CN107267412B

CN107267412B - Bacillus methylotrophicus and application thereof

Info

Publication number: CN107267412B
Application number: CN201710371468.5A
Authority: CN
Inventors: 谢晓梅; 廖敏; 吕婷; 方至萍
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-05-24
Filing date: 2017-05-24
Publication date: 2020-04-14
Anticipated expiration: 2037-05-24
Also published as: CN107267412A

Abstract

The bacillus methylotrophicus CGMCC No.13541 has an antagonistic effect on various plant pathogenic bacteria such as botrytis cinerea, fusarium graminearum and rhizoctonia solani, is an antagonistic strain with obvious effect of preventing and treating plant diseases and is environment-friendly, and has good development and application prospects. The fermentation liquor obtained by culturing the beef extract peptone liquid medium can be used for preventing and treating plant diseases, has the bacteriostasis rate of up to 84 percent to botrytis cinerea of strawberry which is about 8 percent higher than that of common biocontrol bacteria, and lays a foundation for the development and utilization of new disease-preventing microbial resources. Strong stress resistance and wide application range. The growth speed is high, a large amount of colonization can be realized rapidly, and the biological control preparation is convenient to prepare and popularize in the field.

Description

Bacillus methylotrophicus and application thereof

(I) technical field

The invention relates to bacillus methylotrophicus (Bacillus methylotrophicus) LM-N separated from strawberry greenhouse soil and application thereof in preventing and treating strawberry gray mold.

(II) background of the invention

Gray mold caused by Botrytis spp is an important plant disease, and is often damaged on fruits and vegetables such as strawberries, grapes, tomatoes, lettuce, cucumbers and the like. The outbreak of the disease in different periods can cause damping-off of plants in seedling stages or rot of flowers and fruits, and has developed into one of important diseases affecting the preservation and freshness during the production or storage of protected vegetables, fruits and flowers. The gray mold can reduce the yield of vegetables in a protected area by more than 20 percent, and serious areas with improper disease source control or cultivation measures are even faced with dead production. The strawberry is listed as one of ten major industries for developing agricultural production in China, and the economic and nutritional values of the strawberry are high. The strawberry cultivation adopts a greenhouse technology, so that the growth period is shortened, the yield is improved, however, the microclimate environment in the greenhouse is generally high in humidity, the average temperature in winter and spring seasons is higher than that outdoors, the environmental conditions are very favorable for the occurrence of the gray mold disease, the gray mold disease becomes an important factor for limiting the quality and the yield improvement of the strawberries, and the prevention and control measures of the gray mold disease of the strawberries directly determine the yield and the economic benefit of the strawberries.

At present, the prevention and the treatment of the gray mold of the strawberry are simultaneously carried out by several approaches of disease-resistant breeding, agricultural cultivation prevention and treatment, chemical pesticide prevention and biological prevention and treatment. As the gray mold is a facultative parasitic bacterium and also has a saprophytic stage, and an antigen of the gray mold is not found at present, the implementation of disease-resistant breeding is difficult; the agricultural cultivation prevention and control principle mainly strengthens the management of water and fertilizer in the field, adjusts the temperature, the humidity, the illumination and the like, so that complete control facilities of field conditions are required and cannot be realized under the existing conditions of strawberry farmers in China; at present, chemical pesticide control is still the main means for controlling strawberry gray mold in China, but the continuous use of chemical agents, especially the continuous use of single agents, causes gray mold pathogen to gradually generate drug resistance, the control effect of the chemical pesticide is also reduced year by year, pesticide spraying cannot be avoided when the chemical pesticide is applied to the surfaces of fruits, and pesticide pollution of the fruits is caused by residual agents on the fruits. In recent years, people pay attention to the development and utilization of beneficial microorganisms and metabolites thereof to prevent and treat gray mold, and because the beneficial microorganisms and the metabolites thereof are safe to the environment, have obvious effect and are slightly influenced by environmental conditions, the biological prevention and treatment method for controlling strawberry gray mold is gradually developed into an important and potential prevention and treatment way, the application prospect is wide, and the biological prevention and treatment method is one of inevitable trends for preventing and treating strawberry gray mold in the future.

Disclosure of the invention

The invention aims to provide a strain, namely Bacillus methylotrophicus (LM-N), with good antagonistic action on various plant pathogenic bacteria.

The technical scheme adopted by the invention is as follows:

the invention provides a Bacillus methylotrophicus (Bacillus methylotrophicus) LM-N separated from rhizosphere soil of a plant with strawberry gray mold, and the morphological characteristics of the strain are as follows: the shape is rod-shaped, gram-positive, spore ellipse, and the bacterial colony is circular, milky white, opaque, with center raised and wrinkled, and the edge is jagged after culturing for 48h at 30 ℃ on beef extract peptone medium. Deposited in China general microbiological culture Collection center, address: the microbial research institute of the national academy of sciences, No. 3, west road, north chen, chaoyang, beijing, 100101, accession number: CGMCC No.13541, preservation date: year 2017, month 01, day 06.

The invention also provides an application of the bacillus methylotrophicus LM-N in preparation of a plant pathogenic bacterium bactericide, wherein the plant pathogenic bacterium is botrytis cinerea, fusarium graminearum or rhizoctonia solani.

Further, the bactericide contains fermentation liquor obtained by fermenting and culturing bacillus methylotrophicus LM-N.

Further, the fermentation liquor is prepared by the following method: (1) inoculating bacillus methylotrophicus LM-N to a beef extract peptone culture medium, and culturing at 30 ℃ for 20-24 hours to obtain slant thalli; the beef extract peptone culture medium comprises the following components: 3g/L of beef extract, 5g/L of peptone, 3g/L of sodium chloride, 20g/L of agar, distilled water as a solvent, 7.0-7.4 of pH value, and performing moist heat sterilization (121 ℃, 20 min);

(2) selecting slant thallus, inoculating to LB liquid culture medium, and culturing at 30 deg.C for 24 hr to obtain seed liquid; the LB liquid culture medium comprises: 10g/L of peptone, 5g/L of sodium chloride, 10g/L of yeast extract, distilled water as a solvent, 7.0-7.4 of pH value, and performing moist heat sterilization (121 ℃, 20 min);

(3) fermentation culture: inoculating the seed solution to a fermentation culture medium by an inoculation amount with the volume concentration of 4%, and fermenting for 3 days at 30 ℃ and 180rpm to obtain a fermentation liquid; the fermentation medium comprises the following components: 40g/L soybean cake powder (commercially available, special for the culture medium), 10g/L glucose, 3g/L peptone, 2g/L NaCl, 2g/L CaCO₃The solvent is distilled water, the pH value is 7.0-7.4, and the wet heat sterilization is carried out (121 ℃, 20 min);

further, the content of LM-N cells of the bacillus methylotrophicus in the antibacterial agent is 1.0 x 10⁶～1.0×10⁸cfu/mL。

Compared with the prior art, the invention has the beneficial effects that:

1. the bacillus methylotrophicus CGMCC No.13541 has antagonistic action on various plant pathogenic bacteria, such as Botrytis cinerea, Gibberella zeae and Rhizoctonia solani, is an antagonistic strain with obvious effect of preventing and treating plant diseases and environment-friendly, and has good development and application prospects.

2. The bacillus methylotrophicus CGMCC No.13541 is simple and convenient to apply, the strain body and fermentation liquor obtained by culturing in a beef extract peptone liquid culture medium can be used for preventing and treating plant diseases, the bacteriostasis rate of the bacillus methylotrophicus CGMCC No.13541 to botrytis cinerea is up to 84 percent and is about 8 percent higher than that of common biocontrol bacteria, and a foundation is laid for development and utilization of new disease-preventing microorganism resources.

3. The bacillus methylotrophicus CGMCC No.13541 produces spores, has strong stress resistance and wide application range.

4. The bacillus methylotrophicus CGMCC No.13541 has high growth speed, can be fast colonized in a large amount, and is convenient for preparing biological control preparations to be popularized and applied in fields.

(IV) description of the drawings

FIG. 1 is a scanning electron micrograph of strain LM-N.

FIG. 2 is a colony morphology of strain LM-N.

FIG. 3 shows a strain LM-N phylogenetic tree constructed based on the 16S rDNA sequence.

FIG. 4 shows the effect of LM-N on the growth of Botrytis cinerea (A), Rhizoctonia solani (B), and Gibberella cerealis (C).

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1

1 materials and methods

1.1 media and reagents

Beef extract peptone medium: 0.3g of beef extract, 0.5g of peptone, 0.3g of NaCl, 2g of agar and 100mL of water, wherein the pH value is 7.2-7.4, and the beef extract is used for culturing antagonistic bacteria.

PDA culture medium: 20g of potato, 2g of glucose, 2g of agar and 100mL of water, and the pH is natural. It can be used for the culture of pathogenic fungi.

Enrichment culture medium: potato200g of potato, 10g of glucose, 3g of beef extract, 5g of peptone, 5g of NaCl and KH₂PO₄2g，MgSO₄1g, agar 20g, water 1L, pH 7.2. Used for confrontation culture and bacteriostasis rate determination.

LB liquid medium: 10g/L of peptone, 5g/L of sodium chloride, 10g/L of yeast extract, distilled water as a solvent and 7.0 of pH value. The method is used for preparing seed liquid.

1.2 dominant degradation Strain Breeding

1.2.1 sources of strains

Collecting rhizosphere soil of Fengbu strawberry greenhouse strawberry plants in Nibo city of Zhejiang province.

1.2.2 isolation, purification and screening of degrading strains

Weighing 10g of soil sample, adding 90mL of sterile water, oscillating for 2h to form suspension, adding 1mL of liquid into a test tube containing 9mL of sterile water, and oscillating to form 10g of suspension^-2Diluting, 10 times serial dilution to 10^-60.1mL of each gradient dilution was pipetted onto a beef extract peptone medium plate and the broth was smeared onto the beef extract peptone medium plate with a sterile glass smear bar. And (3) culturing the smeared flat plate in a 30 ℃ constant temperature box for 48h, picking out single colonies from the flat plate, repeatedly carrying out flat plate streaking separation culture until the single colonies are obtained by screening, inoculating the pure colonies onto a slant culture medium, and storing in a 4 ℃ refrigerator.

Inoculating 24h activated Botrytis cinerea (Botrytis cinerea) fungus cakes with diameter of 6mm (provided by plant pathology laboratories of Zhejiang university, known as common germs stored in each plant pathology laboratory) in the center of a PDA (personal digital assistant) plate, inoculating the separated and purified bacteria into the PDA plate in a triangular shape by using an inoculating needle, wherein the distance from the center is about 2cm, and placing the PDA plate in a constant-temperature incubator at 30 ℃ for observation and opposite culture bacteriostasis. And (4) storing the strain with obvious bacteriostatic action for later use, and screening to obtain the strain LM-N.

1.2.3 Observation of morphological characteristics of Strain and measurement of physiological and biochemical characteristics

1.2.3.1 morphological identification: gram stain and colony morphology

Taking a single bacterial colony of the strain LM-N cultured for 24 hours, smearing the single bacterial colony on a glass slide for gram staining, and observing the single bacterial colony under a microscope;

taking a strain LM-N glutaraldehyde stationary liquid cultured for 24 hours for overnight culture, carrying out subsequent treatment in an electron microscope chamber, and observing morphological characteristics of the strain by a scanning electron microscope. The results are shown in FIGS. 1 and 2.

As can be seen from fig. 1 and 2: the strain LM-N is rod-shaped bacteria, gram-positive bacteria, the diameter of a bacterial colony is 2-3mm, the bacterial colony is circular, milky white and opaque, the center of the bacterial colony is raised and provided with folds, and the edge of the bacterial colony is serrated.

1.2.3.2 characterization of physiological and biochemical Properties

The physiological and biochemical characteristics are determined according to the handbook of identification of common bacterial systems (Dongxu pearl, Chuia Miao Yin).

The results of the measurement were as follows: the most suitable culture medium of the strain LM-N is a beef extract peptone culture medium, the culture temperature is 30 ℃, the suitable growth pH is 6-7, glucose, sucrose, starch, mannitol, maltose, lactose and xylose can be used as carbon sources, gelatin can be liquefied, a methyl red test is positive, and other physiological and biochemical characteristics are shown in Table 1.

TABLE 1 physiological and biochemical Properties of Strain LM-N

Note: + positive reaction; negative reaction

1.3 molecular biological identification

The 16S r DNA gene sequence (1437bp, SEQ ID NO.1) of the strain is shown as follows:

GGCGGCGTGCTATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAG

the strain is obtained by screening collected strawberry greenhouse plant rhizosphere soil in Shanbo city of Shanghai county, Zhejiang province, 16S rDNA sequencing is used for identifying a gene sequence of the strain, the gene sequence is compared with the similarity of the existing 16S r DNA in an NCBI database, the similarity is 99%, a phylogenetic tree is constructed after analysis, the strain LM-N is identified to be Bacillus methylotrophicus (Bacillus methylotrophicus) in Bacillus (Bacillus), the strain is named as Bacillus methylotrophicus (Bacillus methylotrophicus) LM-N, and the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the address is as follows: the microbial research institute of the national academy of sciences, No. 3, west road, north chen, chaoyang, beijing, 100101, accession number: CGMCC No.13541, preservation date: year 2017, month 01, day 06. .

Example 2 antagonistic Properties and inhibitory rates of the Strain LM-N against Botrytis cinerea, Rhizoctonia solani and Gibberella cerealis

Measurement of

The Botrytis cinerea, Gibberella tritici and Rhizoctonia solani described in this example are provided by the Phytopathology research laboratory of the institute of agriculture and biotechnology, Zhejiang university (generally known).

Pathogenic fungi (Botrytis cinerea), Rhizoctonia solani (Rhizoctonia solani) and Gibberella graminis (Fusarium head bright) which are commercially available and commonly stored in various plant pathology laboratories are activated on a PDA plate for 48 hours at 28 ℃, cakes are prepared at the symmetrical positions of the edges of colonies by using a puncher (with the diameter of 6mm), the mycelia are inoculated to the center of an enrichment medium plate in a downward mode until the diameter of the cakes reaches 2cm by dark culture at 28 ℃, single-layer filter paper sheets are sterilized by the same aperture, soaked in bacterial liquid (LB liquid medium LM-N) of biocontrol bacteria (methylotrophic bacillus LM-N) to be tested for 24 hours at 30 ℃, and evenly placed on the PDA plate. The filter paper sheets are symmetrically arranged 30mm away from the center of the fungus cake of the pathogenic fungus to be tested, and the colony diameter of the pathogenic fungus to be tested and the inhibition rate of the biocontrol fungus to be tested on the pathogenic fungus to be tested are measured after 4 days. Clear water was used as control. Inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%. The test results are shown in FIG. 4 and Table 2, and the plant pathogenic bacteria in FIG. 4 are Botrytis cinerea, Rhizoctonia solani and Gibberella cerealis from left to right.

TABLE 2 inhibition rate of LM-N strain on various plant pathogenic bacteria

The results show that: the methylotrophic bacillus CGMCC No.13541 has the strongest inhibition rate of 84 percent on botrytis cinerea, has the second inhibition effect of 72 percent on rhizoctonia solani and has the inhibition rate of 68 percent on wheat scab. Comprehensively, the bacillus methylotrophicus CGMCC No.13541 has stronger inhibition effect on various plant pathogenic bacteria and has great popularization value.

Example 3 field effect verification of Bacillus methylotrophicus CGMCC No.13541 fermentation broth on strawberry gray mold

3.1 test agent

Bacillus methylotrophicus CGMCC No.13541 fermentation liquor; wettable powder of 50% prohyacin (commercially available).

The fermentation liquor is prepared by the following method:

(1) inoculating bacillus methylotrophicus LM-N to a beef extract peptone culture medium, and culturing at 30 ℃ for 20-24 hours to obtain slant thalli; the beef extract peptone culture medium comprises the following components: 3g/L of beef extract, 5g/L of peptone, 3g/L of sodium chloride, 20g/L of agar, distilled water as a solvent and 7.0 of pH value;

(2) selecting slant thallus, inoculating to LB liquid culture medium, and culturing at 30 deg.C for 24 hr to obtain seed liquid; the LB liquid culture medium comprises: 10g/L of peptone, 5g/L of sodium chloride, 10g/L of yeast extract, distilled water as a solvent and 7.0 of pH value;

(3) fermentation culture: inoculating the seed solution to a fermentation culture medium at a volume concentration of 4%, and fermenting at 30 deg.C and 200rpm for 3 days to obtain a fermentation solution with thallus content of 1.0 × 10⁷cfu/mL; the fermentation medium comprises the following components: 40g/L soybean cake powder (commercially available, special for fermentation medium), 10g/L glucose, 3g/L peptone, 2g/L NaCl, 2g/L CaCO₃The solvent is distilled water, and the pH value is 7.0;

3.2 test article and control object

Test work: strawberry of red color

The control object is: gray mold

3.3 general description of the test

The test site is arranged in a Shanghua brook strawberry planting base in Nibo, Zhejiang, and a test for preventing and controlling the gray mold of the strawberries is carried out in a strawberry planting greenhouse in the test site in 2016. The soil types and the fertilization and irrigation measures of all the test districts are consistent, and the method accords with the local agricultural production standard.

3.4 design of the test

The experiment designs the Bacillus methylotrophicus CGMCC No.13541 fermentation liquor (the thallus content is 1.0 multiplied by 10)⁷cfu/mL) diluted 400 times, 600 times and 800 times with clear water, and 5 treatments of 50% wettable powder diluted 800 times with clear water and clear water control, 8m for each cell²Repeat 3 times.

3.5 test investigation and statistical method

The disease index is investigated 10 days before and after the spraying of the medicament, and the investigation is carried out for 2 times in the test period. The investigation was performed using a random sampling method. 5 plants are randomly selected in each cell, 3 branch marks are selected from each plant, and 5 leaves are counted from the fully unfolded leaf to the lower part before and after application for investigation and recording. During the application of the agent, it was observed whether or not a phytotoxicity was generated.

The classification criteria are as follows:

level 0: no disease spots;

level 1: the lesion area accounts for less than 5% of the whole leaf area;

and 3, level: the lesion area accounts for 6 to 10 percent of the whole leaf area;

and 5, stage: the lesion area accounts for 11 to 20 percent of the whole leaf area;

and 7, stage: the lesion area accounts for 21-50% of the whole leaf area;

and 9, stage: the lesion area accounts for more than 51 percent of the whole leaf area.

Calculating disease index and prevention and treatment effect according to the following formula:

disease index ∑ (number of diseased leaves at each stage × relative stage)/(number of investigated diseased leaves × maximum representative extremum) × 100

Control effect (%) [1- (control area pre-drug disease means x treatment area post-drug disease means)/(control area post-drug disease means x treatment area pre-drug disease means) ] × 100

3.6 prevention and treatment effects of the test agent on strawberry Botrytis cinerea

TABLE 3 field effect of Bacillus methylotrophicus CGMCC No.13541 fermentation liquid on strawberry gray mold

Test of

The test result shows that: 10 days after the application, the prevention and treatment effect is the best when the fermentation liquor of the bacillus methylotrophicus CGMCC No.13541 is diluted by 400 times, and is 75.33 percent; the control effects of the CGMCC No.13541 fermentation liquor diluted 600 and 800 times are not greatly different, namely 71.24 percent and 670.25 percent respectively; the control effect of 50% peyrone diluted by 800 times is obviously lower than that of the treatment of CGMCC No.13541 fermentation liquor.

4 small knot

From the aspect of disease index and prevention and treatment effect of strawberry gray mold, the bacillus methylotrophicus CGMCC No.13541 fermentation liquid has better prevention and treatment effect on strawberry gray mold, and the effect can reach 75.33%. Is harmless to human and fruits, is environment-friendly and is worthy of development and popularization.

SEQUENCE LISTING

<110> Zhejiang university

<120> Bacillus methylotrophicus and application thereof

<130>

<160>1

<170>PatentIn version 3.5

<210>1

<211>1437

<212>DNA

<213>Bacillus methylotrophicus

<400>1

ggcggcgtgc tataatgcag tcgagcggac agatgggagc ttgctccctg atgttagcgg 60

cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc 120

ggggctaata ccggatggtt gtctgaaccg catggttcag acataaaagg tggcttcggc 180

taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag 240

gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300

agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc 360

aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa 420

gtgccgttca aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac 480

gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 540

gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 600

attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 660

aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 720

gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780

aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa 840

gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg 900

cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960

acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg 1020

catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080

cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg 1140

gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200

tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt 1260

tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc 1320

ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac 1380

gagagtttgt aacacccgaa gtcggtgagg taacctttat ggagccagcc gccgaag 1437

Claims

1. Bacillus methylotrophicus (Bacillus methylotrophicus) LM-N is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No.13541, preservation date: year 2017, day 01, 06, address: the microbiological research institute of the national academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

2. The use of Bacillus methylotrophicus LM-N according to claim 1 in the preparation of a bactericide against plant pathogenic bacteria, wherein the plant pathogenic bacteria are Botrytis cinerea, Gibberella graminearum or Rhizoctonia solani.

3. The use according to claim 2, wherein the bactericide comprises a fermentation broth of Bacillus methylotrophicus LM-N obtained by fermentation culture.

4. Use according to claim 3, characterized in that the fermentation broth is prepared as follows:

(1) inoculating bacillus methylotrophicus LM-N to a beef extract peptone culture medium, and culturing at 30 ℃ for 20-24 hours to obtain slant thalli; the beef extract peptone culture medium comprises the following components: 3g/L of beef extract, 5g/L of peptone, 3g/L of sodium chloride, 20g/L of agar, distilled water as a solvent and 7.0-7.4 of pH value;

(2) selecting slant thallus, inoculating to LB liquid culture medium, and culturing at 30 deg.C for 24 hr to obtain seed liquid; the LB liquid culture medium comprises: 10g/L of peptone, 5g/L of sodium chloride, 10g/L of yeast extract, distilled water as a solvent and 7.0-7.4 of pH value;

(3) fermentation culture: inoculating the seed solution to a fermentation culture medium by an inoculation amount with the volume concentration of 4%, and fermenting for 3 days at 30 ℃ and 180rpm to obtain a fermentation liquid; the fermentation medium comprises the following components: 40g/L soybean cake powder, 10g/L glucose, 3g/L peptone, 2g/L NaCl, 2g/L CaCO₃The solvent is distilled water, and the pH value is 7.0-7.4.

5. The use according to claim 3, wherein the antibacterial agent contains 1.0X 10 cells of Bacillus methylotrophicus LM-N⁶～1.0×10⁸cfu/mL。