Bacillus subtilis, microbial preparation and preparation method and application thereof

Abstract

The invention relates to bacillus subtilis, a microbial preparation as well as a preparation method and application of the bacillus subtilis and the microbial preparation, the bacillus subtilis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31166. Compared with the prior art, microorganisms contained in the microbial preparation generate richer antibacterial substances through competition of nutrition and space sites, the disease-resistant potential of plants is induced, the immunity and disease resistance of the plants are enhanced, the gummosis of stone fruit trees can be remarkably controlled, the field experiment control effect reaches 80% or above, and the application prospect is wide. The production cost is greatly saved, and the method has a good market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Bacillus subtilis, a microbial preparation, and a preparation method and application thereof. Background Art

[0002] Stone fruit trees (such as peach, cherry, plum, prune, apricot, etc.) are often accompanied by gummosis during their growth. The harm of gummosis of fruit trees mainly includes affecting tree vigor, reducing fruit quality, reducing yield, shortening the life of fruit trees, etc. At present, the main technical means for the prevention and control of gummosis of stone fruit trees are as follows:

[0003] (1) Relying on chemical agents, such as the Chinese invention patent "A new organic trisulfide compound and its application" with application number CN202380016038.5 (publication number CN118556042A), the Chinese invention patent "A method for delaying the flowering period of peach trees and increasing the fruit setting rate" with application number CN202211487418.0 (publication number CN115708451A), etc. However, this method has drug resistance and causes food safety problems, and will have an adverse impact on the ecological environment of the orchard.

[0004] (2) Natural plant sources, such as the Chinese invention patent "A preparation and use method of a mixture for preventing and treating peach gummosis" with application number CN202111499627.2 (publication number CN114208850A), the Chinese invention patent "A Chinese medicine extract for preventing and treating plant bacterial diseases and its application" with application number CN202110171442.2 (publication number CN112806393A), etc. However, there are many varieties of natural plant sources, and the price of authentic medicinal materials is high. At the same time, due to different growing geographical conditions, their active ingredients vary greatly, resulting in high costs, small applicable areas, unstable efficacy, and other problems, so the promotion effect is poor.

[0005] (3) Microbial technology, such as the Chinese invention patent with application number CN201910729690.7 (publication number CN110343644A) "A bacterial agent and its cultivation method and application", the Chinese invention patent with application number CN201910369359.9 (publication number CN110122507A) "A method and agent for preventing and controlling gummosis of apricot trees", the Chinese invention patent with application number: CN201910331670.4 (publication number CN111838190A) "A biocontrol agent for preventing and controlling stem base rot and gummosis, its preparation method and application", and the Chinese invention patent with application number CN201310294764.1 (publication number CN103451128A) "A strain of Bacillus amyloliquefaciens and its application in preventing and controlling peach gummosis". However, the microbial preparations prepared by this method have poor stability, and most of the known results are experimental results, and the actual use effect cannot be effectively guaranteed. In addition, there are problems such as inconvenience in use and poor versatility. Summary of the invention

[0006] The first technical problem to be solved by the present invention is to provide a Bacillus subtilis (HZBS15082) with good prevention and treatment effect of gummosis of stone fruit trees in view of the existing technology.

[0007] The second technical problem to be solved by the present invention is to provide a microbial preparation comprising the above-mentioned Bacillus subtilis in view of the prior art.

[0008] The third technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned microbial preparation in view of the prior art.

[0009] The fourth technical problem to be solved by the present invention is to provide an application of the above-mentioned Bacillus subtilis in the prevention and treatment of gummosis of stone fruit trees in view of the existing technology.

[0010] The fifth technical problem to be solved by the present invention is to provide an application of the above-mentioned microbial preparation in the prevention and treatment of gummosis of stone fruit trees in view of the existing technology.

[0011] The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: a Bacillus subtilis, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31166.

[0012] The technical solution adopted to further solve the above second technical problem is: a microbial preparation, characterized in that it includes the Bacillus subtilis as described above.

[0013] Furthermore, the microbial preparation includes the following components and their mass percentages: 0.5% to 50% of Bacillus subtilis, 0.5% to 40% of Bacillus licheniformis, 0.5% to 60% of Bacillus velezensis, 0.5% to 60% of Trichoderma aspergillus spore powder, 20% to 50% of nutrient carrier, 3% to 10% of dispersant, and 5% to 15% of wetting agent.

[0014] Among them, 0.5% to 50% of Bacillus subtilis is added in the form of concentrated spore liquid, and the effective live bacteria count is: 400 million / g to 40 billion / g; 0.5% to 40% of Bacillus licheniformis is added in the form of spore powder, and the effective live bacteria count is: 500 million / g to 40 billion / g; 0.5% to 60% of Bacillus velezensis is added in the form of spore powder, and the effective live bacteria count is: 500 million / g to 60 billion / g; 0.5% to 6% of Trichoderma aspergillus spore powder is added in the form of spore powder, and the effective live bacteria count is: 100 million / g to 1.2 billion / g.

[0015] Further, Bacillus licheniformis is a kind of Bacillus, and the thalli will produce active substances such as bacteriocin during the growth process, and these active substances have obvious inhibitory effects on pathogenic bacteria or conditional pathogens of endogenous infection. Bacillus Velezii can activate defense enzymes such as phenylalanine ammonia lyase, peroxidase, superoxide dismutase, chitinase and polyphenol oxidase in plants, thereby promoting plants to produce induced systemic resistance, enhancing the disease resistance of plants, and resisting the invasion of pathogens. The spore powder of Trichoderma spinulosum can induce the expression of plant immune system genes, produce a large number of plant effector molecules, stimulate the immune response of plants, enhance plant disease resistance, and achieve the prevention and control of diseases. The above-mentioned strains and the combination of Bacillus subtilis HZBS15082 in the present invention have obvious superposition and complementary effects in anti-gummosis.

[0016] Furthermore, the nutrient carrier includes at least one of fulvic acid, amino acids, glucose, sucrose or alginic acid.

[0017] Furthermore, the dispersant includes at least one of alkyl sodium salts, lignin sulfonates and their derivatives, sodium naphthalenesulfonate formaldehyde condensates, alkylphenol polyoxyethylene and formaldehyde condensate sulfates, polycarboxylates and their derivatives, cresol-formaldehyde and sodium sulfite condensates, alkylaryl sulfonate phenethylphenol polyoxyethylene ethers and their derivatives.

[0018] Furthermore, the wetting agent includes any one of polyvinyl alcohol, Morwet D-425, Morwet D-110, TERSPENSE 2425 or TERSPENSE 2700.

[0019] The technical solution adopted to further solve the third technical problem is: a method for preparing the microbial preparation as described above, characterized in that it comprises the following steps:

[0020] (1) Preparation of Bacillus subtilis HZBS15082 spore fluid:

[0021] Add an appropriate amount of LB liquid culture medium into the culture container, pick a single colony of Bacillus subtilis HZBS15082 and inoculate it into the culture container, and shake and culture to obtain a liquid fermentation seed liquid;

[0022] Adding the required culture medium into a liquid fermentation tank, inoculating the liquid fermentation seed liquid, and culturing to obtain Bacillus subtilis fermentation liquid;

[0023] The obtained Bacillus subtilis fermentation liquid is concentrated to obtain concentrated microbial spore liquid;

[0024] (2) Preparation of carrier composition:

[0025] According to the above mass percentages, Bacillus licheniformis, Bacillus Velezii, Trichoderma aculeatus spore powder, a nutrient carrier, a dispersant and a wetting agent are uniformly mixed to obtain a carrier composition;

[0026] (3) Preparation of microbial preparations:

[0027] At a low temperature of 30-35° C., the concentrated microbial spore fluid is sprayed onto the above-mentioned carrier composition in the form of a spray according to the above-mentioned mass percentage, so that the microbial spore fluid and the carrier composition are fully mixed and uniformly prepared to prepare a microbial preparation containing the above-mentioned Bacillus subtilis.

[0028] The technical solution adopted to further solve the fourth technical problem is: an application of the Bacillus subtilis as described above in the prevention and control of gummosis of stone fruit trees.

[0029] Preferably, the use of Bacillus subtilis in the prevention and control of gummosis of stone fruit trees is carried out at a concentration of 4×10 8 ~4×10 10 cfu / ml. Within this concentration range, it can take into account both the prevention and control effect and economic benefits of gummosis of stone fruit trees.

[0030] The technical solution adopted to further solve the fifth technical problem is: an application of the microbial preparation as described above in the prevention and control of gummosis of stone fruit trees.

[0031] Compared with the prior art, the advantages of the present invention are: the Bacillus subtilis HZBS15082 screened and obtained by the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31166, a deposit address of No. 3, Yard No. 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and a deposit date of July 4, 2024.

[0032] Furthermore, the present invention utilizes the above-mentioned Bacillus subtilis HZBS15082 to prepare a microbial preparation for the prevention and control of gummosis of drupe fruit trees. The microorganisms contained in the microbial preparation produce more abundant antibacterial substances through competition for nutrients and spatial sites, induce the plant's own disease resistance potential, enhance the plant's immunity and disease resistance, and can significantly control the occurrence of gummosis of drupe fruit trees. The prevention effect in field experiments reaches more than 80%, which is much higher than the existing chemical agent treatment, greatly saves production costs, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a morphological photograph of the Bacillus subtilis strain HZBS15082 in Example 2 of the present invention;

[0034] Figure 2 The control effect of treatment group 1 on peach gummosis in Example 4 of the present invention;

[0035] Figure 3 The control effect of treatment group 2 on peach gummosis in Example 4 of the present invention;

[0036] Figure 4 The control effect of treatment group 3 on peach gummosis in Example 4 of the present invention;

[0037] Figure 5 The control effect of comparison group 1 on peach gummosis in Example 4 of the present invention;

[0038] Figure 6 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the accompanying drawings.

[0040] The culture medium involved in the following examples:

[0041] PDA medium: 200 g potato, 20 g glucose, 15 g agar powder, 1 L deionized water, sterilized at 121 °C for 20 min.

[0042] LB culture medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, dilute to 1 L with deionized water, sterilize at 121 °C for 20 min, pH 7.2.

[0043] NA agar medium: 2.5 g glucose, 10 g tryptone, 5 g sodium chloride, 3 g beef extract, 12 g agar powder, 1 L deionized water, sterilized at 121 °C for 20 min, pH 7.0.

[0044] Test strains: Peach gummosis pathogen Botryosphaeria spp., the isolates were donated by the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences.

[0045] Example 1: Collection of indigenous microorganisms, isolation of strains, and morphological observation and identification

[0046] The indigenous microbial community was collected using the "rice culture medium" method. On April 6, 2023, the "rice culture medium" was buried in the mountain soil less disturbed by human activities near Tangxi Jinshan Tea Art Farm in Yinzhou District, Ningbo City, Zhejiang Province for cultivation. After 6 to 7 days, the surrounding indigenous microorganisms infiltrated into the rice, forming colonies and turning the rice into a thin and soft state. The indigenous microbial community was obtained when it was retrieved.

[0047] Weigh 1g of the "rice" sample containing indigenous microbial communities, add it to 10mL of 0.7mol / L sodium chloride solution, shake the solution for 1min to make a suspension, and then dilute the suspension with 0.7mol / L sodium chloride solution to a concentration of 10 -2 , 10 -3 and 10 -4 gradient.

[0048] To screen for antagonistic bacteria against Botryosphaeria spp., the liquid cultured Botryosphaeria spp. was first broken into mycelial fragments in a homogenizer, and 100 μL of mycelial fragments were spread on a PDA plate. After removing excess water in a fume hood, different gradients (10 -2 , 10 -3 and 10 -4 ) of the above suspension (100 μL / plate). They were placed in a 28°C constant temperature incubator for culture, colonies with significant inhibition zones were selected every 24 hours, potential antagonistic bacteria were streaked and purified on LB plates, and the purified strains were stored at -80°C for future use.

[0049] Example 2: Morphological observation and identification of antagonistic bacterial strains of Botryosphaeria spp.

[0050] The antagonistic strain was cultured on LB plates at a constant temperature (28°C) for 24 hours. Figure 1 As shown, the colonies are round, dry and wrinkled, and white and opaque.

[0051] The above antagonistic strains were entrusted to the Microbiological Product Quality Inspection and Testing Center of the Ministry of Agriculture and Rural Affairs (Wuhan) for identification: 2.1 Identification principles and methods

[0052] Principle: The use of molecular biological methods to clarify the taxonomic relationship between microbial populations from the perspective of genetic evolution is a commonly used identification method in microbial taxonomy research. Bacteria are analyzed by 16S rDNA.

[0053] Test method: Refer to "6.5" in NY / T 1736-2009.

[0054] 2.2 Identification results

[0055] The specific gene fragment of the strain was amplified by PCR, and the amplification primers were as follows:

[0056] 27F (SEQ ID No. 2): AGAGTTTGATCCTGGCTCAG; 1492R (SEQ ID No. 3): ACGGCTACCTTGTTACGACTT), the amplified product was recovered and sequenced, and the sequence was compared with the sequence registered in the GenBank database for homology. The 16S rDNA sequence of the strain was 100% homologous to the standard strain DSM10 of Bacillus subtilis. The homology with the standard strain DSM of Bacillus subtilis was 100%, and its 16S rDNA sequence (SEQ ID No. 1) was as follows:

[0057] ACAAACTCTCGTGGTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCG

[0058] CGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGAC

[0059] TGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGC

[0060] CCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATT

[0061] TGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAA

[0062] CTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACAT

[0063] CTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGG

[0064] GACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGT

[0065] TGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTG

[0066] AGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGC

[0067] ACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACC

[0068] AGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACC

[0069] AGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACA

[0070] CGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCC

[0071] CGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTAC

[0072] GCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGT

[0073] AGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTA

[0074] CTTGTTTCTCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGG

[0075] CGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCCGTA

[0076] GGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTAC

[0077] GCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCA

[0078] TCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACC

[0079] ATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCAC

[0080] GTGTTACTCACCCGTCCGCCGCTAACA

[0081] Example 3: Preparation of a microbial preparation containing Bacillus subtilis HZBS15082

[0082] 3.1 Preparation of Bacillus subtilis HZBS15082 spore fluid:

[0083] 3.1.1 Add an appropriate amount of LB liquid culture medium to a 250 mL Erlenmeyer flask, pick a single colony of Bacillus subtilis HZBS15082 and inoculate it into the Erlenmeyer flask, shake and culture at 30°C and 180 rpm for 24 h to obtain liquid fermentation seed liquid.

[0084] 3.1.1 In a 50L liquid fermentation tank, add a culture medium consisting of 2kg corn flour, 1.2kg soybean flour, 1kg yeast culture medium, 0.2kg potassium nitrate, 0.03kg magnesium sulfate, 0.01kg manganese sulfate and 25.5kg water;

[0085] 3.1.1 Inoculate (1.1) 500 mL of liquid fermentation seed solution, and culture at 28°C and 280 rpm for 24 hours to obtain Bacillus subtilis HZBS15082 fermentation solution;

[0086] 3.1.1 The fermentation broth is concentrated by a concentration technique (using existing concentration techniques) to obtain concentrated microbial spore fluid. 3.2 Preparation of carrier composition:

[0087] Bacillus licheniformis, Bacillus velez, Trichoderma aculeatus spore powder, nutrient carrier, dispersant and wetting agent are added in proportion (0.5%-50% of Bacillus subtilis, 0.5%-40% of Bacillus licheniformis, 0.5%-60% of Bacillus velez, 0.5%-6% of Trichoderma aculeatus spore powder, 20%-50% of nutrient carrier, 3%-10% of dispersant and 5%-15% of wetting agent), and the carrier composition is obtained after uniform mixing.

[0088] 3.3 Preparation of microbial preparations:

[0089] Under a low temperature state of 30 to 35° C., the concentrated microbial spore fluid is directly sprayed onto the above-mentioned carrier composition in the form of a spray, so that the microbial spore fluid and the carrier composition are fully and evenly mixed, and the spore bodies and thick-walled spores of the relevant microorganisms are effectively preserved, so that the product maintains a high biological activity, and a microbial preparation containing Bacillus subtilis (HZBS15082) of the present invention is prepared.

[0090] The specific formula of the microbial preparation used in treatment group 2, treatment group 3, treatment group 4 and comparative group in the following Example 4 is as follows:

[0091] Treatment group 2: the mass percentage of Bacillus subtilis was 15% and the effective viable count was 12 billion / g; the mass percentage of Bacillus licheniformis (purchased from Kunhe Biotechnology Co., Ltd., Tianjin Development Zone) was 15% and the effective viable count was 15 billion / g; the mass percentage of Bacillus velez (purchased from Kunhe Biotechnology Co., Ltd., Tianjin Development Zone) was 15% and the effective viable count was 15 billion / g; the mass percentage of Trichoderma acanthoides spore powder (purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.) was 6% and the effective viable count was 800 million / g; the mass percentage of nutrient carrier (mineral potassium humate) was 46%; the mass percentage of dispersant (alkylphenol polyoxyethylene and formaldehyde condensate sulfate) was 2%; the mass percentage of wetting agent (Morwet D-425, purchased from Akzo Nobel) was 6%.

[0092] Treatment group 3: The mass percentage of Bacillus subtilis is 10% and the effective viable count is 8 billion / gram; the mass percentage of Bacillus licheniformis (purchased from Kunhe Biotechnology Co., Ltd., Tianjin Development Zone) is 10% and the effective viable count is 10 billion / gram; the mass percentage of Bacillus velez (purchased from Kunhe Biotechnology Co., Ltd., Tianjin Development Zone) is 10% and the effective viable count is 10 billion / gram; the mass percentage of Trichoderma acanthosporium spore powder (purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.) is 6% and the effective viable count is 800 million / gram; the mass percentage of nutrient carrier (mineral potassium humate) is 50%; the mass percentage of dispersant (alkylphenol polyoxyethylene and formaldehyde condensate sulfate) is 3%; the mass percentage of wetting agent (Morwet D-425, purchased from Akzo Nobel) is 6%.

[0093] Comparison Group 1: The mass percentage of Bacillus subtilis was 20% and the effective viable count was 16 billion / g; the mass percentage of Bacillus licheniformis (purchased from Tianjin Development Zone Kunhe Biotechnology Co., Ltd.) was 10% and the effective viable count was 10 billion / g; the mass percentage of Bacillus Velez (purchased from Tianjin Development Zone Kunhe Biotechnology Co., Ltd.) was 10% and the effective viable count was 10 billion / g; the mass percentage of the nutrient carrier (mineral potassium humate) was 50%; the mass percentage of the dispersant (alkylphenol polyoxyethylene and formaldehyde condensate sulfate) was 3%; the mass percentage of the wetting agent (Morwet D-425, purchased from Akzo Nobel) was 6%.

[0094] Comparison Group 2: The mass percentage of Bacillus subtilis was 30% and the effective viable count was 24 billion / g; the mass percentage of Trichoderma acanthosporium spore powder (purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.) was 6% and the effective viable count was 800 million / g; the mass percentage of nutrient carrier (mineral potassium humate) was 50%; the mass percentage of dispersant (alkylphenol polyoxyethylene and formaldehyde condensate sulfate) was 3%; the mass percentage of wetting agent (Morwet D-425, purchased from Akzo Nobel) was 6%.

[0095] Example 4: Application of microbial preparation containing Bacillus subtilis HZBS15082

[0096] 4.1 Materials and methods

[0097] 4.1.1 Overview of the test site

[0098] The test site is located at Longguan Yongming Peach Farm in Haishu District, Ningbo City, Zhejiang Province. The test site is a terraced field with two rows per terrace, a row spacing of 3m×4m, loamy soil, and grass between rows. The soil fertility and fertilizer and water management of the entire test site are basically the same.

[0099] The test variety is "Lake View Honeydew", which is 5 years old. In recent years, peach gummosis has occurred seriously in the test area.

[0100] Before the experiment was carried out, from October to December, winter fertilizer was applied per plant, with 20 to 30 kg of organic fertilizer per adult tree and 0.4 kg of compound fertilizer (NPK is 17-17-17) per plant.

[0101] 4.1.2 Experimental design and treatment

[0102] Two pesticide treatment groups were set up, namely, one chemical agent treatment group (treatment group 1) and two microbial preparation treatment groups of the present invention (treatment group 2, treatment group 3), and two comparison groups (comparison group 1 does not contain Trichoderma spinulosa spore powder compared with the treatment group, and comparison group 2 does not contain Bacillus licheniformis and Bacillus velez compared with the treatment group) and a water control group (CK), as shown in Table 1 and Table 2. Each group was sprayed with 18 plants, and the water consumption of the pesticide in each treatment group (18 peach trees) was 30L (the control group was sprayed with the same volume of water), and a backpack electric sprayer was used. The first spraying time was February 18, 2023, and the whole plant was sprayed. The second spraying time was March 12, 2023, focusing on the trunk spraying, the third spraying time was May 16, 2023, and the fourth spraying time was June 16, 2023, focusing on the trunk spraying.

[0103] Table 1 Test drugs

[0104]

[0105] Table 2 Experimental design

[0106] Experimental Group Prescription plan Treatment Group 1 29% Tebuconazole·Azoxystrobin SC 1000 times diluted Treatment Group 2 42 billion / g microbial preparation diluted 120 times Treatment Group 3 28 billion / g microbial preparation diluted 120 times Comparison Group 1 36 billion / g microbial preparation diluted 120 times Comparison Group 2 22 billion / g microbial preparation diluted 120 times Control group Shimizu

[0107] 4.1.3 Experimental investigation and data analysis

[0108] 4.1.3.1 Before spraying, a total of 9 representative peach trees in each test group were marked as investigation trees, divided into 3 replicates, with 3 trees in each replicate, to investigate the number of gum flow points or the percentage of gum flow area in the trunk. Because the gum flow of peaches in different treatments was relatively serious during the first spraying, the first investigation began before the third spraying (May 12), the second investigation was conducted before the fourth spraying (June 14), and the third investigation of peach gum flow was conducted on July 9.

[0109] 4.1.3.2 Safety Investigation

[0110] During the entire test period, observe whether each combination of pesticides has any adverse effects on peach leaves, branches, fruits, etc. If pesticide damage occurs, record the type and degree of damage.

[0111] 4.1.3.3 The classification standard of peach gummosis is based on the classification standard of peach gummosis disease formulated by Luo Jianghui (“Research on the Occurrence and Pathogens of Peach Gumosis” [D]. Chongqing; Southwest University, 2006). The details are as follows:

[0112] Level 0: The whole plant is normal, with no gum flow and no disease spots;

[0113] Level 1: There are 1 to 9 gum flow spots on the whole tree, and the diseased area accounts for less than 5% of the tree area;

[0114] Level 2: There are 10 to 19 gum flow spots on the whole tree, and the diseased area accounts for 5% to 10% of the trunk area;

[0115] Level 3: There are 20 to 29 gum flow spots on the whole tree, and the diseased area accounts for 10% to 20% of the trunk area;

[0116] Level 4: There are 30 or more gum flow spots on the whole tree, and the affected area accounts for more than 20% of the trunk area.

[0117] Investigate whether each treatment has phytotoxicity, count the number of diseased plants and the disease situation, and calculate the disease index and relative control effect. The experimental data were processed using Excel 2013.

[0118] Disease index = [∑(disease level × number of diseased plants)] / [highest disease level × total number of investigated plants] × 100;

[0119] Relative protective effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0120] 4.2 Results and Analysis

[0121] 4.2.1 Safety of handling of test reagents

[0122] During the entire experimental period, the treatments with various combinations of agents had no adverse effects on peach leaves, branches, fruits, etc., indicating that each agent or combination had good safety.

[0123] 4.2.2 Effects of different pesticides on the prevention and control of peach gummosis

[0124] Table 3 Disease index and relative control effect of peach gummosis treated with each agent

[0125]

[0126] As shown in Table 3 and Figure 2 , Figure 3 , Figure 4 , Figure 5 as well as Figure 6As shown, from the test results of this embodiment, it can be seen that the disease index of peach gummosis treated with chemical agents exceeds 30 and gradually increases, indicating that the gummosis of peach in the test site is serious, and the spraying treatment can only alleviate the degree of peach gummosis and reduce the growth rate of peach gummosis. The field prevention effect of the microbial preparation in the present invention is good for the prevention of peach gummosis, effectively controls the degree of peach gummosis, and the prevention and control effect is better than that of chemical agent treatment. Among them, Bacillus licheniformis, Bacillus velez, Trichoderma spinulosum spore powder, etc. cooperate with Bacillus subtilis HZBS15082 in the present invention in the aspect of anti-gumosis, which has important superposition and complementary effects.

Claims

1. A Bacillus subtilis, deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.31166.

2. A microbial preparation, characterized in that: The invention comprises the Bacillus subtilis as claimed in claim 1.

3. The microbial preparation according to claim 2, characterized in that The invention comprises the following components and their mass percentages: 0.5% to 50% of Bacillus subtilis, 0.5% to 40% of Bacillus licheniformis, 0.5% to 60% of Bacillus velez, 0.5% to 6% of Trichoderma aculeatus spore powder, 20% to 50% of nutrient carrier, 3% to 10% of dispersant, and 5% to 15% of wetting agent; Among them, the effective live bacteria count of Bacillus subtilis is 400 million / gram to 40 billion / gram, the effective live bacteria count of Bacillus licheniformis is 500 million / gram to 40 billion / gram, the effective live bacteria count of Bacillus Velezii is 500 million / gram to 60 billion / gram, and the effective live bacteria count of Trichoderma aspergillus spore powder is 100 million / gram to 1.2 billion / gram.

4. The microbial preparation according to claim 3, characterized in that The nutrient carrier includes at least one of fulvic acid, amino acids, glucose, sucrose or alginic acid.

5. The microbial preparation according to claim 3, characterized in that The dispersant comprises at least one of alkyl sodium salts, lignin sulfonates and their derivatives, sodium naphthalene sulfonate formaldehyde condensates, alkylphenol polyoxyethylene and formaldehyde condensate sulfates, polycarboxylates and their derivatives, cresol-formaldehyde and sodium sulfite condensates, alkyl aryl sulfonate phenethylphenol polyoxyethylene ethers and their derivatives.

6. The microbial preparation according to claim 3, characterized in that The wetting agent includes any one of polyvinyl alcohol, Morwet D-425, Morwet D-110, TERSPENSE 2425 or TERSPENSE 2700.

7. A method for preparing a microbial preparation according to any one of claims 3 to 6, characterized in that The following steps are involved: (1) Preparation of Bacillus subtilis HZBS15082 spore fluid: Add an appropriate amount of LB liquid culture medium into the culture container, pick a single colony of Bacillus subtilis HZBS15082 and inoculate it into the culture container, and shake and culture to obtain a liquid fermentation seed liquid; Adding the required culture medium into a liquid fermentation tank, inoculating the liquid fermentation seed liquid, and culturing to obtain Bacillus subtilis fermentation liquid; The obtained Bacillus subtilis fermentation liquid is concentrated to obtain concentrated microbial spore liquid; (2) Preparation of carrier composition: According to the above mass percentages, Bacillus licheniformis, Bacillus Velezii, Trichoderma aculeatus spore powder, a nutrient carrier, a dispersant and a wetting agent are uniformly mixed to obtain a carrier composition; (3) Preparation of microbial preparations: At a low temperature of 30-35° C., the concentrated microbial spore fluid is sprayed onto the carrier composition in the form of a spray, so that the microbial spore fluid and the carrier composition are fully and evenly mixed to prepare a microbial preparation containing the Bacillus subtilis.

8. Use of the Bacillus subtilis according to claim 1 in the prevention and control of gummosis of stone fruit trees.

9. The use of Bacillus subtilis in the prevention and control of gummosis of stone fruit trees according to claim 8, characterized in that: The concentration of Bacillus subtilis is 4×10 8 ~4×10 10 cfu / ml.

10. Use of the microbial preparation according to any one of claims 2 to 6 in the prevention and control of gummosis of stone fruit trees.

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