CN112195137A - Bacillus belgii for antagonizing magnaporthe grisea and producing gamma-polyglutamic acid and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and discloses a strain capable of antagonizing rice blast germs and producing gamma-polyglutamic acid. The strain is Bacillus belgii SS-20, has been preserved in China center for type culture Collection with a preservation date of 26 days 10 months 2020 and a preservation number of CCTCC NO: m20200637. The strain has the capability of antagonizing rice blast germs and producing gamma-polyglutamic acid, can be applied to the fields of plant disease control, microbial fertilizer, food preservation and preservation, medicine, environmental protection and the like, and has very great application value and market development potential.

Description

Bacillus belgii for antagonizing magnaporthe grisea and producing gamma-polyglutamic acid and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus beiLeisi capable of antagonizing rice blast germs and producing gamma-polyglutamic acid and application thereof.

Background

Rice blast (rice blast disease) is one of the most serious rice diseases, and can cause 10-30% of yield reduction each year. The rice blast fungus (Magnaporthe oryzae) is a pathogenic bacterium causing rice blast. Conidia of rice blast fungus are pear-shaped and are induced to form a specialized infected cell-attachment cell (apressorium) after falling on the surface of rice tissue, the attachment cell generates huge turgor pressure by precipitating a layer of thick melanin on the inner side of a cell wall and synthesizing a large amount of glycerol in the cell, and the attachment cell invades the rice tissue to grow and expand in the rice tissue, so that scabs are generated. The infected hyphae can extend from the lesion to the air to form conidiophores, and the conidiophores are differentiated from the tail ends to carry out another round of infection. In addition to infecting rice, the filamentous fungi may also infect certain weeds in other plants, such as barley, wheat, and grasses.

At present, chemical control is still the conventional control method for controlling rice blast, and the generation of the rice blast is mainly inhibited by inhibiting the biosynthesis pathway of melanin. However, the long-term use of chemical agents on a large scale is not economical and safe, and may cause pathogenic bacteria to develop resistance. The biological control has the advantages of difficult resistance generation, high targeting property, good specificity and the like, and is environment-friendly, which depends on the screening and the discovery of microbial resources with biological control effect.

The main microbial pesticide includes bacteria, actinomycetes and fungi. The bacillus is a typical representative of antagonistic bacteria, and the action mechanism of the bacillus generally has the aspects of antagonism, competition, parasitism, induced resistance and the like. Antagonism refers to the fact that biocontrol bacteria secrete extracellular metabolites to directly kill or inhibit the growth of pathogenic bacteria, or form an environmental condition unfavorable to pathogenic bacteria to indirectly inhibit the growth of pathogenic bacteria. The competitive action refers to that the biocontrol microorganism weakens or eliminates pathogenic bacteria in the same ecological environment by competing for nutrient substances, robbing water, space, physical and biological sites and the like, thereby playing a role in preventing and treating diseases. After entering the plant body, the bacillus preferentially occupies the invasion site of the pathogenic bacteria and competes for nutrition with the pathogenic bacteria. Parasitic action refers to the phenomenon that one organism invades into another organism to obtain nutrition so as to obtain survival and development, and has various forms of adsorption growth, winding, invasion, digestion and the like. After the pathogenic bacteria are colonized by the antagonistic microorganisms, the required growth environment and nutrients are destroyed and thus cannot grow and die. The induction of plant resistance generally includes both forms of systemic acquired resistance and induction of systemic resistance. Systemic acquired resistance refers to that pathogenic bacteria or attenuated strains infect plants to cause the plants to generate anaphylactic reaction and plant protective hormone. The induction of resistance refers to a process that biological or non-biological factors act on a host plant to activate a physical or chemical resistance mechanism of the plant to generate systemic resistance, and the action is characterized in that a non-pathogenic substance induces the plant body to generate a disease-resistant response, but an inducer directly kills or inhibits the pathogenic substance. The bacillus can induce the host plant to generate physical structural resistance, such as inducing the plant to form a structurally compact protective layer at the cell front edge invaded by the pathogen, or inducing the structural change of the host cell wall, such as the formation of papilla on the cell wall and the enhancement of the mechanical strength of the cell, thereby limiting the further expansion of the pathogen, and also inducing the physiological and biochemical activity change of the host plant (including inducing the host plant to accumulate related proteins, synthesize phytoalexin and other metabolites, generate hydrolase and oxidase and the like).

The mechanism of the bacillus for preventing and controlling plant diseases mainly comprises the following steps: the biological control agent can generate a plurality of antibiotics, effectively colonize and compete rhizosphere nutrition, degrade enzymes of microorganisms and induce system resistance, so that the biological control agent can be developed into a series of biological control products such as microorganism viable bacteria preparations and the like, and can be widely applied to the agricultural fields and the food field such as biological fertilizers, biological feeds, biological pesticides and the like. In recent years, researches have found that Bacillus belgii is widely distributed in nature, has good characteristics of rapid growth and stability, is easy to separate and culture, is harmless to human and animals, and does not pollute the environment. The compound has certain functions in the aspects of promoting plant growth, antagonizing pathogenic bacteria, resisting stress, brewing and the like, and has very important theoretical research and practical application values.

The antibacterial substances produced by bacillus mainly comprise proteins, polypeptides, enzymes and the like, and most of the antibacterial substances are peptide antibiotics. The synthesis of bacillus lipopeptides is a non-ribosomal pathway, i.e. a series of biosyntheses is accomplished by macromolecular complex enzyme-non-ribosomal synthetic peptide synthetases (NRPS), the so-called "sulfur template polymerase mechanism". The special biosynthesis mechanism determines that the non-ribosomal peptide has the characteristics of complex amino acid composition and molecular form, small molecular weight, stable physicochemical property, various biological activities and the like. The lipopeptide antibiotics mainly include surfactin lipopeptide (surfactin), iturin (iturin), and fengycin (fengycin). The Iturin family members include Iturin A, B, C, D, E, Bacillus antimycin (Bacillus) D, F, L, and the enzyme subtilin (Mycosubtilin), among others. The non-peptide antibacterial substance also comprises macrolides, phenols, isocoumarins, polyenes, aminoglycosides containing disaccharide, etc. Biological agriculture made of the bacillus and natural antibiotics thereof is an ideal substitute of chemical agents.

Gamma-polyglutamic acid (PGA) is an anionic polypeptide polymer formed by connecting a plurality of glutamic acid monomers (D type or L type) through gamma-amide bonds, can be synthesized by a chemical method or microbial fermentation, has a molecular weight of more than 100-1000 KDa and an average molecular weight of about 1.23 multiplied by 10⁴KDa. PGA has no typical peptide chain structure, and the basic skeleton is in the form of a linear fiber. PGA as a novel environment-friendly high-molecular green biological product has unique physical and chemical properties such as degradability, film forming property, fiber forming property, plasticity, cohesiveness and the like, and can be applied to a plurality of fields such as medicine manufacturing, food processing and fresh keeping, cosmetics, manufacturing industry, agriculture and the likeA domain.

In the aspect of medicine, the PGA has a large number of free carboxyl groups in the molecular structure, so that the PGA has better water solubility and drug loading rate. In addition, self-assembly of PGA with chitosan (or other oligomers) or derivatives thereof can yield pH-sensitive biodegradable nanoparticles that can be used as drug carriers and vaccines for delivery to function in vivo. PGA as biological adhesive can be used for controlling the continuous bleeding of tissue, preventing the leakage of sealing gas and liquid in the body, and repairing the cutting of aorta, and is a novel safe biological adhesive tape. In sewage treatment, free carboxyl groups of PGA are bonded with cations to influence the charging property thereof, thereby further changing the intermolecular force of PGA, and causing coagulation thereof into a flocculent aggregate to cause flocculation. In the aspect of health care, the calcium supplement agent formed by chelating PGA and Ca has obvious slow release effect through detection, the slow release capability does not depend on gastric acid, and Ca can be slowly released to different degrees under the acidic and neutral conditions to achieve the calcium supplement effect. In the agricultural field, PGA can improve the water retention, water permeability and air permeability of soil, and finally improve the soil quality; can be used as a corrosion inhibitor to improve the utilization rate of fertilizers and pesticides; a moisture-preserving film can be formed outside the seeds, which is beneficial to the germination, seedling emergence and rooting of the seeds; can prolong the fresh-keeping and quality-guaranteeing period of fruits, vegetables and the like.

The existing strains can only be used for antibiosis or can only be used for producing PGA, or the effects of the two are limited, the problems of high production cost, low raw material utilization rate, limited application range and the like exist, and the large-scale production and the wide application are difficult to carry out. The strain SS-20 of the invention has the functions of inhibiting rice blast bacteria (producing 3 antibacterial lipopeptides and 1 antibacterial protease) and PGA, and widens the application range of the strain. Therefore, the strain SS-20 has great application value and market development potential.

Disclosure of Invention

The invention aims to provide a bacillus beijerinckii strain for antagonizing rice blast bacteria and producing gamma-polyglutamic acid and application thereof. The method solves the problems that the application range of the existing strain is limited, the production cost is high, and the large-scale production is difficult.

The purpose of the invention is realized by the following technical scheme: the invention provides a strain for antagonizing rice blast germs and producing gamma-polyglutamic acid, wherein the strain SS-20 is preserved in China center for type culture collection, the preservation date is 2020, 10 and 26 days, and the preservation number is CCTCC NO: m20200637, accession number: wuhan university school in Wuhan City of Hubei province. SS-20 grows well on LB plate, the colony is milky white, irregular, opaque, have wrinkles. The cell is rod-shaped, the size is 1.9-2.4 μm, and the two ends are blunt. Can produce spores and is gram-positive.

The strain is determined to be Bacillus belgii through morphological identification and 16s rRNA amplification sequence development tree analysis, and according to the international naming rule: the strain is named by the name of the genus, the name of the seed and the name of the strainBacillus、velezensisAnd SS-20, namedBacillus velezensisSS-20. The Bacillus belgiiBacillus velezensisThe 16S rDNA sequence of SS-20 is shown in SEQ ID NO. 1.

The physiological and biochemical identification result of the strain SS-20 provided by the invention is as follows: esterase (C4), naphthol-AS-Bi-phosphohydrolase, alpha-galactosidase and beta-galactosidase were all positive. Nitrate reduction: positive, acidification of glucose: positive, escin hydrolysis: positive, gelatin hydrolysis: positive, glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, citric acid, phenylacetic acid assimilation: positive, in addition cytochrome oxidase: and (4) positive.

The application of the strain in antagonism of rice blast bacteria is provided.

The application of the strain in preparing rice blast resistant pesticide.

The fermentation product of the strain is applied to antagonizing rice blast germs.

The fermentation product contains iturin, fengycin, surfactin and spore lysin.

The strain is applied to the production of gamma-polyglutamic acid.

The strain is applied to agriculture.

The gene information of the antibacterial substance of the strain SS-20 provided by the invention is as follows:

strain SS-20 has iturin synthase: (ituA) A fengycin synthetase (b)fenB) Surfactant synthetase regulation: (sfP) Partial synthetic gene of Bacillus lysin synthetase Bacilysin: (bacAB) The sizes of PCR products are 1149bp, 1401bp, 675bp and 749bp respectively, and the sequences are shown as SEQ ID NO. 2-5 respectively. After BLAST, the two are respectively compared withBacillus amyloliquefaciensThe similarity of Lx-11 bacillus D synthase A gene (JN 086148.1) is 99.74 percent,Bacillus amyloliquefaciens JT84 FenB (fenB) gene (KX 351428.1) has a similarity of 99.93%,Bacillus amyloliquefaciens JT84 phospho naphthyl transferase (sfp) gene (KX 346253.1) has a similarity of 100.00%,Bacillus subtilis a1/3 bacilysin gene (AF 396778.1) had a similarity of 98.00%.

The strain SS-20 provided by the invention can also be cultured in a fermentation medium at 37 ℃ and 200 rpm for 3 d to produce PGA.

The invention has the beneficial effects that: the invention provides a Bacillus beiLeisi strain for inhibiting rice blast bacteria and producing PGA, which can greatly reduce the production cost, improve the production efficiency and widen the application range of the strain. The strain provided by the invention belongs to internationally recognized safe strains. The strain SS-20 provided by the invention can be used for preparing a biocontrol preparation for inhibiting plant diseases, can also be used for producing microbial fertilizers, additives and the like, and can also be used for producing pharmaceutical and cosmetic additives, food preservatives, environment-friendly preparations and the like.

Drawings

FIG. 1 is a drawing of the present inventionBacillus velezensis Colony morphology of SS-20 on LB plates.

FIG. 2 shows the present inventionBacillus velezensis Scanning electron micrograph of SS-20.

FIG. 3 shows the present inventionBacillus velezensis Spore staining pattern of SS-20.

FIG. 4 shows the present inventionBacillus velezensis SS-20 map for inhibiting rice blast bacteria.

FIG. 5 shows the present inventionBacillus velezensis The effect of SS-20 fermentation product on inhibiting rice blast germs is shown in the figure.

FIG. 6 shows the present inventionBacillus velezensis The SS-20 fermentation product inhibits the morphogram of the magnaporthe grisea mycelium (20 times objective).

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The invention is further illustrated by the following examples.

Example 1: separation and screening of SS-20

Sampling points are arranged outdoors around laboratories of institute for ecological environment protection of agriculture academy of sciences of Shanghai city to collect air bacteria, and an LB culture medium is exposed in the air for 30 min. The cultures were subsequently incubated at 37 ℃ for 24 h, and single colonies were isolated and numbered. Single colonies were inoculated in duplicate into fresh medium and pure cultures were isolated again by gradient dilution. And carrying out proliferation culture on the pure culture strains, observing the characteristics of the colony such as morphology, color, transparency and the like, and selecting a single colony for storage for later use.

Example 2: SS-20 species identification

The morphological observation and physiological and biochemical characteristic analysis of the strain are carried out according to the manual of identifying common bacteria systems. The strain SS-20 has round, opaque, milky white, viscous and irregular edge colonies on LB plates (see figure 1).

The cells were rod-shaped (see FIG. 2), gram-positive, spore-forming (see FIG. 3).

The physiological and biochemical identification results show that: esterase (C4), naphthol-AS-Bi-phosphohydrolase, alpha-galactosidase and beta-galactosidase were all positive. Nitrate reduction: positive, acidification of glucose: positive, escin hydrolysis: positive, gelatin hydrolysis: positive, glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, citric acid, phenylacetic acid assimilation: positive, in addition cytochrome oxidase: and (4) positive.

Bacterial genome DNA extraction kit is used for extracting bacterial genome DNA, and universal primers 27F/1492R are used as primers to amplify 16S rRNA of the bacterial strain. And detecting the PCR product by 1% agarose gel electrophoresis, recovering the gel, and purifying and sequencing the PCR product by Shanghai biological engineering Co. The sequencing results (SEQ ID NO. 1) were BLAST aligned in the NCBI database. The PCR product of SS-20 strain is purified and sequenced to obtain 1385 bp sequence, and the 16S rRNA sequence of SS-20 is found by BLAST comparisonBacillus velezensis FZB42 (NR_075005.2)、Bacillus velezensisThe homology of CBMB205 (NR _ 116240.1) reaches 99.93% and 99.86%.

The strain SS-20 is Bacillus belgii (Bacillus subtilis) by comprehensive analysisBacillus velezensis）。

Example 3: the SS-20 strain has the effect of inhibiting rice blast germs

The test was carried out by plate-confrontation culture, and the solid plate was PDA medium (containing 200 g of boiled filtrate of potato, 20 g of glucose, 20 g of agar per liter, natural pH). The method comprises the steps of inoculating rice blast fungus 856 (provided by plant pathology laboratories of the institute of ecological environmental protection of academy of agricultural sciences of Shanghai city) on PDA, inoculating the prepared SS-20 strains on the corresponding two sides (about 1 cm from the edge of a flat plate) in a point-grafting manner, culturing at 27 ℃ in a confrontation manner, measuring the inhibition capacity of the SS-20 on the growth of the rice blast fungus, and taking the treatment without point-grafting bacteria as a control. After 7 d, the colony diameter of the rice blast germs is observed and measured, and the hypha growth inhibition rate is calculated. The fungal hyphal growth inhibition rate was calculated as follows: the fungal hypha growth inhibition rate = [1- (experimental group fungal hypha growth diameter/control group fungal hypha growth diameter) ] × 100%.

The results showed that SS-20 had a significant inhibitory effect against Pyricularia oryzae, whereas the fungal hyphae of the control group grew well (FIG. 4). The culture time is prolonged to 15 days, and the SS-20 bacteriostatic effect is not weakened. Through measurement, the bacteriostatic efficiency of SS-20 on rice blast fungi can reach 77.46%, which shows that SS-20 can obviously inhibit the growth of rice blast fungi hypha.

Example 4: determination of bacteriostatic activity of SS-20 fermentation product

The SS-20 strain obtained by streak culture is selected and inoculated into MOLP culture medium (100 mL), the fermentation temperature is 37 ℃, the rotating speed of a shaker is 200 rpm, and the fermentation time is 2 d. The MOLP culture medium formula is as follows: 30 g/L of peptone, 20 g/L of sucrose, 7 g/L of yeast extract and KH₂PO₄1.9 g/L, 1 per mill of trace elements and the balance of distilled water, and the pH is 7.2.

And (3) measuring the antibacterial activity of the SS-20 fermentation product by adopting an Oxford cup method. Specifically, SS-20 fermentation product is obtained according to the above method, and the supernatant is extracted with methanol to obtain SS-20 fermentation product. The central part of the PDA culture medium is inoculated with rice blast fungus 856, 200 mu L of SS-20 fermentation product is added into sterile oxford cups at two sides, the mixture is placed at 27 ℃ for opposite culture, and the inhibition capacity of the SS-20 fermentation product on the growth of the rice blast fungus is measured. Sterile methanol was used as a control and fermentation product containing SS-20 was used as a parallel treatment. 3 replicates per treatment set, and after 7 days the growth of rice blast germs was observed and measured (FIG. 5).

The results of the confrontation culture show that the rice blast germs are obviously inhibited by SS-20 fermentation products, and the bacteriostasis rate can reach 71.79 percent through measurement. Shows that SS-20 live bacteria cells have the effect of inhibiting rice blast germs, and the extracted fermentation products can also obviously inhibit the growth of rice blast germs hypha. The fungus hypha morphology after SS-20 fermentation product treatment changed significantly, the hypha quantity decreased, the hypha shriveled, the hypha end expanded and denatured, and the cyst was severe (fig. 6).

Example 5: amplification of genes related to SS-20 antibacterial substances

PCR amplification of genes related to antibacterial substances was performed using the SS-20 bacterial genomic DNA as a template and the primers listed in Table 1. After the PCR was completed, the detection was carried out by 1% agarose gel electrophoresis. The results show that it is possible to display,ituA，fenB，sfP，bacABall amplification succeeds, which shows that the SS-20 has the capability of synthesizing iturin, fengycin, surfactin and bacilysin.

Strain SS-20 has iturin synthase: (ituA) A fengycin synthetase (b)fenB) Surfactin lipopeptide antibiotic synthetase regulation: (sfP) Partial synthetic gene in bacillus lysin synthetase BacilysinbacABThe sizes of the PCR products of the related genes are 1149bp, 1401bp, 675bp and 749bp respectively, and the PCR products are respectively matched with the PCR products of the related genes after BLASTBacillus amyloliquefaciens The similarity of Lx-11 bacillus D synthase A gene (JN 086148.1) is 99.74 percent,Bacillus amyloliquefaciensJT84 FenB (fenB) gene (KX 351428.1) has a similarity of 99.93%,Bacillus amyloliquefaciensJT84 phospho naphthyl transferase (sfp) gene (KX 346253.1) has a similarity of 100.00%,Bacillus subtilisa1/3 bacilysin gene (AF 396778.1) had a similarity of 98.00%.

TABLE 1 primer information table of related genes

。

Example 6: production of PGA by SS-20 fermentation

Culturing SS-20 in fermentationNutrient medium (glucose 40 g/L, sodium glutamate 40 g/L, yeast extract 5 g/L, MgSO)₄ 0.25 g/L，K₂HPO₄2 g/L, pH = 7.2), culturing at 37 deg.C for 3 d, centrifuging at 12000 rpm for 10 min to remove thallus, and collecting the fermentation supernatant. Adding 3 times volume of precooled absolute ethyl alcohol, uniformly oscillating, standing overnight at 4 ℃, and collecting precipitate. Dissolving with deionized water to obtain PGA crude extract.

The PGA solution may be reacted with a sodium hydroxide solution of the cationic surfactant cetyltrimethylammonium bromide (CTAB) at a suitable concentration to form a suspension. In the experiment, the absorbance of the suspension formed by PGA and CTAB at 250 nm is in direct proportion to the concentration of PGA, and has a good linear relationship. Preparing 8% NaOH solution, and using the NaOH solution as a solvent to prepare 8% CTAB-NaOH solution. Preparing a series of gradient PGA standard solutions, and drawing a standard curve of y =0.0166x-0.134 (R)²=0.9996）。

Diluting the PGA crude extract by a proper amount to obtain a solution to be detected, taking 2 mL of the solution to be detected, accurately adding 2 mL of CTAB-NaOH solution, oscillating and mixing uniformly, standing for 3 min, and measuring the absorbance at the wavelength of 250 nm. The absorbance of the precipitate at 250 nm was found to be completely consistent with that of the standard by analysis of the precipitate. The concentration of fermented PGA was found to be >5.0 g/L by standard curve. Indicating that the strain SS-20 has the ability to produce PGA.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

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acgggtgaat tattgcttgc cgcgttaagc cttgcgctga acagatggac tggaaacgag 780

acattcaaaa tcagcatgga aggccatgga agggaagagc atctggagca tcttgatatc 840

agcagaacca tcggctggtt tacgtcaatt tatccggtat tggttgacgc aagttttcaa 900

gatcagactg atgacggcga gcagctcggc tatcacatta agcggacaaa agacatgatg 960

cgccgcattc cgcataaagg agcgggatac ggggtgctga agtatataag caaactctgg 1020

gaagagactg aatctgatgc tcctgaaatc agttttaact atctggggca gttcgaccgg 1080

gaaatccgtt ccagcggctt cggtgtttct cccgtaaaag cgggaaatga agtcagcccg 1140

gactgggaac gtccgtacac acttgatatc agcggctccg tatcgtcgga atgtctcagc 1200

atgcatgtcg tgtacaaccg ttttcagtat caaaaagaaa cgattgaagt tttgacggga 1260

cattttcatg cctttctcaa gcagatcatc acgcattgca caggcaaaga ggagcgtgaa 1320

tggagcgccg ctgatttcag tgacgaggag ttaacgctgg aagatctgag tgacattatg 1380

ggagccgtca acaaactata g 1401

<210> 4

<211> 675

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 4

ttataaaagc tcttcgtacg ttttcatctc aatcccgtca caaaaatcgg gatgcgccgc 60

acaaacggcc agcttatact cctcgtcagc ctcatatgtg cggatgaaac aaggttcatg 120

tccgtccggg agctcaatgg acacatggcc gtcgtctttg aggcggacgc tgaatgaatc 180

aagcggcagg gaaagccctt ttccggcctg tttgataaag ctttctttca tcgaccacag 240

gtggtaaaaa taatcggtct gctgatcggg gtgtttcgct tgcagatcac tgtattccgt 300

cggcgaaaaa aaccgtttgg cgatatcaat cgtgccgggc ttcatttttt caatatcaat 360

gccgatcggt tttgaatcaa cggcgcacac gatccagcga ccggagtggg aaatattaaa 420

gtgcatgtcc ggaagcgcgg ggatgtacgg ctttccgtat tcctgaacgc tgaatgaaat 480

cccggccgga tcaagtccgt aagccttcgc cgcagcggtg cggatcagca tgtcgccgat 540

caaggtgcgg tgagcatcct ccttatggta aaagcgccgg catttttccc gcttttcggc 600

ggacacggcc gccatcatcc gatcctcttc ccctgcagaa agcgggcggt ccatataaat 660

tccgtaaatc ttcat 675

<210> 5

<211> 749

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 5

ccatgatgcc ttcgatgctg atatagtcgg aataaccgct tgtttcgtac cagtcgtcat 60

actcgccctg taaaaattct tccgcgataa acggcgcttc aaacgtgacc gctttcggta 120

cgttgatcga cttcagatat tcattgactc tgttaaactc ggcttcggcc gtttccatct 180

ctttgataag cgccacgccg atggagctcg ccagatatgt aggcttcaga atgagcggcg 240

ttccgatttc ctgaagcgcg gcgcggaaat cttccagcgt cgtcacccgt ttgtttttga 300

tggacttgac gccggcccgg ttgaaggcgg ctctcatttt gtttttatct ctggcatttt 360

cagcggcctg tacgcccgct ccccgcaggc cgagacgttc acacgctttt gccataggag 420

cgataaacag ttcattgttg gtcgtaatgg cgtcaaccgc gaacatgccg gccaccttga 480

cgatttcttc gacgacttct tcctcaggtt tgtcatgatc ttcatgagcc cagtaaatcg 540

aatcgggatg ctcaaaatca gccagactct taaaatagtc tttatcttta atgaccgcga 600

ccgagtattt ttcaatcaaa gctgcatgag aggctgtaat tgcaaaaggt ctcggaataa 660

agctgacgag gttgtatttt tcggctgcgc ttttgtaaaa catatgcggc gggcatcccc 720

caaggtcagc gataaccaat actgttttt 749

Claims (9)

1. A strain for antagonizing magnaporthe grisea and producing gamma-polyglutamic acid is Bacillus belgii (Bacillus subtilis)Bacillus velezensis) SS-20, which is preserved in China center for type culture Collection with a preservation date of 26 months and 10 months in 2020, and a preservation number of CCTCC NO: m20200637, accession number: wuhan university school in Wuhan City of Hubei province.

2. The strain of claim 1, wherein the Bacillus belgii is bacillus spBacillusBacillus velezensisThe 16S rDNA sequence of SS-20 is shown in SEQ ID NO. 1.

3. The strain of claim 1, wherein the strain has an iturin synthase gene, the sequence of which is shown in SEQ ID No. 2; a fengycin synthetase gene, the sequence of which is shown as SEQ ID NO. 3; the sequence of the surfactant synthetase regulating gene is shown as SEQ ID NO. 4; partially synthetic gene of spore lysin synthetasebacABThe sequence is shown in SEQ ID NO. 5.

4. Use of the strain of claim 1 for antagonizing Pyricularia oryzae.

5. The use of the strain of claim 1 for the preparation of a pesticide resistant to rice blast.

6. Use of a fermentation product of the strain of claim 1 for antagonizing Pyricularia oryzae.

7. The use of claim 5, wherein the fermentation product comprises iturin, fengycin, surfactin and lysin.

8. The use of the strain of claim 1 for the production of gamma-polyglutamic acid.

9. The strain of claim 1 for use in agriculture.

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