CN107523525B

CN107523525B - Bacillus belgii and application thereof in prevention and treatment of walnut canker

Info

Publication number: CN107523525B
Application number: CN201710958436.5A
Authority: CN
Inventors: 段春华; 李宗泰; 牛赡光; 张淑静; 刘慇; 王清海; 曲永赟
Original assignee: Shandong Academy of Forestry
Current assignee: Shandong Academy of Forestry
Priority date: 2017-10-16
Filing date: 2017-10-16
Publication date: 2020-04-17
Anticipated expiration: 2037-10-16
Also published as: CN107523525A

Abstract

The invention discloses Bacillus belgii and application thereof in preventing and treating walnut canker. The strain is Bacillus subtilis LH3-2 with the preservation number of CGMCC No. 14744. Experiments show that the Bacillus belgii strain has the characteristics of high growth speed, high spore yield, strong stress resistance, capability of fast and mass colonization on the surface of walnut, and the like, so that the Bacillus belgii strain has a good application prospect. The biological control preparation prepared from the Bacillus belgii can efficiently control walnut canker, and is a biological control preparation with great application prospect.

Description

Bacillus belgii and application thereof in prevention and treatment of walnut canker

Technical Field

The present invention belongs to the field of biological plant disease preventing and controlling technology. Specifically, the invention relates to Bacillus belgii and application thereof in prevention and treatment of walnut canker, and a biological prevention and treatment preparation prepared from the Bacillus belgii.

Background

The walnut is an important strategic tree species of woody grain and oil in China, and the fruit of the walnut is rich in nutrition and has higher economic value. In recent years, the walnut industry in China is rapidly developed and becomes the largest walnut producing country in the world. The walnut planting area is rapidly increased, the centralized cultivation scale is continuously enlarged, but the traditional extensive mode is still continued on the aspect of cultivation management, so that the trunk diseases and insect pests represented by walnut canker and the like are rapidly propagated and developed. Walnut canker, also known as canker and black water disease, is a common disease on walnut trunks, has been found in the state of california in the united states in 1915 of the last century, is distributed in provinces such as shanxi, northriver, southeast, Shandong, Jiangsu, Anhui and the like in China, and has pathogenic bacteria of the disease, namely dothiorella subsp (dothiorella Sacc), and a sexual generation of the disease is Botryosphaeria dothidea, which mainly damages main trunks and lateral branches of walnut trees, and causes withering in severe cases, reduced fruiting capacity, even death of the whole walnut trees, and huge economic loss is caused.

For the prevention and treatment of walnut canker, forestry cultivation management measures and chemical pesticides are mainly used for prevention and treatment in the current production. However, the disease has the characteristics of long incubation period, long disease period and multiple hazards, and the hazards can be well controlled only by carrying out multiple treatments in one growth period and applying chemical pesticides for multiple times in the prevention and treatment process. Has the problems of high prevention and treatment cost, long time consumption, environmental pollution and the like. Therefore, the search for new economic, efficient, green and environment-friendly prevention and treatment technologies and ways becomes a problem to be urgently solved in the prevention and treatment of the walnut canker at present.

The bacillus is a general name of aerobic or facultative anaerobic bacillus producing gram-positive bacillus, has rich and various physiological characteristics, widely exists in nature, is an important microbial population in the rhizosphere of soil and plant body surfaces, is non-toxic and harmless to people and livestock, does not pollute the environment, can produce various antibiotics and enzymes, and has broad-spectrum antibacterial activity. The mechanism of using bacillus as biocontrol bacteria to prevent and treat plant diseases mainly comprises: antibiotic action, nutrition competition, space competition, heavy parasitism, induction of plant resistance and other mechanisms, and the main biocontrol mechanism is to generate antagonistic substances to inhibit the growth of harmful pathogenic bacteria or kill pathogenic bacteria. Most of the substances are peptide antibiotics, and have the advantages of high efficiency, low residue, environmental friendliness and the like.

Disclosure of Invention

The invention aims to provide a new microorganism, namely a Bacillus belgii strain LH3-2 for efficiently preventing and treating walnut canker. The Bacillus belgii strain has the characteristics of high growth speed, large spore yield, strong stress resistance, capability of fast and mass colonization on the surface of a plant and the like, thereby having good application prospect.

The above purpose of the invention is realized by the following technical scheme: the strain provided by the invention is a Bacillus velezensis (Bacillus velezensis) strain LH3-2, which is separated from the small clear river water in Jinan City, and has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: Hongyang district Tunglu in Beijing, China academy of sciences microbial research institute) in 2017, 26 months and the preservation number is CGMCC No. 14744. It has the following biological properties: bacterial colonies on a beef extract culture medium are milky white, viscous and wrinkled and convex, and the bacterial cells are rod-shaped and can move under microscopic examination; gram-positive (E.coli control); and observing the spores to be oval or columnar after the spores are dyed.

The culture method or the propagation method of the Bacillus belgii (B.velezensis) strain LH3-2 comprises the following steps:

(1) the common culture and preservation beef extract culture medium comprises the following components: 3g of beef extract, 2.5g of glucose, 5g of sodium chloride, 17g of agar and 1000mL of distilled water, and the pH value is adjusted to 7.2-7.4.

(2) The laboratory liquid culture adopts a beef extract liquid culture medium, and the formula is as follows: 3g of beef extract, 2.5g of glucose, 5g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.2-7.4.

(3) The solid culture medium formula comprises: the solid material and the inorganic salt are mixed according to the mass ratio of 98.7: 1.3; the solid materials are calculated according to the mass ratio of rice hull, corn flour, soybean meal and bran to be 52: 30: 10: 8; the inorganic salt comprises, by mass, 12% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 73% of light calcium carbonate.

(4) The formula of the mass fermentation culture comprises: the formula of the solid culture medium in the step (3).

The invention also provides a biological control preparation for controlling walnut canker, which comprises the Bacillus belgii (B.velezensis) strain LH 3-2.

The invention also provides a biological control method for preventing and treating the walnut canker, which comprises the step of applying the bacillus beileyanensis (B.velezensis) strain LH3-2 or the biological control preparation to walnuts with the canker.

The invention also provides application of the bacillus beilesensis or the biological control preparation in preventing and treating walnut canker.

The preparation method of the biological control preparation comprises the following steps:

(1) preparing seed solution of Bacillus belgii strain LH 3-2;

(2) inoculating the seed liquid prepared in the step (1) into a solid culture medium, and culturing at a constant temperature of 27-30 ℃;

(3) and (3) adding sterile water into the culture cultured in the step (2), mixing, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 27-30 ℃ and the relative humidity of more than 85%.

In one embodiment of the present invention, the preparation method comprises the steps of:

(1) transplanting spores of the Bacillus belgii strain LH3-2 into a beef extract liquid culture medium, and performing shake culture on a shaker at 27-30 ℃ for 2-3 d to obtain a seed solution;

(2) inoculating the seed solution prepared in the step (1) into a solid culture medium according to the mass ratio of 10%, and performing shake culture at 27-30 ℃ for 3-5 days;

(3) adding sterile water into the culture cultured in the step (2) according to the mass ratio of 1: 15, mixing, filtering, inoculating the filtrate to a large amount of fermentation medium according to the volume ratio of 1: 6, and performing fermentation culture in a fermentation chamber with the room temperature of 27-30 ℃ and the relative humidity of more than 85% for 8-9 days.

The beef extract liquid culture medium in the step (1) is prepared from 3g of beef extract, 2.5g of glucose, 5g of sodium chloride and 1000mL of distilled water, and the pH value is adjusted to 7.2-7.4.

The solid culture medium in the step (2) consists of solid materials and inorganic salt, wherein the mass ratio of the solid materials to the inorganic salt is 98.7: 1.3; the solid material comprises, by mass, rice hulls, corn flour, soybean meal and bran in a ratio of 52: 30: 10: 8; the inorganic salt comprises, by mass, 12% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 73% of light calcium carbonate.

The mass fermentation medium in the step (3) is synchronous with the solid medium in the step (2).

Experiments show that the Bacillus belgii strain has the characteristics of high growth speed, high spore yield, strong stress resistance, capability of fast and mass colonization on the surface of walnut, and the like, so that the Bacillus belgii strain has a good application prospect. The biological control preparation prepared from the Bacillus belgii can efficiently control walnut canker, and is a biological control preparation with great application prospect.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications are within the scope of the invention.

Example one

1. Isolation and purification of B.velezensis LH3-2

The bacillus belgii (B.velezensis) LH3-2 is obtained by separating the water of the Minqing river (117.09E,36.73N) from the Minqing river of the Minnan city in 2013 and 5 months by adopting a dilution plate method and a plate scribing method, wherein the separation method comprises the following steps:

(1) isolation of Bacillus

The sample is collected from the small clear river in the city of Jinan, the sample is sampled by using a sterilized mineral water bottle, the distance between two sides of the river is 2m from the river bank, the sample is 3 x 500mL, the sample is uniformly mixed, and the mixture is brought back indoors and temporarily stored in a 4 ℃ incubator. Weighing 10g of water sample in 90mL of sterile water, placing the water sample in a shaking table at 30 ℃ and shaking at 150rpm for 10min, then placing the water sample in a water bath kettle at 60 ℃ and incubating for 30min, and taking 100 mu L of 10^-2、10^-3、10^-4And coating the diluent on an LB (Langmuir-Blodgett) culture medium plate, coating three layers of the diluent in parallel in each gradient, culturing at 28 ℃ for 2 days, picking microbial colonies with different forms on the beef extract culture medium, scribing on the beef extract culture medium plate, and regularly observing the growth condition of the colonies. Then, a plate marking method is adopted to purify the bacillus strains, and the bacillus strains are respectively numbered and stored.

(2) Screening of efficient antagonistic bacillus for walnut canker

① preliminary screening, preparing PDA (Potato Dextrose agar) plate by using a contraposition culture method, taking fungus cakes with diameter of 5mm from the edges of bacillus and walnut canker by using a puncher, respectively transplanting the fungus cakes into the centers of two opposite sides of the plate, culturing at constant temperature of 28 ℃, and observing the inhibition effect of the bacillus on pathogenic bacteria day by day.

② rescreening the screened bacillus strains with high-efficiency antagonistic activity, mainly screening bacillus strains with better tolerance through temperature resistance, acid and alkali resistance and drug resistance tests, and performing pot control tests and field tests.

The inventor obtains a Bacillus belgii (B.velezensis) LH3-2 strain capable of efficiently preventing and treating walnut canker through a large amount of screening work. Experiments prove that the Bacillus beilesensis raw powder shows very high-efficiency prevention and treatment effects in preventing and treating walnut canker. Therefore, the Bacillus belgii is a novel strain of Bacillus belgii with wide application prospect, and can be used for preparing biological control preparations for preventing and treating walnut canker.

2. Identification of strains

(1) Microbiological characteristics

Bacterial colonies on a beef extract culture medium are milky white, viscous and wrinkled and convex, and the bacterial cells are rod-shaped and can move under microscopic examination; gram-positive (E.coli control); and observing the spores to be oval or columnar after the spores are dyed.

(2) Molecular biological Properties

The 16s rRNA gene sequence determination results of Bacillus velezensis (Bacillus velezensis) strain LH3-2 are as follows:

CCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCC

(3) results of cell morphology and physicochemical experiments

TABLE 1 cell morphology and results of physicochemical experiments on B.velezensis LH3-2

Experimental project	Results	Experimental project	Results
				Gram stain	Positive for	Acid production from carbohydrates	+
Cell shape	Rod-shaped	Glucose	+
				The diameter of the cells is more than 1 mu m	-	Xylose	+
Form spores	+	L-arabinose	+
				Enlargement of spore	-	Mannitol	-
Spore is round	-	Lactose	-
				Parasporal crystal	-	Fermentation of glucose to produce gas	+
Contact enzyme	+	Using citric acid salts	+
				Oxidase enzyme	+	Growth at 50 deg.C	+
Anaerobic growth	-	Growth at pH 5.7	+
				VP assay	+	Growth with 7% NaCl	+
VP﹤pH 6	+	Starch hydrolysis	+
				VP﹥pH 7	-	Decomposition of casein	+
Nitrate reduction	+	Hydrolyzed gelatin	+

Example two

1. Bacillus belgii fermentation process

The formula of the beef extract liquid culture medium is as follows: 3g of beef extract, 2.5g of glucose, 5g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.2-7.4.

The solid material is as follows: according to the mass ratio, the ratio of the rice hulls to the corn flour to the soybean meal to the bran is 52: 30: 10: 8; the inorganic salt comprises the following components in percentage by mass: 12% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 73% of light calcium carbonate; the mass ratio of the solid material to the inorganic salt is 98.7: 1.3.

A large-scale solid fermentation process of bacillus belgii (b. velezensis) strain LH 3-2:

① culture of strain seed liquid

Selecting a small amount of bacteria from a test tube inclined plane by using a B.velezensis strain LH3-2, transferring the bacteria into a beef extract liquid culture medium, and carrying out shake culture on a shaking table at 28 ℃ for 2-3 days to obtain a seed solution.

② cultivation of solid producer strains

Inoculating the seed solution into a solid culture medium (500mL triangular flask) according to the mass ratio of 10%, culturing at the constant temperature of 28 ℃ for 3-5 days, and shaking for multiple times in the middle.

③ fermentation of large amount of solid

Diluting a culture obtained by solid culture in ② with sterile water according to the proportion of 1: 15, filtering with sterile gauze, removing coarse residues to obtain a production bacterial liquid, inoculating the production bacterial liquid into a large amount of fermentation culture medium according to the proportion of 1: 6, placing the inoculated raw materials into a fermentation chamber (at 28 ℃ and relative humidity of more than 85%) for fermentation culture for 8-9 days to obtain a fermentation liquid, wherein the active bacteria in the fermentation liquid can reach 40 hundred million/g, and the active bacteria can reach 200 plus 300 hundred million/g after the raw powder is further prepared.

In order to determine the prevention and treatment effect of Bacillus belezii (B.velezensis) LH3-2 fermentation liquor on walnut canker, a basis is provided for popularization and application of the Bacillus belezensis LH3-2 fermentation liquor. We performed field experiments in 3-6 months in 2016 in Shandong province, Linyi City, and the results are reported as follows:

1 test materials and methods

1.1 test agent

B.velezensis strain LH3-2 fermentation broth (40 hundred million/g viable bacteria) produced by the fermentation method of the second embodiment of the invention; 80% ethylicin emulsifiable concentrate (commercially available from Jiangsu New agricultural chemical Co., Ltd.).

1.2 test materials and control objects

The test material is walnut; the object for preventing and treating walnut canker is walnut canker.

1.3 conditions of the test

The test site is arranged in a peony hickory producing area in the prefecture of Linyi city. In this area, the incidence of ulcer disease was 15.3% and disease index was 6.8. The test soil type is brown soil, the organic matter content is 0.75%, the pH value is 6.8, the cultivation conditions and management measures of all test districts are consistent, and the walnut canker is in the early stage of disease development during application.

1.4 test design and arrangement

The experiment has 3 treatments, namely 10 times of fermentation liquor of a B.velezensis strain LH3-2, 300 times of 80% ethylicin missible oil and no application of clear water as a control, wherein each treatment is repeated for 4 times, and each treatment is 60 strains. Spraying once in 2016 each of 3 months and 15 days, 4 months and 11 days, 5 months and 16 days and 6 months and 15 days, wherein the spraying device is an agricultural-16 type sprayer.

1.5 test investigation and calculation method

1.5.1 weather conditions

The medicine for the 1 st application (3 months and 15 days) is cloudy in the day, the wind power is 3 grades, the highest air temperature is 13 ℃, the lowest air temperature is 2 ℃, and the relative humidity is 56%. The medicine is applied for the 2 nd time (4 months and 11 days), the day is cloudy, the wind power is 3 grades, the highest temperature is 21 ℃, the lowest temperature is 9 ℃, and the relative humidity is 55%; the 3 rd application (5 months and 16 days) is carried out on the sunny day, the wind power is 3 grades, the highest temperature is 26 ℃, the lowest temperature is 13 ℃, and the relative humidity is 60 percent; the 4 th application (6 months and 15 days) is carried out on the sunny day, the wind power is 3 grades, the highest temperature is 32 ℃, the lowest temperature is 20 ℃, and the relative humidity is 67%.

1.5.2 drug efficacy and safety investigation

And (3) drug effect investigation: investigation was performed 14d after the last application. Each treatment investigated 50 plants, the bark in the lower part of the trunk, the incidence, the progression of the disease and the disease index.

Safety investigation: and (5) observing the safety of the walnuts at 7d and 14d after the first pesticide application, if the pesticide damage occurs, describing the pesticide damage symptoms in detail and determining the pesticide damage degree according to the grading standard of the pesticide damage degree.

Grading method (with single plant as unit):

level 0: non-sick class

Level 1: the lesion area is below 1/4 of the whole trunk area.

And 2, stage: the lesion area accounts for 1/4-2/4 of the whole trunk area.

And 3, level: the lesion area accounts for 2/4-3/4 of the whole trunk area.

4, level: the lesion area accounts for more than 3/4 of the whole trunk area.

1.5.3 method for calculating drug effect

The drug effect is calculated according to the formulas (1) and (2):

in the formula: CK (CK)₀-pre-drug condition index in the placebo zone;

CK₁-disease index after drug administration in the placebo zone;

PT_{0————}pre-drug treatment disease index;

PT_{1————}disease index after drug treatment application.

1.5.4 Effect on Sophora japonica growth

Observing whether the pesticide has phytotoxicity on the walnuts, and recording the type and degree of the phytotoxicity. In addition, other beneficial effects on the walnuts (e.g., growth stimulation, etc.) were also noted.

Phytotoxicity was recorded in the following manner:

(a) if the phytotoxicity can be measured or calculated, it is expressed in absolute terms, for example, as plant height.

(b) In other cases, the extent and frequency of phytotoxicity can be estimated in two ways:

① the phytotoxicity degree of walnut is recorded according to phytotoxicity grading method, and is represented by —, + + + + + + + +, +.

The phytotoxicity grading method comprises the following steps:

and (2) preparing: no chemical injury;

+: slight phytotoxicity does not affect the growth of the forest;

++: moderate phytotoxicity, can be recovered, and can not cause the yield reduction of the trees;

+++: moderate phytotoxicity affects the normal growth of the trees and causes certain loss on the yield and quality of the trees;

++++: serious phytotoxicity, blocked growth of forest trees and serious loss of yield and quality.

② the percentage of phytotoxicity is evaluated by comparing the chemical treatment area with the blank control area, and the phytotoxicity symptoms (dwarfing, chlorosis, malformation, etc.) of the crops are accurately described.

2 results

2.1 prevention and treatment effects of the test agent on walnut canker

The disease conditions of walnut canker before the three-agent treatment are shown in table 2, and the control effect of the three-agent treatment on the walnut canker is shown in table 3. The results in table 3 show that the fermentation liquor of the B.velezensis strain LH3-2 and 80% of ethylicin missible oil have good control effects on walnut canker, wherein the control effect of the B.velezensis strain LH3-2 is over 70%. From the disease index of the walnut ulcer disease field test (table 3), 14 days after the last application, the control effect of bacillus belief (b.velezensis) LH3-2 is 75.37%, which is obviously higher than 57.25% of 80% ethylicin missible oil.

TABLE 2 disease condition of walnut ulcer before three kinds of drug treatment

TABLE 3 preventive and therapeutic effects on walnut ulcer by three drug treatments

2.2 walnut safety survey

Through the observation of the application of the medicines for 7d and 14d, compared with the control area, the walnut growth is normal, no phytotoxicity is generated in each medicine treatment area, and the safety of the tested concentration of the Bacillus beilisensis fermentation liquor on the walnut is proved.

3 small knot

3.1 from the view of disease index and prevention and treatment effect, the Bacillus belgii (B.velezensis) strain LH3-2 (with the preservation number of CGMCC No.14744) has good prevention and treatment effect on walnut canker, the prevention and treatment effect of 14 days after the last application reaches more than 70%, and is obviously better than that of a control medicament, and the difference is obvious.

3.2 from the growth vigor of walnuts, the Bacillus belgii (B.velezensis) strain LH3-2 has an obvious growth promoting effect on walnuts, has no phytotoxicity, and is safe and reliable.

SEQUENCE LISTING

<110> scientific research institute of forestry in Shandong province

<120> Bacillus belgii and application thereof in prevention and treatment of walnut ulcer disease

<130>0

<160>1

<170>PatentIn version 3.3

<210>1

<211>1376

<212>DNA

<213> 16s rRNA gene sequence of Bacillus velezensis strain LH3-2

<400>1

cctcaccgac ttcgggtgtt acaaactctc gtggtgtgac gggcggtgtg tacaaggccc 60

gggaacgtat tcaccgcggc atgctgatcc gcgattacta gcgattccag cttcacgcag 120

tcgagttgca gactgcgatc cgaactgaga acagatttgt gggattggct taacctcgcg 180

gtttcgctgc cctttgttct gtccattgta gcacgtgtgt agcccaggtc ataaggggca 240

tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtca ccttagagtg 300

cccaactgaa tgctggcaac taagatcaag ggttgcgctc gttgcgggac ttaacccaac 360

atctcacgac acgagctgac gacaaccatg caccacctgt cactctgccc ccgaagggga 420

cgtcctatct ctaggattgt cagaggatgt caagacctgg taaggttctt cgcgttgctt 480

cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcctt tgagtttcag 540

tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt agctgcagca ctaaggggcg 600

gaaaccccct aacacttagc actcatcgtt tacggcgtgg actaccaggg tatctaatcc 660

tgttcgctcc ccacgctttc gctcctcagc gtcagttaca gaccagagag tcgccttcgc 720

cactggtgtt cctccacatc tctacgcatt tcaccgctac acgtggaatt ccactctcct 780

cttctgcact caagttcccc agtttccaat gaccctcccc ggttgagccg ggggctttca 840

catcagactt aagaaaccgc ctgcgagccc tttacgccca ataattccgg acaacgcttg 900

ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtggctttct ggttaggtac 960

cgtcaaggtg ccgccctatt tgaacggcac ttgttcttcc ctaacaacag agctttacga 1020

tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag actttcgtcc attgcggaag 1080

attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag tgtggccgat 1140

caccctctca ggtcggctac gcatcgtcgc cttggtgagc cgttacctca ccaactagct 1200

aatgcgccgc gggtccatct gtaagtggta gccgaagcca ccttttatgt ctgaaccatg 1260

cggttcagac aaccatccgg tattagcccc ggtttcccgg agttatccca gtcttacagg 1320

caggttaccc acgtgttact cacccgtccg ccgctaacat cagggagcaa gctccc 1376

Claims

1. Bacillus belgii（Bacillus velezensis）The strain LH3-2, wherein the preservation number of the strain is CGMCCNo.14744.

2. The use of the bacillus beilesensis strain LH3-2 of claim 1 for preventing and treating walnut canker.

3. A biocontrol formulation comprising bacillus beijerinckii strain LH3-2 of claim 1.

4. A process for the preparation of a biocontrol agent as claimed in claim 3, characterized in that it comprises the steps of:

(1) preparing seed solution of Bacillus belgii strain LH 3-2;

(3) adding sterile water into the culture cultured in the step (2), mixing, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 27-30 ℃ and the relative humidity of more than 85%;

the solid culture medium in the step (2) consists of solid materials and inorganic salt, wherein the mass ratio of the solid materials to the inorganic salt is 98.7: 1.3; the solid material comprises, by mass, rice hulls, corn flour, soybean meal and bran = 52: 30: 10: 8; the inorganic salt comprises 12% of monopotassium phosphate, 5% of magnesium sulfate, 10% of ammonium sulfate and 73% of light calcium carbonate by mass percent;

5. The method of claim 4, comprising the steps of:

(1) transplanting spores of the Bacillus belgii strain LH3-2 into a beef extract liquid culture medium, and performing shake culture on a shaker at 27-30 ℃ for 2-3 d to obtain a seed solution; the beef extract liquid culture medium comprises: 3g of beef extract, 2.5g of glucose, 5g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.2-7.4;

(3) adding sterile water into the culture cultured in the step (2) according to the mass ratio of 1: 15, mixing, filtering, inoculating the filtrate to a large amount of fermentation medium according to the volume ratio of 1: 6, and performing fermentation culture in a fermentation chamber with the temperature of 27-30 ℃ and the relative humidity of more than 85% for 8-9 days.

6. The use of the biocontrol formulation of claim 3 for the control of walnut canker.

7. A biocontrol method for walnut canker, characterized in that a bacillus beijerinckii strain LH3-2 as claimed in claim 1 or a biocontrol agent as claimed in claim 3 is applied to walnuts having canker.