method of the fermentation medium in step (2) is as follows: soybean meal powder, corn starch and water are mixed, protease is added for enzymolysis at 40-45°C for 2-3h, amylase is added for enzymolysis at 65-70°C for 2-3h, corn steep liquor, beef extract, peptone, glucose, sodium chloride, calcium carbonate, dipotassium hydrogen phosphate, ferric chloride and manganese sulfate are added in sequence, mixed, sterilized under high pressure at 115-121°C for 15-25min, and cooled to room temperature; the fermentation medium is: soybean meal powder 65-7 0g / L, corn starch 82-87g / L, corn steep liquor 5-10g / L, beef extract 1-3g / L, peptone 5-10g / L, glucose 8-15g / L, sodium chloride 2-5g / L, calcium carbonate 1-4g / L, dipotassium hydrogen phosphate 0.5-1.5g / L, ferric chloride 0.2-0.6g / L, manganese sulfate 0.03-0.06g / L, protease 1-4g / L, amylase 1-4g / L, the rest is water, pH is 6.8-7.2.

[0021] Preferably, the fermentation medium is: 68 g / L soybean meal, 85 g / L corn starch, 6 g / L corn steep liquor, 2.5 g / L beef extract, 9 g / L peptone, 10 g / L glucose, 3 g / L sodium chloride, 2.5 g / L calcium carbonate, 0.8 g / L dipotassium hydrogen phosphate, 0.5 g / L ferric chloride, 0.05 g / L manganese sulfate, 2.5 g / L protease, 2.5 g / L amylase, and the rest is water, with a pH of 7.0.

[0022] Furthermore, the culture method in step (3) is as follows: 1-5% of the seed solution of Bacillus velezensis ZX101 is inoculated into the fermentation medium at a volume ratio and cultured at 35-40° C. for 55-65 h until the fermentation liquid concentration reaches 1×10 8 -1×10 9 cfu / mL.

[0023] Furthermore, the culture method of step (4) is: adding 2-5% by volume of the budding Aureobasidium pullulans seed liquid into the fermentation medium after the culture in step (3), and culturing for 35-40 hours at 25-30°C and 100-130r / min until the pullulan content in the fermentation liquid is 4-8g / L.

[0024] Furthermore, in step (5), the added amounts of trehalose and potassium humate are 2-4% and 1-3% by volume respectively.

[0025] The invention provides an application of a Bacillus velez ZX101 biological bacterial agent in the prevention and treatment of tea tree leaf blight caused by Diaporthehongkongensis.

[0026] The invention also provides a foliar fertilizer with Bacillus velezensis ZX101 as an effective ingredient for use in preventing and treating tea tree leaf blight caused by Diaporthe hongkongensis.

[0027] The specific method of the above application is as follows: mix foliar fertilizer and water in a volume ratio of 1:10-20, add 1-3‰ Tween-80, and stir thoroughly to obtain the solution; use the spray method to apply the pesticide in a forward-moving manner, with a usage amount of 55-65kg / mu of solution, apply the pesticide for the second time 10-15d after application, and apply the pesticide 2-3 times in total.

[0028] Beneficial effects:

[0029] (1) Bacillus velezensis ZX101 can significantly inhibit the growth of Diaporthe hongkongensis and reduce the occurrence of tea leaf blight. This strain is isolated from healthy tea leaves, has strong adaptability to the tea garden environment, can persist in the tea garden for a long time, has strong secondary infection ability, and is suitable for biological control of tea leaf blight.

[0030] (2) The present invention co-cultivates Aureobasidium short-stalked and Bacillus velezensis ZX101, which allows the generated pullulan to fully contact with Bacillus velezensis ZX101 in the fermentation liquid, prompting it to form a sufficient protective film on the surface of all Bacillus velezensis ZX101 cells, promoting the adsorption of cells on the leaf surface, and solving the problem of low attachment rate of microorganisms on the leaf surface caused by rain erosion. Trehalose improves the tolerance of cells to leaf illumination and dryness, and improves the survival rate of microorganisms on the leaf surface; and potassium humate promotes photosynthesis by increasing leaf chlorophyll and further improves the disease resistance of crops. The foliar fertilizer prepared by the present invention with Bacillus velezensis ZX101 as the effective ingredient achieves the synergistic effect of various material components, further improves the control effect of Bacillus velezensis on tea tree leaf blight, and can be used as a green and environmentally friendly biological control method. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The colony morphology and microscopic morphology of Bacillus velez ZX101; a is the colony image, b is the Gram staining image, and c is the microscopic morphology image;

[0032] Figure 2 is the phylogenetic tree of Bacillus velez ZX101 based on 16S rRNA sequences; Z-1 represents Bacillus velez ZX101;

[0033] Figure 3 This is a diagram showing the antibacterial effect of Bacillus Velez ZX101 on Diaporthe hongkongensis; a is the control group and b is the Bacillus Velez ZX101 group. DETAILED DESCRIPTION

[0034] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The formula of the culture medium described in the following examples is:

[0036] ①LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, adjust pH to 7.0 with 5 mol / L NaOH, and dilute to 1 L with deionized water;

[0037] ②Potato glucose medium: 200g potatoes, 20g glucose, 1L distilled water, pH 7;

[0038] ③ Nutrient agar medium: peptone 10g, beef extract 3g, NaCl 5g, agar 15g, distilled water 1L, pH 7.0;

[0039] ④PDA medium: 200g potatoes, 20g glucose, 18g agar, 1L distilled water, pH 7.0;

[0040] Trehalose was purchased from Meihua Biotechnology Group Co., Ltd., potassium humate was purchased from Xinjiang Shengda Yifang Biotechnology Co., Ltd.; Aureobasidium pullulans was purchased from China General Microbiological Culture Collection Center with the collection number CGMCC No.33984; Diaporthe hongkongensis and Bacillus velez ZD1002 were isolated and preserved in the School of Bioengineering, Qilu University of Technology.

[0041] Example 1 Isolation and purification of Bacillus velezensis ZX101

[0042] 1. Isolation and purification of strains

[0043] (1) Separation source

[0044] The Bacillus velezensis ZX101 involved in the present invention is separated from tea leaves in Rizhao tea garden in Shandong Province.

[0045] (2) Separation time and place

[0046] Isolated and obtained from the School of Bioengineering of Qilu University of Technology in February 2022.

[0047] (3) Separation and purification

[0048] Cut healthy tea leaves into several tissue blocks with a length and width of 5 mm, disinfect the leaf tissue blocks with 0.2% sodium hypochlorite solution and 75% alcohol for 30 seconds, then wash them with sterile water twice, dry the surface of the tissue blocks with sterile absorbent paper, place them in LB solid culture medium with tweezers, and culture them at 28°C for 48 hours, selectively select colonies with different morphologies on the solid culture medium for purification culture, repeatedly pick single colonies for streaking culture, and obtain a single purified strain. Pick the purified bacterial strain and inoculate it into LB liquid culture medium, culture it in a shaking table at 28°C and 180r / min for 24 hours, and when the OD600 value of the fermentation liquid is 0.6-0.7, use the glycerol tube preservation method to preserve the strain and store it in a -80°C refrigerator for use.

[0049] 2. Results Analysis

[0050] According to the above separation and purification method, a total of 5 bacterial strains were isolated from tea tree leaves in Rizhao tea garden in Shandong Province.

[0051] Example 2 Screening of Bacillus velezensis ZX101

[0052] (1) Preparation of standard bacterial solutions of different strains to be tested

[0053] The above bacterial strains were inoculated into LB liquid culture medium and cultured at 28°C and 180 r / min for 48 h to prepare fermentation broths of different strains. The fermentation broths of the strains to be tested were prepared into a concentration of 1.0×10 9 cfu / mL standard bacterial solution.

[0054] (2) Preparation of Diaporthe hongkongensis spore suspension

[0055] Diaporthe hongkongensis was inoculated on PDA solid medium and cultured at 30°C for 5 days. The conidia were washed with sterilized 0.1wt% Tween 80. The obtained conidia of Diaporthe hongkongensis were filtered through four layers of filter paper and placed in a 10mL centrifuge tube to obtain a spore suspension. The concentration of 1×10 7 cfu / mL conidia suspension of Diaporthe hongkongensis.

[0056] (3) Screening of Bacillus velezensis ZX101

[0057] The plate confrontation culture method was used to screen the inhibitory bacteria of Diaporthe hongkongensis. 10 μL of the Diaporthe hongkongensis spore suspension obtained above was spotted in the center of the PDA plate, and 10 μL of different bacterial solutions were spotted at an equal distance of 3 cm. 10 μL of LB liquid culture medium was dropped in one of the places as a blank control group. After the liquid was dried, the plate was sealed and inverted at 30°C for 5 days. After observation, the strain with better inhibitory effect on Diaporthe hongkongensis was selected and named ZX101. The ZX101 strain was suspended in 33% glycerol and frozen in a -80°C refrigerator for later use.

[0058] Example 3 Identification of Bacillus velezensis ZX101

[0059] (1) Colony culture characteristics: Bacillus velezensis ZX101 strain preserved in 33% glycerol was inoculated on a nutrient agar plate by the plate streak method and cultured at 30°C for 24 hours, and then the morphological characteristics of the colonies were observed. The colonies of strain ZX101 were milky white and opaque, with a sticky surface, rough and wrinkled skin, and uneven edges. Single colonies were irregular in shape, such as Figure 1 As shown in a.

[0060] (2) Microscopic observation: Gram staining of strain ZX101 was performed and observed under a microscope. Figure 1 The strain ZX101 shown in b and c is a Gram-positive bacterium with a rod shape.

[0061] (3) Molecular biological characteristics

[0062] The genome of strain ZX101 was extracted using a genomic DNA extraction kit, and the 16SrRNA gene of strain ZX101 was amplified by PCR. After identification by agarose gel electrophoresis, it was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The 16SrRNA sequencing sequence was submitted to the GenBank database for BLAST comparison analysis. The phylogenetic tree was constructed using MEGA software. The phylogenetic tree is shown in Figure 2 .

[0063] The 16S rDNA gene sequence of the strain is as follows (SEQ ID NO: 1):

[0064] TGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGA

[0065] GTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATA

[0066] CCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTA

[0067] CAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGAT

[0068] GCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTC

[0069] CTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG

[0070] CCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTG

[0071] CCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGT

[0072] GCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAG

[0073] GGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGT

[0074] CATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGG

[0075] TGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAA

[0076] CTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCA

[0077] CGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAA

[0078] CGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGA

[0079] CGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT

[0080] TACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGA

[0081] GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG

[0082] CAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACT

[0083] GCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACC

[0084] TGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAG

[0085] CCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGC

[0086] TGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTAC

[0087] ACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTA

[0088] GGAGCCAGCCGCCGAAGG

[0089] Through microscopic observation and molecular biological characteristics research, it was identified that strain ZX101 belongs to the same branch as Bacillus velezensis and has the closest affinity. The strain ZX101 was identified as Bacillus velezensis, which was deposited in the China Center for Type Culture Collection on March 14, 2024, with the deposit number: CCTCC NO: M2024470.

[0090] Example 4 Test on the antibacterial rate of Bacillus velez ZX101 against Diaporthe hongkongensis

[0091] Bacillus Velez ZX101 was inoculated into a conical flask containing 200 mL of LB medium at a 1% inoculum amount, and cultured in a shaker at 28°C and 180 rpm for 48 h. The supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization to obtain a sterile supernatant of Bacillus Velez ZX101, and a 1×10 9 cfu / mL sterile supernatant of Bacillus velezensis ZX101.

[0092] Using the pour plate method, take 1mL of sterile supernatant of Bacillus velez ZX101, pour it into the unsolidified PDA medium at 45℃, mix well, and quickly pour the plate. After the PDA medium solidifies, drop 10μL of Diaporthe hongkongensis spore suspension in the center of the PDA medium, replace the supernatant with sterile blank medium as a control, and culture it in a constant temperature incubator at 30℃ for 5 days. Observe the growth of mycelium every other day until the mycelium fills the plate. Each test was repeated 3 times. The diameter of Diaporthe hongkongensis was measured using a vernier caliper using the cross-cross method, and the average value was taken as the measurement result. The inhibition rate was calculated according to the following formula:

[0093] Inhibition rate (%) = (colony growth diameter of control group - colony growth diameter of treatment group) / colony growth diameter of control group × 100.

[0094] The results showed that the inhibition rate of Bacillus velez ZX101 against Diaporthe hongkongensis was 87%.

[0095] Example 5 Preparation and antibacterial test of Bacillus velezensis ZX101 biological agent

[0096] (1) Preparation of Bacillus Velezii ZX101 biological agent

[0097] Under sterile conditions, Bacillus Velez ZX101 was streaked on LB medium and activated at 28°C for 48 hours, while observing for contamination. After activation, a loop of single colony was picked up with an inoculation loop and inoculated into LB liquid medium, and cultured at 37°C and 180 rpm for 24 hours to obtain the biological agent of Bacillus Velez ZX101 with a concentration of 1×10 9 cfu / mL.

[0098] (2) Antibacterial test of Bacillus velezensis ZX101 biological agent

[0099] The plate confrontation culture method was used for screening. The spore suspension of Diaporthehongkongensis obtained above was dropped in the center of the PDA culture medium plate. 10 μL of Bacillus velezensis ZX101 biological inoculant was dropped around it at an equal distance of 3 cm. The standard bacterial solution was replaced with a sterile blank culture medium as a control. After the liquid dried on the PDA culture medium, it was placed in a constant temperature incubator and cultured at 30°C. The antibacterial effect was observed after 5 days.

[0100] like Figure 3 As shown, the results showed that the biological agent Bacillus Velez ZX101 had a significant inhibitory effect on Diaporthehongkongensis.

[0101] Example 6 Experiment on the inhibition of infection of Diaporthe hongkongensis by Bacillus velez ZX101 biological agent

[0102] Select the leaves of the sun-lit green tea tree without damage or disease, wipe them with clean water, wipe and disinfect the leaves with 1% sodium hypochlorite, and then rinse them with sterile water. The treatment group wiped the leaves with the Velezacacillus ZX101 biological bacterial agent obtained in Example 5 for 1 min, and the control group replaced the bacterial solution with sterile water. After the tea leaves were naturally air-dried, the surfaces of the leaves of the treatment group and the control group were evenly punctured and inoculated with 0.1 mL of 1×10 7 cfu / mL of Diaporthe hongkongensis; After inoculation, the tea leaves of the treatment group and the control group grew naturally for 6 days, and the growth of the leaves was observed. Each experiment contained 6 replicates, each experiment contained 3 parallels, and each parallel contained 6 leaves.

[0103] The results showed that the leaves of the tea trees in the control group showed yellowing and withering symptoms, while the leaves of the treatment group did not develop the disease, indicating that the Bacillus Velez ZX101 biological bacterial agent prepared by the method of the present invention can significantly inhibit the tea leaf blight caused by the pathogenic bacteria of the genus Diaporthehongkongensis.

[0104] Example 7 Preparation of Bacillus velezensis ZX101 foliar fertilizer

[0105] A method for preparing a foliar fertilizer with Bacillus velezensis ZX101 as an effective ingredient is as follows:

[0106] (1) Bacillus velezensis ZX101 was transferred to LB medium, cultured at 37°C for 24 hours, and used as seed solution; Aureobasidium pullulans with a deposit number of CGMCC No. 33984 was transferred to potato glucose medium, cultured at 28°C for 48 hours, and used as seed solution;

[0107] (2) Preparation of fermentation medium: soybean meal powder, corn starch and water were mixed, protease was added for enzymatic hydrolysis at 43°C for 2.5 h, and then amylase was added for enzymatic hydrolysis at 68°C for 2.5 h. Corn syrup, beef extract, peptone, glucose and inorganic salt were added, mixed, and sterilized at 121°C for 20 min. The fermentation medium contained 68 g / L soybean meal, 85 g / L corn starch, 6 g / L corn steep liquor, 2.5 g / L beef extract, 9 g / L peptone, 10 g / L glucose, 6.85 g / L inorganic salt (3 g / L sodium chloride, 2.5 g / L calcium carbonate, 0.8 g / L potassium dihydrogen phosphate, 0.5 g / L ferric chloride, 0.05 g / L manganese sulfate, 2.5 g / L protease (enzyme activity was 1800-2000 U / g, Nanning Dongheng Huadao Biotechnology Co., Ltd.), 2.5 g / L amylase (enzyme activity was 1800-2000 U / g, Nanning Dongheng Huadao Biotechnology Co., Ltd.), and the rest was water, and the pH was 7.

[0108] (3) The seed solution of Bacillus velezensis ZX101 prepared in step (1) was added to the fermentation medium prepared in step (2) at a volume ratio of 2%, and cultured at 37° C. for 60 h until the fermentation solution concentration reached 1×10 9 cfu / mL;

[0109] (4) After step (3) is completed, the budding Aureobasidium pullulans seed solution prepared in step (1) is added to the fermentation tank of step (3) at a volume ratio of 2.5%, and cultured at 28° C. and 120 r / min for 36 hours until the pullulan content in the fermentation liquid is 5 g / L;

[0110] (5) Add trehalose and potassium humate to the fermentation liquid obtained in step (4) to final concentrations of 3% and 2%, respectively, to obtain foliar fertilizer.

[0111] Example 8 Preparation of foliar fertilizer

[0112] A preparation method of foliar fertilizer is as follows:

[0113] Steps (1)-(2) are the same as those in Example 7 (1)-(2);

[0114] (3) The seed solution of Bacillus velezensis ZX101 obtained in step (1) was added to the fermentation medium obtained in step (2) at a volume ratio of 2%, and cultured at 37° C. for 60 h until the fermentation solution concentration reached 1×10 9 cfu / mL;

[0115] (4) adding 2.5% by volume of the Aureobasidium pullulans seed solution obtained in step (1) to the fermentation medium obtained in step (2), and culturing at 28° C. and 120 rpm for 36 h until the pullulan content in the fermentation liquid reaches 5 g / L;

[0116] (5) mixing the fermentation broths obtained in (3) and (4) at a ratio of 1:1 to obtain a mixed fermentation broth;

[0117] (6) Add trehalose and potassium humate to the mixed fermentation liquid obtained in step (5) to a final concentration of 3% and 2%, respectively, to obtain a foliar fertilizer.

[0118] Example 9 Preparation of foliar fertilizer

[0119] A preparation method of foliar fertilizer is as follows:

[0120] (1)-(3) are the same as steps (1)-(3) in Example 7;

[0121] (4) After step (3), add pullulan to the fermentation broth obtained in step (3) to a final concentration of 5 g / L;

[0122] (5) Add trehalose and potassium humate to the fermentation liquid obtained in step (4) to a final concentration of 3% and 2%, respectively, to obtain a foliar fertilizer.

[0123] Example 10 Preparation of foliar fertilizer

[0124] A preparation method of foliar fertilizer is as follows:

[0125] (1)-(3) are the same as steps (1)-(3) in Example 7;

[0126] (4) After step (3) is completed, trehalose and potassium humate are added to the fermentation liquid obtained in step (3) to a final concentration of 3% and 2%, respectively, to obtain foliar fertilizer.

[0127] Example 11 Preparation of foliar fertilizer

[0128] A preparation method of foliar fertilizer is as follows:

[0129] (1)-(4) are the same as steps (1)-(4) in Example 7;

[0130] (5) After step (4) is completed, trehalose is added to the fermentation liquid obtained in step (4) to a final concentration of 3% to obtain foliar fertilizer.

[0131] Example 12 Preparation of foliar fertilizer

[0132] A preparation method of foliar fertilizer is as follows:

[0133] (1)-(4) are the same as steps (1)-(4) in Example 7;

[0134] (5) After step (4) is completed, potassium humate is added to the fermentation liquid obtained in step (4) to a final concentration of 2% to obtain foliar fertilizer.

[0135] Example 13 Preparation of foliar fertilizer

[0136] A preparation method of foliar fertilizer is as follows:

[0137] (1)-(4) are the same as steps (1)-(4) in Example 7 to obtain foliar fertilizer.

[0138] Example 14 Preparation of foliar fertilizer

[0139] A preparation method of foliar fertilizer is as follows:

[0140] (1) The same process as step (2) of Example 7 for preparing the fermentation medium;

[0141] (2) After step (1) is completed, trehalose and potassium humate are added to the fermentation liquid of step (1) to a final concentration of 3% and 2%, respectively, to obtain foliar fertilizer.

[0142] Example 15 Preparation method of foliar fertilizer

[0143] A preparation method of foliar fertilizer is as follows:

[0144] (1) Bacillus velezensis ZD1002 was transferred to LB medium, cultured at 37°C for 24 hours, and used as seed solution; Aureobasidium pullulans with a deposit number of CGMCC No. 33984 was transferred to potato glucose medium, cultured at 28°C for 48 hours, and used as seed solution;

[0145] (2) Preparation of fermentation medium: soybean meal powder, corn starch and water were mixed, protease was added for enzymatic hydrolysis at 43°C for 2.5 h, and then amylase was added for enzymatic hydrolysis at 68°C for 2.5 h. Corn syrup, beef extract, peptone, glucose and inorganic salt were added, mixed, and sterilized at 121°C for 20 min. The fermentation medium contained 68 g / L soybean meal, 85 g / L corn starch, 6 g / L corn steep liquor, 2.5 g / L beef extract, 9 g / L peptone, 10 g / L glucose, 6.85 g / L inorganic salt (3 g / L sodium chloride, 2.5 g / L calcium carbonate, 0.8 g / L potassium dihydrogen phosphate, 0.5 g / L ferric chloride, 0.05 g / L manganese sulfate, 2.5 g / L protease (enzyme activity was 1800-2000 U / g, Nanning Dongheng Huadao Biotechnology Co., Ltd.), 2.5 g / L amylase (enzyme activity was 1800-2000 U / g, Nanning Dongheng Huadao Biotechnology Co., Ltd.), and the rest was water, and the pH was 7.

[0146] (3) The seed solution of Bacillus velezensis ZD1002 prepared in step (1) was added to the fermentation medium prepared in step (2) at a volume ratio of 2%, and cultured at 37°C for 60 h until the fermentation solution concentration reached 1×10 9 cfu / mL;

[0147] (4) After step (3) is completed, the budding Aureobasidium pullulans seed solution prepared in step (1) is added to the fermentation tank of step (3) at a volume ratio of 2.5%, and cultured at 28° C. and 120 r / min for 36 hours until the pullulan content in the fermentation liquid is 5 g / L;

[0148] (5) Add trehalose and potassium humate to the fermentation liquid obtained in step (4) to final concentrations of 3% and 2%, respectively, to obtain foliar fertilizer.

[0149] Note: The difference between this example and Example 6 is that Bacillus velezensis ZX101 is replaced by Bacillus velezensis ZD1002 preserved in the laboratory, and Bacillus velezensis ZD1002 has no significant inhibitory effect on Diaporthe hongkongensis.

[0150] Example 16: Field tea tree disease resistance test using foliar fertilizer with Bacillus velez ZX101 as active ingredient

[0151] The experimental tea garden is a tea base in Rizhao City, Shandong Province. The medicine was prepared in medicine barrels, with a liquid dosage of 60 kg per mu. Preparation method: add 1 kg of foliar fertilizer to every 15 kg of water, and add 1‰ Tween-80, stir well, and apply the medicine by spraying forward. The foliar fertilizer obtained in Examples 7-15 was used as the treatment group. Three replicate plots were set up for each treatment group, with 50 tea trees in each plot and an area of ​​30 m 2 .

[0152] Treatment process: 2 days after the first application, simulated rainfall was carried out according to 60 kg (containing 1‰ Tween-80) per mu, and tap water was sprayed artificially using a sprayer to simulate rainfall. The second application was carried out 10 days after the first application, and the application was carried out twice in total; 2 days after the second application, the second simulated rainfall was carried out using a sprayer; 2 days after the second simulated rainfall, the tea tree leaf blight fungus Diaporthe hongkongensis spores (1 kg of spores containing 10 spores were added to every 15 kg of water) were sprayed with a sprayer. 6 cfu / mL of fermentation liquid) once, observe the incidence of tea leaf blight under different treatments, and make statistics.

[0153] The incidence of tea leaf blight was statistically analyzed, and the results are shown in Table 1:

[0154] Table 1 The incidence of tea leaf blight under different foliar fertilizer treatments

[0155] Serial number Treatment Group Processing conditions Incidence (%) 1 Example 7 This program 21.00±5.00 2 Example 8 Pullulan separate fermentation 47.00±7.00 3 Example 9 Pullulan alone 51.00±6.00 4 Example 10 Pullulan Free 70.00±6.00 5 Embodiment 11 No Potassium Humic Acid 56.00±6.00 6 Example 12 No trehalose 54.00±4.00 7 Embodiment 13 Potassium humate and trehalose are free 61.00±4.00 8 Embodiment 14 No bacteria 82.00±3.00 9 Embodiment 15 Strain replacement 52.00±5.00

[0156] As shown in Table 1, compared with Example 7, Example 14 does not contain Bacillus Velezii ZX101, and the incidence rate of tea trees after artificial infection with Diaporthe hongkongensis is 82%, and Bacillus Velezii ZX101 has a significant preventive and therapeutic effect on tea leaf blight. The difference between Example 15 and Example 7 is that the strains of Bacillus Velezii are different, and the incidence rate of tea leaf blight increases from 21% to 52%, indicating that the isolated strain of the present invention has a better preventive and therapeutic effect on tea leaf blight caused by Diaporthe hongkongensis.

[0157] As shown in Table 1, compared with Example 7, Example 13 lacks the protective agent trehalose of Bacillus Velez ZX101 and potassium humate that promotes leaf photosynthesis, and the incidence rate is greatly increased; compared with Example 13, Examples 11 and 12 have a slightly improved protective effect of Bacillus Velez ZX101 on tea trees due to the presence of trehalose or potassium humate. It can be seen that the addition of trehalose and potassium humate can effectively enhance the prevention and control effect of Bacillus Velez ZX101 on tea tree leaf blight.

[0158] As can be seen from Table 1, the difference between Example 10 and Example 7 is that the foliar fertilizer does not contain pullulan. Due to the influence of artificial spraying, a large amount of Bacillus Velez subtilis ZX101 cannot be colonized on the leaf surface, which ultimately leads to a decrease in its disease resistance. Compared with Example 7, Example 8 has the same ingredients in the foliar fertilizer, except that Bacillus Velez subtilis ZX101 and budding short-stalked mold are cultured separately; and the difference between Example 9 and Example 7 is that pullulan is added alone. It can be seen from the results that co-culturing budding short-stalked mold and Bacillus Velez subtilis ZX101 can promote the protective effect of pullulan on Bacillus Velez subtilis ZX101.

[0159] In summary, the foliar fertilizer with Bacillus velezensis ZX101 as the effective ingredient provided by the present invention has a good control effect on tea leaf blight caused by the pathogenic bacteria of the genus Diaporthe hongkongensis, and can be used as a green and environmentally friendly biological control method for tea leaf blight.

Claims

1. A Bacillus velezensis ZX101, the deposit number of which is CCTCCNO: M2024470.

2. A biological agent of Bacillus Velez ZX101, characterized in that: The Bacillus Velezii ZX101 described in claim 1 is used as an effective ingredient.

3. The method for preparing the biological agent of Bacillus Velez ZX101 according to claim 2, characterized in that: Under sterile conditions, the activated Bacillus Velez ZX101 was inoculated into LB liquid culture medium and cultured at 35-40°C and 150-200rpm for 20-30h to prepare a biological agent of Bacillus Velez ZX101 with a concentration of 1×10 8 -1×10 9 cfu / mL.

4. A foliar fertilizer comprising the Bacillus Velez subtilis ZX101 according to claim 1 as an effective ingredient.

5. The preparation method of foliar fertilizer according to claim 4, characterized in that, The specific steps include: (1) Preparation of seed liquid of Bacillus velez ZX101 and Aureobasidium pullulans with a deposit number of CGMCC No. 33984; (2) Preparation of fermentation medium; (3) inoculating the Bacillus velezensis ZX101 seed solution obtained in step (1) into the fermentation medium obtained in step (2) for culturing; (4) adding the budding Aureobasidium pullulans seed solution prepared in step (1) to the fermentation medium obtained in step (3) and continuing to culture to obtain a mixed fermentation solution; (5) Adding trehalose and potassium humate to the mixed fermentation liquid obtained in step (4) to obtain foliar fertilizer.

6. The preparation method according to claim 5, characterized in that: The preparation method of the Velez subtilis ZX101 seed liquid in step (1) is as follows: inoculate the Velez subtilis ZX101 into LB culture medium, and culture it at 35-40° C. for 20-30 hours to prepare the Velez subtilis ZX101 seed liquid; the preparation method of the budding Aureobasidium pullulans seed liquid in step (1) is as follows: inoculate the budding Aureobasidium pullulans into potato glucose culture medium, and culture it at 25-30° C. for 45-50 hours to prepare the budding Aureobasidium pullulans seed liquid.

7. The preparation method according to claim 5, characterized in that: The preparation method of the fermentation medium in step (2) is as follows: soybean meal powder, corn starch and water are mixed, protease is added for enzymolysis at 40-45°C for 2-3h, amylase is added for enzymolysis at 65-70°C for 2-3h, corn steep liquor, beef extract, peptone, glucose, sodium chloride, calcium carbonate, dipotassium hydrogen phosphate, ferric chloride and manganese sulfate are added in sequence, mixed, sterilized under high pressure at 115-121°C for 15-25min, and cooled to room temperature; the fermentation medium is: soybean meal powder 65-70g / L, corn starch 82-87g / L, corn steep liquor 5-10g / L, beef extract 1-3g / L, peptone 5-10g / L, glucose 8-15g / L, sodium chloride 2-5g / L, calcium carbonate 1-4g / L, dipotassium hydrogen phosphate 0.5-1.5g / L, ferric chloride 0.2-0.6g / L, manganese sulfate 0.03-0.06g / L, protease 1-4g / L, amylase 1-4g / L, the rest is water, pH is 6.8-7.

2.

8. The preparation method according to claim 5, characterized in that: The culture method of step (3) is as follows: 1-5% of the seed solution of Bacillus velezensis ZX101 is added to the fermentation medium at a volume ratio and cultured at 35-40°C for 55-65h until the fermentation liquid concentration reaches 1×10 8 -1×10 9 cfu / mL; The culture method of step (4) is as follows: adding 2-5% of the seed liquid of Aureobasidium pullulans to the fermentation medium after the culture in step (3) at a volume ratio, and culturing for 35-40 hours at 25-30° C. and 100-130 r / min until the pullulan content in the fermentation liquid is 4-8 g / L; The added amounts of trehalose and potassium humate in step (5) are 2-4% and 1-3% by volume respectively.

9. Use of the Bacillus Velez ZX101 biological agent according to claim 2 in the prevention and treatment of tea leaf blight.

10. Use of the foliar fertilizer according to claim 4 in preventing and treating tea leaf blight.

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