CN109370939B

CN109370939B - Bacillus belgii and separation method and application thereof

Info

Publication number: CN109370939B
Application number: CN201811313257.7A
Authority: CN
Inventors: 陈颖; 邹丽芳; 杨瑞环; 李生樟; 刘昭; 张翠萍; 陈功友
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2020-06-02
Anticipated expiration: 2038-11-06
Also published as: CN109370939A

Abstract

The invention relates to biocontrol bacillus belgii and a separation method and application thereof. The Bacillus beleisi strain is deposited in the China center for type culture Collection in 2018, 7 and 25 months under the deposit name: the Bacillus 504 or Bacillus velezensis 504 has a preservation number of CCTCC NO: M2018493. The Bacillus belgii has obvious inhibition effect on rice bacterial streak blight, banana bacterial blight, pepper spot, walnut bacterial black spot, cowpea blight and the like, and has broad-spectrum antibacterial activity and important biocontrol application prospect.

Description

Bacillus belgii and separation method and application thereof

Technical Field

The invention belongs to the field of microbial technology application, and particularly relates to a bacillus beiLeisi, a separation method and application thereof.

Background

Bacterial Blight of rice (BB) and Bacterial leaf streak of rice (BLS) are important Bacterial diseases commonly seen in rice production, and are caused by fusarium oxysporum (xanthomonas oryzae pv. oryzae, Xoo) and rice leaf streak germ (x.oryzae pv. oryzae, Xoc) (variegate, zhou dan, liu, pinus sylvestris, chen gongyou. the establishment of xanthomonas oryzae pathopoiesia-associated gene insertion mutant system [ J ] phytopathology report, 2012, 42(02):176-185 ]. The infection and propagation of xanthomonas mainly depend on toxic factors such as polysaccharide, adsorbance, type iii secretion system and lipopolysaccharide secreted in host tissues (daqinghua, Zhang Shiqing, daoxiang, Zhuhui, Eichio yuensis, Stalactia mustacea rights. research progress of xanthomonas toxicity factor regulation [ J ] biological disaster science, 2012, 35(02): 134-141.). The disease incidence range of the rice bacterial leaf blight is extremely wide and the rice bacterial leaf blight is spread in all large rice production areas around the world; after rice suffers from bacterial leaf blight disease, gray white disease spots are generated along veins, the yield of the rice is generally reduced by 20-30%, and even in severe cases, the rice is harvested absolutely (Dianzhai literature, Verlihuang. research and molecular breeding of rice bacterial leaf blight resistance genes, China journal of bioengineering.1999). Bacterial leaf streak of rice is an important quarantine bacterial disease in rice production, and is popular locally in southern China and southeast Asia countries; after rice is infected with stripe disease, stripe symptoms appear on leaves, and when the stripe disease is serious, the yield reduction of the rice reaches 40-60%, and the damage is more serious than bacterial blight (Zhangsheng, etc., the research on bacterial stripe disease of the rice progresses, Jiangsu agricultural science (JiangsuJ. of agr. Sci.), 2014, 30 (4): 901-908).

At present, the two methods for preventing and treating rice diseases in production mainly comprise chemical prevention and agricultural prevention and treatment. The chemical prevention has the defects of environmental pollution, harm to human and animal health, easy mutation of pathogenic bacteria to generate drug resistance and the like; agricultural prevention and control are difficult to popularize on a large scale. Therefore, biological control using beneficial microorganisms has become a major trend in agricultural production (zong ying, zhao yue ju, liu yang, populi. a study of bacillus beilaisi on inhibiting fusarium graminearum [ J ]. nuclear agro-papers 2018, 32(02): 310-317.). Bacillus, one of the hot spots in the field of biological control research, can inhibit the growth of plant pathogenic bacteria by secreting antibacterial substances such as antibacterial proteins, lipopeptides and polyketides, thereby better promoting the growth of plants (sang Jian Wei, Yang, Chen Yipeng, Cai Ji Miao, Lu Cui Mei, Huang Gui Xia. endophytic bacillus amyloliquefaciens BEB17 lipopeptide and polyketide antibacterial activity analysis [ J ] Phytopathology report, 2018, 48(03): 402-412.). Not only ensures the safety of the environment and people and livestock, but also can effectively prevent pathogenic bacteria from generating resistance, and simultaneously meets the multiple requirements of consumers on the yield, the quality and the safety of agricultural products. Therefore, the bacillus has great application prospect in modern agricultural production (Chen Shi Ying, Liu Yong Feng, Liu Zi Zhou, Zhangiang Sheng. research progress of the plant disease biocontrol bacillus [ J ]. Jiangsu agricultural science report, 2012, 28(05): 999-1006.).

Bacillus belgii (Bacillus velezensis) is a new species of Bacillus, widely distributed in various ecological environments (Cai Gaili, Zhang Fang, Europe friendship, Zhao Chang Song, Penxuan He, Jiang Aiming) such as soil, plant rhizosphere and interior, river and the like [ J Bacillus velezensis ] research progress]Northern horticulture, 2018(12): 162-167). As a biocontrol bacillus, the bacillus has the effects of promoting plant growth and resisting pathogenic microorganisms, has broad-spectrum antibacterial activity and can be used as a biological control regulator in agricultural production. The mechanism of the Bacillus belgii for inhibiting the growth of phytopathogens mainly comprises the following steps: producing antibacterial protein and lipopeptide antibiotics, synthesizing antibiotics by using non-ribosomal polypeptide synthetase and polyketide, and inducing plants to generate systemic resistance; by generating IAA, NH₃And ACC deaminase to promote plant growth.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a bacillus belgii strain, a separation method and application thereof.

The Bacillus belgii has obvious antagonistic action on rice bacterial blight.

The purpose of the invention can be realized by the following technical scheme:

the first aspect of the present invention is: the Bacillus belgii is separated from soil of a water spinach field in Yuxi county of Sanming City of Fujian province, and is named as Bacillus 504 or Bacillus velezensis 504, and the Bacillus belgii strain is preserved in China center for type culture Collection in 2018, 7 and 25 months, and has the preservation number of CCTCC NO: M2018493. The preservation address is as follows: wuhan university school (the first small facing of Wuhan university), Wuhan university collection, eight Wuhan district 299, Wuhan City, Hubei province.

Performing plate culture on a solid culture medium, and observing that the colony color of the Bacillus belgii is milky white, the edge is not smooth and irregular, and the surface is dry, rough and opaque; the bacterial body of the strain is short rod-shaped through microscope observation, and spores can be generated.

Through physiological and biochemical tests, the results are clearBacillus belgii is a gram-positive bacterium which produces H₂S, capable of secreting gelatinase, incapable of hydrolyzing beta-galactosidase, arginine bihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease, capable of producing acetyl methyl methanol and indole by using 3-hydroxy butanone, capable of oxidizing sucrose, incapable of oxidizing glucose, mannitol, inositol, sorbitol, rhamnose, melibiose and amygdalin, and capable of utilizing 17 carbon sources such as glycerol, glucose, fructose, mannose, inositol, mannitol, sorbitol and the like.

The xanthomonas oryzae comprises two pathogenic varieties of xanthomonas oryzae Xoc and xanthomonas oryzae Xoo.

The antagonism of the bacillus beilesiensis 504 on rice bacterial blight germ and rice bacterial streak germ is detected by a plate confronting culture method, and the antagonism is proved to have broad-spectrum antagonism activity on 2 types of pathogenic bacteria.

Furthermore, the antagonism of the Bacillus belgii to other 9 pathogenic xanthomonas bacteria is detected, and the Bacillus belgii is proved to have antagonistic activity to 9 plant pathogenic bacteria such as banana bacterial wilt pathogen, pepper spot pathogen, walnut bacterial black spot pathogen and cowpea wilt pathogen.

The second aspect of the present invention is: providing a method for isolating said bacillus belgii: the method comprises the steps of adding sterile water to a soil sample by adopting a gradient dilution plate coating method, dissolving and oscillating for 15min, sequentially diluting the soil sample into soil bacterium suspensions with different gradients, respectively coating the soil bacterium suspensions on an NA (NA) plate inoculated with rice streak pathogens, observing whether a bacteriostatic zone is generated, scribing bacterial colonies capable of generating an obvious bacteriostatic zone on the NA plate to obtain single bacterial colonies, carrying out PCR (polymerase chain reaction) amplification by utilizing 16S rRNA (ribonucleic acid) genes and gyrA (gyrA) genes of bacteria, and constructing a phylogenetic tree to determine the classification status of the bacterial strains.

The invention uses 16S rRNA gene and gyrA gene to respectively construct phylogenetic trees, carries out the comparative analysis of genetic relationship, and combines the physiological and biochemical characteristics of the strain to prove that the strain is Bacillus velezensis.

Wherein, the sequence of the 16S rRNA gene is shown as SEQ ID NO.1, and the sequence of the gyrA gene is shown as SEQ ID NO. 2.

The third aspect of the present invention is: applications of the bacillus are provided, which comprise the following applications:

in one embodiment of the invention, the Bacillus belgii 504 has a broad spectrum antagonistic effect against both Physalospora oryzae Xoc and Physalospora oryzae Xoo.

In one embodiment of the present invention, the Bacillus belgii 504 has a significant antagonistic effect against rice bacterial blight.

In one embodiment of the present invention, the rice bacterial blight includes 8569, YC2, AH1, YC6, YC11, YN04-1, LYG46, JL3, PXO99^A、YC18、XZ35、YC7。

In one embodiment of the present invention, the Bacillus belgii 504 has an antagonistic effect on rice streak pathogens.

In one embodiment of the invention, the rice streak germ comprises HNB07-3, RS105, RS85, HNB3-17, HANB12-26, ZJB01-25, HANB1-19, JSB1-39, AHB3-7, HNB8-47, AHB1-58 and YNB 01-3.

In one embodiment of the invention, the Bacillus belgii 504 is directed against a variety of other plant pathogenic bacteria of the genus Xanthomonas, including banana bacterial blight (X.campestris pv. muraearum), pepper spot (X.campestris pv. vesicoria), walnut bacterial black spot (X.campestris pv. Juglans), cowpea blight (X.axonopsis pv. vignicola), bean blight (X.axonopsis pv. phaseoli), onion bacterial leaf blight (X.axonopsis pv. allii), tomato bacterial wilt (Ralstonia solanacearum), sugarcane flow-gum pathogen (X.axonopsis pv. vascurvulgarum), cotton bacterial angular leaf blight (X.axonopsis pv. mura).

Compared with the prior art, the Bacillus belgii provided by the invention has obvious antagonistic action on both rice bacterial blight and rice streak disease, has antagonistic effect on various common pathogenic bacteria of xanthomonas at present, is an ideal biocontrol microorganism resource, and shows great application prospect in the field of biological control of plant diseases. Provides a new resource for the biological control of various plant bacterial diseases in the agricultural production nowadays.

Drawings

FIG. 1 is a photograph of Bacillus belgii 504 observed by a microscope (1000X) and its colony morphology.

FIG. 2. Bylella belgii 504 phylogenetic tree constructed based on the 16S rRNA gene.

FIG. 3A phylogenetic tree of Bacillus beleisi 504 constructed based on the gyr A gene.

FIG. 4.16 gel electrophoresis results of S rRNA gene. Wherein 1 represents Marker; 2 represents the product of the 16S rRNA gene.

FIG. 5 shows the results of gel electrophoresis of the gyrA gene. Wherein 1 represents Marker; 2 represents the product of the gyrA gene.

Fig. 6 is a graph showing the antagonistic effect of bacillus belgii 504 against 12 strains of rice bacterial blight (Xanthomonas oryzae v. oryzae, Xoo), a: 8569; b: YC 2; c, AH 1; d: YC 6; e: YC 11; f: YN 04-1; g: LYG 46; h: JL 3; i: PXO99^A；J：YC18；K：XZ35；L：YC7。

Fig. 7 is a graph showing the antagonistic effect of bacillus belgii 504 against 12 strains of rice streak germ (Xanthomonas oryzae pv. oryzae, Xoc), a: HNB 07-3; b: RS 105; c: RS 85; d: HNB 3-17; e: HANB 12-26; f: ZJB 01-25; g: HANB 1-19; h: JSB 1-39; i: AHB 3-7; j: HNB 8-47; k: AHB 1-58; l: YNB 01-3.

FIG. 8 is a graph of the antagonistic effect of B.belgii 504 on the other 9 plant pathogens Xanthomonas and Laurel, A: banana bacterial wilt (x. campestris pv. musaceae); b: fusarium wilt of cowpea (x. axonopodispv. vignicola); c: walnut bacterial alternaria nigra (Xanthomonas campestris pv. jugladis); d: ralstonia solanacearum (Ralstonia solanacearum); e: bacterial leaf blight of onion (x. axonopodispv. allii); f: pepper spot pathogen (x. campestis pv. vesicatoria); g: cotton bacterial angular leaf spot (x. campestis pv. malvacearum); h: bean wilt pathogen (x. campestris pv. phaseoli); i: gummosis illustrative disease of sugarcane (X.axonopodis pv. vascuorum).

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not to be construed as limiting the invention.

The strain media used in the following examples are as follows:

beef extract peptone medium NA (g/L): 3g of beef extract, 5g of polypeptone, 10g of cane sugar, 1g of yeast powder and 15g of agar powder, adding water to dissolve the mixture, fixing the volume to 1000mL, adjusting the pH value to 7.0-7.2, and sterilizing under high pressure (121 ℃, 20 min).

Example 1 obtaining of Bacillus belgii 504

1. Source of soil

Soil of hollow vegetable field in Yuxi county, Sanming City of Fujian province

2. Screening of strains

(1) Soil sample collection

Carrying out soil sample collection by a Z-shaped 5-point sampling method: 200g of soil sample is collected at each point, the soil samples are uniformly mixed, and 200g of soil sample is taken by a quartering method and is filled into a sterilization bag to be used as one soil sample. 3 soil samples were collected per plot as replicates. The time, place and kind of the sample are recorded. The collected soil sample is stored in a refrigerator at 4 ℃ for bacterial separation.

(2) Isolation of bacteria

Plate dilution method: weighing 10g of soil sample into a conical flask, adding 90mL of sterile water, oscillating in a shaking table at 200rpm and 28 ℃, taking out after 15min, standing at room temperature for 5min, and preparing a soil bacterium suspension stock solution. The soil bacterium suspension stock solution is subjected to gradient dilution to respectively obtain 10^-1、10^-2、10^-3、10^-4、10^-5Total 6 gradient dilutions of bacterial suspension. And uniformly coating 200 mu L of bacterial suspension diluent on an NA plate containing rice streak disease germ RS105, and repeating for 2-3 times in each gradient. Biochemical treatment at 28 deg.CAnd (5) in an incubator, inversely culturing for 24 hours, and observing.

(3) Bacterial purification

Observing and selecting single bacterial colony with bacteriostatic circle, streaking and purifying on NA plate, performing inverted culture in 28 deg.C biochemical incubator, 12 hr later selecting single bacterial colony, and numbering in sequence.

(4) Preservation of bacteria

Inoculating the strain in a liquid NA culture medium, culturing for 12h in a shaking table at 28 ℃ and 180rpm, sucking 1mL of bacterial liquid and 1mL of 50% sterile glycerol, gently shaking and uniformly mixing, and storing at-80 ℃ for a long time.

(5) Screening of antagonistic bacteria

Adopting an oxford cup method: inoculating pathogenic bacteria in NA liquid culture medium, culturing in shaking table at 28 deg.C and 180rpm for 12h, sucking 200 μ L bacterial suspension, mixing with NA solid culture medium, turning, placing Oxford cups with diameter of 8mm at the center of NA plate, and inoculating 10 μ L Bacillus subtilis (OD) to be tested in each Oxford cup₆₀₀About 2.0), repeating the steps for 2-3 pathogenic bacteria, culturing in a biochemical incubator at 28 ℃ for 24h, observing whether a bacteriostatic zone exists or not, recording the serial number of the strain, measuring the size of the bacteriostatic zone, and finishing and photographing.

The screened antagonistic bacteria with the best effect is named as Bacillus velezensis 504, and the colony color of the Bacillus velezensis is observed to be milky white, the edge is not smooth and irregular, and the surface is dry, rough and opaque through solid medium plate culture; the bacterial body of the strain is short rod-shaped through microscope observation, and spores can be generated. FIG. 1 shows the observation photograph (1000X) of Bacillus belgii 504 under a microscope and the colony morphology.

Example 2 16S rRNA Gene identification of Bacillus belgii 504

Genomic DNA of strain 504 was extracted, and primers: 27F

5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R

5'-TACGGCTACCTTGTTACGACTT-3', PCR amplification is carried out using the extracted gDNA as a template to obtain the target fragment. The PCR reaction system is as follows:

TABLE 1 Taq polymerase chain reaction System

The basic conditions of the PCR reaction are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s (1kb/min), pre-extension at 72 ℃ for 8min, and storage at 4 ℃ for 30 cycles. After the reaction, the PCR products were checked by electrophoresis on 1% agarose gel, and the results were detected and recorded by a gel imager (see FIG. 4). The PCR stock was sent to Shitanhua Biotech (Shanghai) Co., Ltd for sequencing. The sequencing result is analyzed by using DNAStar, and BLAST comparison is carried out on an NCBI website to determine the species of the related strains.

The results show that: the 16S rRNA gene of the strain 504 has 99.78% similarity with Bacillus velezensis. A phylogenetic tree with pseudogenes was constructed using MEGA6.0, and the results are shown in FIG. 2.

Example 3 identification of the gyrA Gene of Bacillus belgii 504

Genomic DNA of strain 504 was extracted, and primers: and carrying out PCR amplification by using the extracted DNA as a template to obtain a target fragment, wherein the DNA is GyrA-F5'-CAGTCAGGAAATGCGTACGTCCTT-3' and GyrA-R5'-CAAGGTAATGCTCCAGGCATTGCT-3'. The PCR reaction system is as follows:

TABLE 2 Taq polymerase chain reaction System

Reaction conditions are as follows: 10min at 94 ℃; 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ and 30 cycles; 72 ℃ for 10min, 10 ℃ infinity. After the reaction, the PCR products were checked by electrophoresis on 1% agarose gel, and the results were detected and recorded by a gel imager (see FIG. 5). The PCR stock was sent to Shitanhua Biotech (Shanghai) Co., Ltd for sequencing. The sequencing result is analyzed by using DNAStar, and BLAST comparison is carried out on an NCBI website to determine the species of the related strains.

The results show that: the similarity of the gyrA gene of the strain 504 and the gyrA gene sequence of Bacillus velezensis model bacteria reaches 99 percent, and the similarity of the gyrA gene sequence of Bacillus amyloliquefaciens model bacteria only reaches 96 percent. A phylogenetic tree with pseudogenes was constructed using MEGA6.0, and the results are shown in FIG. 3.

Example 4 physiological and biochemical identification of Bacillus belgii 504

The physiological and biochemical characteristics of the bacillus beleisi 504 are as follows: having generation of H₂S, the ability to secrete gelatinase, unable to hydrolyze β -galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease, able to utilize 3-hydroxybutanone to produce acetyl methyl alcohol, unable to produce indole, able to oxidize sucrose, unable to oxidize glucose, mannitol, inositol, sorbitol, rhamnose, melibiose and amygdalin, and able to utilize 17 carbon sources such as glycerol, glucose, fructose, mannose, inositol, mannitol, sorbitol, see tables 3 and 4.

TABLE 3 physiological and biochemical characteristics of Strain 504-enzyme Activity, carbon Source Oxidation

+: positive reaction; -: negative reaction;

TABLE 4 physiological and biochemical characteristics of Strain 504-production of acid Using carbon sources

+: positive reaction; -: negative reaction; w: weak positive reaction

Example 5 determination of antagonistic Spectrum of Bacillus belgii 504

1) Determination of antagonistic activity of Bacillus belgii 504 on 12 kinds of rice bacterial blight bacteria

Respectively inoculating 12 different rice bacterial blight and test Bacillus belgii 504 into NA liquid culture medium, culturing at 28 deg.C and 180rpm in shaking table for 12 hr, and unifying OD₆₀₀Are all 2.0; respectively sucking 200 mu L of corresponding pathogen bacterial liquid and an NA solid culture medium, fully mixing the pathogen bacterial liquid and the NA solid culture medium, turning the plate, then placing an oxford cup with the diameter of 8mm in the center of an NA flat plate, internally connecting 10 mu L of test bacteria in each oxford cup, repeating each pathogen bacteria for 2-3 times, placing the oxford cup in a biochemical incubator at 28 ℃, observing whether a bacteriostatic circle exists after culturing for 24 hours, recording the size of the bacteriostatic circle, and finishing and photographing.

The antagonistic effect of bacillus belgii 504 against 12 strains of rice bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) is shown in fig. 6, where in fig. 6, a: 8569; b: YC 2; c, AH 1; d: YC 6; e: YC 11; f: YN 04-1; g: LYG 46; h: JL 3; i: PXO99^A(ii) a J: YC 18; k: XZ 35; l: YC 7. The data of the corresponding bacteriostatic effect are shown in table 5.

TABLE 5 bacteriostatic effect of Bacillus belgii 504 on different rice bacterial blight germs

2) Determination of antagonistic activity of Bacillus belgii 504 on 12 rice leaf streak pathogens

Respectively inoculating 12 different rice streak germs and test Bacillus belgii 504 into NA liquid culture medium, culturing at 28 deg.C in a shaking table at 180rpm for 12h, and unifying OD₆₀₀Are all 2.0; respectively sucking 200 mu L of corresponding pathogen bacterial liquid and an NA solid culture medium, fully mixing the pathogen bacterial liquid and the NA solid culture medium, turning the plate, then placing an oxford cup with the diameter of 8mm in the center of an NA flat plate, internally connecting 10 mu L of test bacteria in each oxford cup, repeating each pathogen bacteria for 2-3 times, placing the oxford cup in a biochemical incubator at 28 ℃, observing whether a bacteriostatic circle exists after culturing for 24 hours, recording the size of the bacteriostatic circle, and finishing and photographing.

The antagonistic effect of bacillus belgii 504 against 12 rice leaf spot germs (Xanthomonas oryzae pv. oryzae, Xoc) is shown in fig. 7, in which a: HNB 07-3; b: RS 105; c: RS 85; d: HNB 3-17; e: HANB 12-26; f: ZJB 01-25; g: HANB 1-19; h: JSB 1-39; i: AHB 3-7; j: HNB 8-47; k: AHB 1-58; l: YNB 01-3. The data of the corresponding bacteriostatic effect are shown in table 6.

TABLE 6 bacteriostatic effect of B.beilesiensis 504 on different rice streak pathogens

3) Determination of antagonistic activity of Bacillus belgii 504 against 9 plant pathogens

Respectively inoculating 9 pathogenic bacteria such as banana bacterial wilt pathogen, walnut black spot pathogen, tomato ralstonia solanacearum and the like and the test Bacillus belgii 504 into NA liquid culture medium, culturing for 12h in a shaking table at 28 ℃ and 180rpm, and unifying OD₆₀₀Are all 2.0; respectively sucking 200 mu L of corresponding pathogen bacterial liquid and an NA solid culture medium, fully mixing the pathogen bacterial liquid and the NA solid culture medium, turning the plate, then placing an oxford cup with the diameter of 8mm in the center of an NA flat plate, internally connecting 10 mu L of test bacteria in each oxford cup, repeating each pathogen bacteria for 2-3 times, placing the oxford cup in a biochemical incubator at 28 ℃, observing whether a bacteriostatic circle exists after culturing for 24 hours, recording the size of the bacteriostatic circle, and finishing and photographing.

The antagonistic effect of bacillus belgii 504 against 8 plant pathogens xanthomonas and lawsonia is shown in fig. 8, where in fig. 8, a: banana bacterial wilt (x. campestris pv. musaceae); b: fusarium wilt of cowpea (x. axonopodis pv. vignicola); c: walnut bacterial alternaria nigra (Xanthomonas campestris pv. juglandis); d: ralstonia solanacearum (Ralstonia solanacearum); e: bacterial leaf blight of onion (x. axonopodis pv. allii); f: pepper spot pathogen (x. campestis pv. vesicatoria); g: cotton bacterial angular leaf spot (x. campestis pv. malvacearum); h: bean wilt pathogen (x. campestrispv. phaseoli); i: gummosis illustrative disease of sugarcane (X.axonopodis pv. vascuorum). The data of the corresponding bacteriostatic effect are shown in table 7.

TABLE 7 bacteriostatic effect of B.beilesiensis 504 on 9 pathogenic bacteria

Therefore, the Bacillus belgii 504 provided by the invention has a remarkable antagonistic effect on the rice bacterial blight Xoo, has a broad-spectrum bacteriostatic effect on the rice stripe blotch Xoc, has an antagonistic effect on pathogenic bacteria of various xanthomonas, and provides a new resource for biological control of rice bacterial diseases.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

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gccattccga ccgcaatacc ggcagccccg tttacgagca gattcggaaa tctcgaaggc 540

atgacggcag gctctctttc tgaaccgtca tagttatctt gatagtcaat cgtgtctttc 600

gtaatgtcac gcagaatttc cattgcgatt tttgacattc tcgcttctgt gtaacgcatc 660

gcggccgctg agtcgccgtc aaccgaaccg aagttgccgt gtccgtcaac aagcatgtag 720

cggtagttaa aatcctgcgc cattctgacc attgattcgt aaaccgctga gtcaccgtgc 780

gggtggtact taccgataac ttcaccgacg atacgggcag attttttata tggtttgtca 840

ctggtcatgc ctaaatcatt cattgcgtac aaaatccgtc tgtgaaccgg cttcagaccg 900

tcacgcacat ccggaagcgc ccgggatacg a 931

Claims

1. The Bacillus velezensis is characterized in that the Bacillus velezensis strain is preserved in China center for type culture collection in 2018, 7 and 25 months, and the preservation number is CCTCC NO: M2018493.

2. The Bacillus belgii of claim 1, wherein the Bacillus belgii colonies are opalescent, have rough, irregular edges, dry, rough, and opaque surfaces; the thallus is short rod-shaped and can produce spores.

3. The Bacillus belgii of claim 1, wherein the Bacillus belgii is a gram-positive bacterium, brachypodium, spore-forming, aerobic or facultative anaerobic bacterium; having generation of H₂the S has the ability to secrete gelatinase, cannot hydrolyze β -galactosidase, arginine bihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease, can utilize 3-hydroxy butanone to produce acetyl methyl alcohol, cannot produce indole, can oxidize sucrose, cannot oxidize glucose, mannitol, inositol, sorbitol, rhamnose, melibiose and amygdalin, and can utilize carbon sources including glycerol, glucose, fructose, mannose, inositol, mannitol and sorbitol.

4. The Bacillus belgii of claim 1, wherein the Bacillus belgii is antagonistic to Xanthomonas oryzae.

5. The Bacillus belgii strain of claim 4, wherein the Xanthomonas oryzae comprises two pathogenic varieties Xanthomonas oryzae (Xanthomonas oryzae pv. oryzae, Xoc) and Xanthomonas oryzae (Xanthomonas oryzae) respectively.

6. The Bacillus belgii of claim 1, wherein the Bacillus belgii is antagonistic to rice bacterial blight and rice streak disease.

7. The Bacillus belgii of claim 1, wherein the Bacillus belgii has antagonistic activity against the plant pathogenic xanthomonas.

8. The Bacillus belgii of claim 7, wherein the plant pathogens Xanthomonas comprises banana bacterial wilt, pepper spot, walnut bacterial blackspot and cowpea wilt.

9. Use of bacillus belgii according to claim 1, comprising the following applications:

the application of the antagonistic bacteria of the rice bacterial blight;

the application of the antagonistic bacteria of the rice bacterial streak germ;

the application of the plant pathogenic xanthomonas antagonistic bacterium is provided;

the plant pathogenic xanthomonas includes banana bacterial wilt pathogen, pepper spot pathogen, walnut bacterial black spot pathogen and cowpea wilt pathogen.