CN109355233B

CN109355233B - Bacillus amyloliquefaciens and application thereof

Info

Publication number: CN109355233B
Application number: CN201811473006.5A
Authority: CN
Inventors: 魏婷婷; 王芳; 杨德玉; 赵彭年; 王洪庆; 王远; 李继广
Original assignee: Shenyang Research Institute of Chemical Industry Co Ltd
Current assignee: Shenyang Research Institute of Chemical Industry Co Ltd
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2021-04-09
Anticipated expiration: 2038-12-04
Also published as: CN109355233A

Abstract

The invention relates to the technical field of biological control and biological engineering, in particular to bacillus amyloliquefaciens SY-SF-01 and application thereof. The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens SY-SF-01 preserved in China center for type culture collection, the preservation number is CCTCC M2018264, and the preservation time is 2018, 5 months and 14 days. And further provides a screening method for understanding the bacillus amyloliquefaciens SY-SF-01 and application thereof in inhibiting rhizoctonia solani kuhn, cucumber fusarium wilt, tobacco target spot pathogen, red bean colletotrichum and cotton verticillium wilt. The strain has very obvious effect of preventing and treating rice sheath blight, the living body prevention effect is as high as more than 90%, the tobacco target spot disease is also well prevented and treated, and the in vitro bacteriostasis rate and the living body pot culture prevention effect can be more than 90%.

Description

Bacillus amyloliquefaciens and application thereof

Technical Field

The invention relates to the technical field of biological control and biological engineering, in particular to bacillus amyloliquefaciens SY-SF-01 and application thereof.

Background

The rice sheath blight disease is one of important rice diseases in the world and is one of main diseases of each large rice area in China, the pathogenic bacteria of the rice sheath blight disease is Rhizoctonia solani Kuhn, mycelium or sclerotia is mainly used for overwintering in soil or on diseased straws and other host residues, and the rice sheath blight disease has the characteristics of strong rot and wide parasitism, and favors high temperature and high humidity. The rice sheath blight can occur in the whole growth period of rice, mainly harms leaf sheaths and leaves, and can also damage ears and penetrate into stalks in severe cases. The disease causes the reduction of the maturing rate of rice, the thousand kernel weight and the yield reduction of the rice to be 50 percent.

The tobacco target spot disease is a new disease reported in China for the first time by Wuyuanhua and the like, the disease is one of main diseases of tobacco leaf parts caused by Rhizoctonia solani Kuhn, the disease mainly occurs in the vigorous stage and the mature stage of tobacco, and is a new disease of the domestic tobacco with short incubation period and high popularity, the disease suddenly and widely occurs in tobacco planting areas in Dandong City of Liaoning province in 2005, huge economic loss is brought to tobacco growers, and the disease is successively reported in North Liaoning areas, Guangxi areas, Heilongjiang areas and the like.

Spp. is a bacillus rod-shaped bacterium which is aerobic or facultative anaerobic, gram-positive, and produces spores. The spores generated by the bacillus have strong capability of resisting the external environmental pressure such as drying, heat, ultraviolet radiation and the like, and simultaneously, the bacillus population has a plurality of strains with special functions, so the bacillus population has wide application value in the fields of industry, agriculture, medicine, video processing and the like. The bacillus amyloliquefaciens is a bacillus, is an aerobic bacillus, has wide antibacterial capability, has good application prospect in the environmental protection, planting industry and aquaculture industry, can degrade tanning solid waste, degrade corn straws, relieve broiler liver oxidation damage, prevent and control plant diseases and the like.

At present, the use of bacillus for preventing and treating plant diseases is reported at home and abroad, and the bacillus with bacteriostatic action mainly comprises the following components: bacillus subtilis, bacillus amyloliquefaciens, bacillus thuringiensis, bacillus polymyxa, bacillus megaterium, bacillus pumilus and the like. In the aspect of biological control of fungal diseases such as rice sheath blight, bacillus subtilis is widely researched in China, for example, Bie and the like, fungycin analogues are separated and extracted from bacillus subtilis fermentation liquor, and can obviously inhibit cotton verticillium wilt; Lee.H and the like extract a lipopeptide bamylocin F from fermentation liquor of bacillus subtilis, and the lipopeptide bamylocin F has a good effect on inhibiting corn northern leaf blight. In China, Zhengzhen extracts antibacterial peptide from bacillus subtilis G-7 to prevent and control banana wilt; wangjing et al colonize bacillus amyloliquefaciens (Bacillus. amyloliquefaciens) B6 on tomato roots to prevent and control tomato blight; the populic winter statics and the like utilize the bacillus amyloliquefaciens strain XZ-1 to biologically control the black spot of the sweet potatoes. Therefore, the bacillus has a wide variety of plant diseases and has no foresight and corresponding revelation.

Disclosure of Invention

The invention aims to provide bacillus amyloliquefaciens SY-SF-01 and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a bacillus amyloliquefaciens characterized by: the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens SY-SF-01 preserved in China center for type culture collection, the preservation number is CCTCC M2018264, and the preservation time is 2018, 5 months and 14 days.

The strain SY-SF-01 is bacillus separated from soil of a paddy field in Wuchang city of Heilongjiang province; the strain is identified as bacillus amyloliquefaciens by a 16S rDNA gene sequencing method; morphological characteristics of Bacillus amyloliquefaciens SY-SF-01: the strain SY-SF-01 is a gram-positive aerobic bacterium, a bacterial colony formed on a peptone culture medium plate is round or nearly round, milky white and glossy, the bacterial colony has protrusions along with the prolonging of the culture time, and a thallus rod shape and endogenous spores are observed under a microscope.

Application of the bacillus amyloliquefaciens SY-SF-01 in prevention and treatment of rice sheath blight disease, cucumber fusarium wilt, tobacco target spot disease, red bean anthracnose or cotton verticillium wilt.

A biological control agent contains the bacillus amyloliquefaciens SY-SF-01.

The bacillus amyloliquefaciens is a culture, a culture concentrate, a culture bacterial suspension, a fermentation liquid or a fermentation separation supernatant of the strain.

In the fermentation liquor, the viable count of the bacillus amyloliquefaciens is not less than 1 multiplied by 10⁸cfu/mL。

The fermentation liquor is obtained by activating the bacteria, inoculating the bacteria into a peptone liquid culture medium, and performing shake culture at 25-30 ℃ for 24-72 h at 120-200 r/min;

the culture concentrate is obtained by performing centrifugal separation, cation exchange resin D101 adsorption, 75% ethanol desorption and ethyl acetate extraction on the fermentation liquor.

The culture conditions for culturing the strain of the present invention are not particularly limited, and the culture temperature, pH, shaking speed, liquid loading amount, and the like may be suitable for the growth of the strain. Usually, the temperature is controlled at 25-30 deg.C, pH is 7.0-8.0, the shaking speed is 120-200 r/min, the liquid loading is usually 250mL, 80-120 mL culture medium is loaded, the culture is carried out for 24-72 hours (preferably 48 hours), and the final thallus concentration is 1 × 10⁸～1×10¹⁰cfu/mL。

The application of the biocontrol microbial inoculum, and the application of the biocontrol microbial inoculum in preventing and treating rice sheath blight disease, cucumber fusarium wilt, tobacco target spot disease, red bean anthracnose or cotton verticillium wilt.

The application of the bio-control fungicide is that the bio-control fungicide is sprayed on rice or tobacco plants and is used for preventing and treating rice sheath blight disease or tobacco target spot disease.

The invention has the following advantages and beneficial effects:

the bacillus amyloliquefaciens SY-SF-01 has a wider antibacterial spectrum, has obvious control effect on soil-borne diseases of crops which are difficult to control by chemical agents, has better inhibiting effect on rice sheath blight bacteria, cucumber fusarium wilt bacteria, red bean anthracnose bacteria, tobacco target spot bacteria and cotton fusarium wilt bacteria, can play an important role in planting of pollution-free field crops and vegetables, has low production cost, is easy to produce, is simple in actual application and operation, and has large application potential in the aspect of biological control of plant diseases; the method specifically comprises the following steps:

the bacillus amyloliquefaciens SY-SF-01 has obvious bacteriostatic action on various germs such as rice sheath blight bacteria, cucumber fusarium wilt bacteria, red bean colletotrichum, tobacco target spot bacteria, cotton fusarium wilt bacteria and the like, and has the prevention effect on the rice sheath blight as high as more than 90 percent, the invention also discloses that the bacillus amyloliquefaciens SY-SF-01 has obvious prevention and control effect on the rice sheath blight, the biological prevention and control effect of a living body has the bacteriostatic rate of 93.2 percent, and the indoor pot culture prevention and control effect and the field prevention and control effect on the rice sheath blight can both reach more than 90 percent; in addition, the bacteriostasis rate and the living potted plant control effect on the tobacco target spot are as high as more than 90 percent, and the cucumber fusarium wilt control effect is good.

Detailed Description

The present invention is described in further detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1 isolation, screening and identification of Strain SY-SF-01

Bacillus amyloliquefaciens SY-SF-01 is separated from soil of a paddy field in Wuchang city of Heilongjiang province.

The separation method comprises the following steps: placing 10g of collected soil sample into a 250mL triangular flask filled with 100mL of distilled water, placing the flask in a shaking table, oscillating for 2d at 28 ℃ to prepare a sample suspension, adding 0.5mL of the sample suspension into a sterile water centrifugal tube filled with 4.5mL of sterile water, diluting by 10 times step by step, and taking 0.1mL of 10⁴、10⁵、10⁶、10⁷The diluted solution was spread on peptone medium (sucrose 30g, peptone 5g, FeSO)₄·7H₂O 0.01g，MgSO₄·7H₂O 0.5g，KCl 0.5g，K₂HPO₄1g, 20g of agar powder and 1000ml of distilled water, and the pH value is 7.0-7.2), and culturing for 2d in a constant temperature incubator at 28 ℃ according to the form. Selecting 180 single colonies together, and repeatedly streaking and purifying to obtain pure strains.

The screening method comprises the following steps: the bacteriostatic activity of the screened strains is determined by adopting an in-vitro plate confronting culture method, rhizoctonia solani is inoculated to the central position of a PDA culture medium plate, streaking culture is carried out at a position 2.5cm away from a bacterial cake by using an inoculating loop dipped with the separated strains, the width of a bacteriostatic zone between the pathogenic bacteria and the separated strains is measured after 24 hours of culture at 28 ℃, the strains with the bacteriostatic bandwidth are screened, and the obtained strains with the strongest bacteriostatic ability and easy reproduction are named as SY-SF-01.

The identification method comprises the following steps: the strain SY-SF-01 is a gram-positive aerobic bacterium, a colony formed on a peptone culture medium plate is round or nearly round, milky white and glossy, the colony has protrusions along with the prolonging of the culture time, and a thallus rod shape with the size of 1.0-1.2 microns multiplied by 0.6-0.8 microns is observed under a microscope, and endophytic spores are formed. Amplifying 16S rDNA gene of SY-SF-01 strain, measuring its 16S rDNA gene sequence (see sequence table), and logging inhttp://www.ncbi.nlm.nih.govAnd (3) performing nucleotide comparison in GenBank on a website, wherein the result shows that the genetic relationship between the biocontrol bacterium SY-SF-01 and Bacillus amyloliquefaciens is closest. The strain SY-SF-01 is determined to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) through comprehensive analysis.

The 16S rDNA sequence of the SY-SF-01 strain is as follows:

TGTCACTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTG

TTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGT

ATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGC

AGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGG

CTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGT

GTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCC

TCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCA

ACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG

ACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGG

GACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTC

TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCC

CGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGT

GCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTA

GCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCT

CCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC

GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAA

TTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCC

CCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGC

CCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGC

GGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGG

TGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTAC

GATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGT

CCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTG

TCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC

GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCAT

CTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAA

ACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACA

GGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGC

AAGCTCCCATCTGTCCGCTCGACTGCATGTATAGCACGCCC

example 2 further plate bacteriostatic Effect of Strain SY-SF-01

Fermentation medium: 30g of sucrose, 5g of peptone and FeSO₄·7H₂O 0.01g，MgSO₄·7H₂O0.5g，KCl 0.5g，K₂HPO₄1g, 1000ml of distilled water, pH 7.0-7.2

Preparing a biocontrol bacterium SY-SF-01 fermentation liquor: the SY-SF-01 pure strain obtained in the example 1 is inoculated into a 250ml triangular flask with the liquid containing 50ml of sterilized toothpick, and is subjected to shaking culture for 48 hours on a constant temperature shaking table with the temperature of 28 ℃ and the rotating speed of 160r/min to obtain a fermentation liquid for later use.

Inoculating pathogenic bacteria such as rice sheath blight bacteria, cucumber fusarium wilt bacteria, small red bean anthracnose bacteria, tobacco target spot bacteria and cotton fusarium wilt bacteria on a PDA culture medium, growing the PDA culture medium on a culture dish, punching a bacterial cake from the edge of the bacterial colony by using a puncher with the diameter of 6mm, inoculating the bacterial cake at the central position of a blank PDA culture medium flat plate, inoculating 1 mu l of biocontrol bacteria SY-SF-01 fermentation liquor on two sides with equal distance from the bacterial cake, culturing at the constant temperature of 27 ℃ by taking sterile water as a reference, observing the opposite culture bacteriostasis condition, repeating each treatment for 3 times, measuring the growth radius of the bacterial colony of the pathogenic bacteria when the pathogenic bacteria hypha in the control PDA culture medium flat plate is about to grow on the whole plate, and calculating the relative bacteriostasis rate of the bacterial strain SY-SF-01 to the growth of the pathogenic bacteria by adopting the.

The relative inhibition rate is (control colony radius-treated colony radius)/control colony radius multiplied by 100 percent

Bacillus amyloliquefaciens SY-SF-01 has obvious bacteriostatic action on rice sheath blight bacteria, cucumber fusarium wilt bacteria, red bean colletotrichum, tobacco leaf blight bacteria and cotton fusarium wilt bacteria, the results are shown in Table 1, and the relative bacteriostatic rates are 93.2%, 87.5%, 89.5%, 92.9% and 89.7% respectively.

TABLE 1 bacteriostatic effect of Bacillus amyloliquefaciens SY-SF-01 on different pathogenic bacteria

Example 3 preparation of a fermentation broth concentrate of Strain SY-SF-01

The Bacillus amyloliquefaciens SY-SF-01 obtained in example 1 is inoculated on a peptone solid medium to activate the strain, the activated strain is inoculated on a sterilized toothpick into a 250ml triangular flask with the liquid containing volume of 50ml, and the shaking table is operated at a constant temperature of 28 ℃ and the rotating speed of 160r/minPerforming shake culture for 24h to obtain fermented seed solution, inoculating the seed solution into an enlarged fermentation medium (with the same composition as the fermentation medium in example 2) at an inoculum size of 5% by volume, culturing at 25-30 deg.C and 120-200 r/min for 24-72 h to obtain fermentation liquid, and diluting to obtain a product with a concentration of 1 × 10⁸～1×10¹⁰cfu/mL, centrifuging at 1000r/min, taking the centrifuged supernatant, adsorbing with nonpolar cation exchange resin D101, adding 10g of nonpolar cation exchange resin D101 into each 1000mL triangular flask containing 300mL of the centrifuged supernatant, oscillating for 24h, filtering out the resin, eluting with 75% ethanol until colorless, extracting the eluate with ethyl acetate, removing the extraction solvent with a rotary evaporator to obtain a concentrated solution, and diluting the concentrated solution of the fermentation solution with sterile water to obtain a 10-time diluted solution of the concentrated solution of the fermentation solution and a 100-time diluted solution of the concentrated solution of the fermentation solution for later use.

Peptone solid medium: 30g of sucrose, 5g of peptone and FeSO₄·7H₂O 0.01g，MgSO₄·7H₂O 0.5g，KCl 0.5g，K₂HPO₄1g, 20g of agar powder and 1000ml of distilled water, wherein the pH value is 7.0-7.2

Fermentation medium: 30g of sucrose, 5g of peptone and FeSO₄·7H₂O 0.01g，MgSO₄·7H₂O0.5g，KCl 0.5g，K₂HPO₄1g, 1000ml of distilled water, and pH 7.0-7.2

Example 4 potted plant biocontrol effect of fermentation broth concentrate of Strain SY-SF-01 on Rice sheath blight disease

Inoculating rice sheath blight pathogenic bacteria cake on PDA solid culture medium (potato 200g, glucose 20g, agar powder 18g, 1L distilled water) plate, culturing at constant temperature of 25 deg.C, and standing until a large amount of sclerotium grows out.

Sowing the Yanfeng 47 rice seeds in a seedling pot, and transplanting for later use when the seedlings grow to 3 leaves and one heart stage.

The concentrated fermentation solution (stock solution) fermented for 48 hours, the 10-time diluent of the concentrated fermentation solution and the 100-time diluent of the concentrated fermentation solution in the embodiment 3 are respectively and uniformly sprayed on the rice leaves which grow uniformly and are transplanted for 20 days, each treatment is repeated for 3 times, the blank control group is sprayed with equal amount of distilled water, after 24 hours, the sclerotium of the rhizoctonia solani is inoculated in the leaf sheath, and after 10 days, the disease spot expansion condition is investigated.

Grading according to the harm symptoms of sheath and leaf of rice, wherein the grading standard of rice sheath blight is as follows:

level 0: the whole plant is disease-free;

level 1: the 4 th leaf and the leaf sheaths and leaves below the 4 th leaf (the 1 st leaf is sword leaf);

and 3, level: the 3 rd leaf and the leaf sheaths and leaves below the 3 rd leaf are attacked;

and 5, stage: the 2 nd leaf and the leaf sheaths and leaves below the 2 nd leaf are attacked;

and 7, stage: the scabies of the sword leaf and the scabies and the leaves below the scabies;

and 9, stage: the whole plant is attacked and died in advance.

TABLE 2 prevention and treatment effects of concentrated fermentation broth of SY-SF-01 strain on rice sheath blight

As can be seen from Table 2, the concentrated fermentation broth of the strain SY-SF-01 with the fermentation time of 48h has good control effect on rice sheath blight disease.

Example 5 fermentation time optimization

The experiment in example 3 was repeated with fermentation time of 24h, 36h, 48h, 60h, and 72h for the preparation of the concentrated fermentation solution described in example 2 to obtain concentrated fermentation solution (stock solution), 10-fold diluted fermentation solution, and 100-fold diluted fermentation solution, respectively, at different fermentation times, and the experimental results were as follows:

TABLE 3 control of rice sheath blight disease by fermentation broth concentrate of SY-SF-01 strain at different fermentation times

TABLE 4 prevention and control Effect of fermentation broth concentrate of SY-SF-01 strain on rice sheath blight disease at different fermentation times II

TABLE 5 prevention and control Effect of fermentation broth concentrate of SY-SF-01 strain on rice sheath blight disease at different fermentation time III

TABLE 6 prevention and control effects of concentrated fermentation liquid of SY-SF-01 strain on rice sheath blight disease at different fermentation times IV

TABLE 7 control Effect of fermentation broth concentrate of SY-SF-01 strain on rice sheath blight at different fermentation time V

As can be seen from tables 3 to 7, the fermentation time is 24 to 72 hours, the concentrated fermentation liquor of the strain SY-SF-01 has good control effect on rice sheath blight, and the fermentation time is 48 hours as the best.

Example 6 field biocontrol effect of fermentation broth concentrate of Strain SY-SF-01 on Rice sheath blight disease

The test is carried out in a paddy field in the Wei country of Wuchang city of Heilongjiang province, the test field is a perennial paddy planting field,the rice variety is Liaoxing No. 1 (japonica rice), and rice sheath blight occurs all the year round. The cell area is 320m²(16m multiplied by 20m) and random block arrangement, wherein each treatment is repeated for 3 times, a strain SY-SF-01 fermentation liquid concentrated solution fermented for 48 hours in example 3 and 10-fold and 100-fold diluent thereof are arranged according to a table 8, the concentrated solution is applied by a spraying method, a pefurine is taken as a key spraying part from the middle upper part, the concentrated solution is applied in 8 months and 1 day in 2018, the rice sheath blight disease occurrence condition of each cell is investigated in 9 months and 1 day in 2018, then the disease index and the control effect are calculated, the statistics and the calculation are carried out by referring to' pesticide field efficacy test criterion (I) bactericide control rice sheath blight disease GB/T17980.20-2000, and the investigation result is shown in a table 9.

And (4) classifying according to the symptom degree of the plant as a harmful symptom, taking leaves as a unit, randomly sampling 5 points in each cell, surveying and connecting 4 points at each point, and recording the total leaf number, the diseased leaf number and the disease grade number of the survey.

Grading standard of rice sheath blight

Level 0: the whole plant is disease-free;

level 1: basal lamina sheath disease;

and 2, stage: the disease of each leaf sheath or leaf below the third leaf (the first leaf from the apical leaf);

and 3, level: the disease of each leaf sheath or leaf below the second leaf;

4, level: apical leaf sheath or apical leaf disease;

and 5, stage: the whole plant is attacked and withered.

TABLE 8 test design of test agents

TABLE 9 field control Effect of SY-SF-01 Strain fermentation broth concentrate on Rice sheath blight disease

As can be seen from Table 9, the fermentation broth concentrate of the Bacillus amyloliquefaciens SY-SF-01 strain also has good control effect on rice sheath blight in the field.

Example 7 potted plant biocontrol effect of fermentation broth concentrate of Strain SY-SF-01 on tobacco Targeted leaf spot

Inoculating tobacco target spot pathogenic bacteria cake on PDA solid culture medium (potato 200g, glucose 20g, agar powder 18g, 1L distilled water) plate, and culturing at constant temperature of 26 deg.C until the culture dish is full of the pathogenic bacteria cake.

When the NC89 tobacco grows to the leaf stage of 9, respectively spraying the stock solution of the fermentation liquid concentrated solution, the 10-time diluent of the fermentation liquid concentrated solution and the 100-time diluent of the fermentation liquid concentrated solution fermented for 48 hours in the embodiment 3 on different tobacco plant leaves, spraying an equal amount of clear water as a blank control, repeating the treatment for 10 times, inoculating the tobacco target spot pathogen bacterial cakes by adopting a needle punching method after 24 hours, wherein the diameter of the bacterial cakes is 6mm, inoculating the 5 th and 6 th true leaves for each plant, inoculating 3 bacterial cakes for each plant, preserving moisture by using sterilized cotton, removing the bacterial cakes and the cotton after 48 hours, investigating the morbidity and the disease index after 7 days, and calculating the prevention effect.

A method for investigating the diameter of disease spots by artificially inoculating tobacco leaf blight germs in a greenhouse by referring to Zhao Yanqin and the like comprises the following steps: level 0: 0.0 mm; level 1: 0.0-1.0 mm; grade 3, 1.1-2.0 mm; and 5, stage: 2.0-5.0 mm; and 7, stage: 2.01-9.0 mm; and 9, stage: >9.01 mm.

TABLE 10 prevention and treatment effects of concentrated fermentation broth of SY-SF-01 strain on tobacco target spot

As can be seen from Table 8, the fermentation broth concentrate of the Bacillus amyloliquefaciens SY-SF-01 strain with the fermentation time of 48h also has a good effect of preventing and treating the target leaf spot of tobacco.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> Shenyang chemical research institute Co., Ltd

<120> bacillus amyloliquefaciens and application thereof

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<170> SIPOSequenceListing 1.0

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<213> Bacillus amyloliquefaciens SY-SF-01(Bacillus amyloliquefaciens SY-SF-01)

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tgtcacttcg gcggctggct ccataaaggt tacctcaccg acttcgggtg ttacaaactc 60

tcgtggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat 120

ccgcgattac tagcgattcc agcttcacgc agtcgagttg cagactgcga tccgaactga 180

gaacagattt gtgggattgg cttaacctcg cggtttcgct gccctttgtt ctgtccattg 240

tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc 300

tccggtttgt caccggcagt caccttagag tgcccaactg aatgctggca actaagatca 360

agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca 420

tgcaccacct gtcactctgc ccccgaaggg gacgtcctat ctctaggatt gtcagaggat 480

gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg 540

tgcgggcccc cgtcaattcc tttgagtttc agtcttgcga ccgtactccc caggcggagt 600

gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacactta gcactcatcg 660

tttacggcgt ggactaccag ggtatctaat cctgttcgct ccccacgctt tcgctcctca 720

gcgtcagtta cagaccagag agtcgccttc gccactggtg ttcctccaca tctctacgca 780

tttcaccgct acacgtggaa ttccactctc ctcttctgca ctcaagttcc ccagtttcca 840

atgaccctcc ccggttgagc cgggggcttt cacatcagac ttaagaaacc gcctgcgagc 900

cctttacgcc caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 960

acgtagttag ccgtggcttt ctggttaggt accgtcaagg tgccgcccta tttgaacggc 1020

acttgttctt ccctaacaac agagctttac gatccgaaaa ccttcatcac tcacgcggcg 1080

ttgctccgtc agactttcgt ccattgcgga agattcccta ctgctgcctc ccgtaggagt 1140

ctgggccgtg tctcagtccc agtgtggccg atcaccctct caggtcggct acgcatcgtc 1200

gccttggtga gccgttacct caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg 1260

tagccgaagc caccttttat gtctgaacca tgcggttcaa acaaccatcc ggtattagcc 1320

ccggtttccc ggagttatcc cagtcttaca ggcaggttac ccacgtgtta ctcacccgtc 1380

cgccgctaac atcagggagc aagctcccat ctgtccgctc gactgcatgt atagcacgcc 1440

c 1441

Claims

1. A bacillus amyloliquefaciens characterized by: bacillus amyloliquefaciens deposited in the China center for type culture Collection (Bacillus amyloliquefaciens) (II)Bacillus amyloliquefaciens) SY-SF-01 with preservation number of CCTCC M2018264 and preservation time of 2018, 5 months and 14 days.

2. The application of the bacillus amyloliquefaciens SY-SF-01 disclosed in claim 1 in prevention and treatment of rice sheath blight disease, cucumber fusarium wilt, tobacco target spot disease, red bean anthracnose or cotton verticillium wilt.

3. A biological control agent is characterized in that: the biocontrol microbial inoculum comprises the bacillus amyloliquefaciens SY-SF-01 as claimed in claim 1.

4. The biocontrol microbial inoculum of claim 3 comprising a culture, a suspension of a cultured strain, a fermentation broth of the strain.

5. The biocontrol microbial inoculum of claim 4 wherein the number of viable bacteria of Bacillus amyloliquefaciens in the fermentation broth is not less than 1 x 10⁸cfu/mL。

6. The biocontrol microbial inoculum according to claim 4, wherein the fermentation broth is obtained by activating the bacteria, inoculating the bacteria into a peptone liquid culture medium, and performing shake culture at 25-30 ℃ for 24-72 h at 120-200 r/min.

7. The use of the biocontrol microbial inoculum of claim 3, wherein the biocontrol microbial inoculum is used for preventing and treating rice sheath blight disease, cucumber fusarium wilt, tobacco target spot disease, red bean anthracnose or cotton verticillium wilt.

8. The use of the biocontrol microbial inoculum according to claim 7, wherein the biocontrol microbial inoculum is sprayed on rice or tobacco plants for preventing and treating rice sheath blight diseases or tobacco target spot diseases.