CN111154688B

CN111154688B - Biocontrol bacillus beleisi SF259 and application thereof

Info

Publication number: CN111154688B
Application number: CN202010064158.0A
Authority: CN
Inventors: 邹丽芳; 黄梦桑; 李生樟; 杨瑞环; 陈路生; 屈伊凝; 陈功友
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2021-07-27
Anticipated expiration: 2040-01-20
Also published as: CN111154688A

Abstract

The invention relates to a biocontrol bacillus beleisi SF259 and application thereof. The Bacillus beleisi strain is deposited in the China center for type culture Collection in 2019, 9 months and 20 days, and the deposit name is as follows: bacillus velezensis SF259(Bacillus velezensis SF259) with preservation number of CCTCC M2019735. The Bacillus belgii has a remarkable inhibition effect on rice bacterial streak blight, banana bacterial wilt, cowpea blight, cotton bacterial angular leaf spot, botrytis cinerea, rice blast and the like, and has broad-spectrum antibacterial activity and important biocontrol application prospects.

Description

Biocontrol bacillus beleisi SF259 and application thereof

Technical Field

The invention belongs to the field of application of microbial technology, and particularly relates to biocontrol bacillus belgii and application thereof.

Background

Rice is an extremely important food crop in the world, especially in asian regions. Two pathogenic varieties of xanthomonas oryzae x.oryza pv. oryza (Xoo) and x.oryza pv. oryzicola (Xoc) are capable of infecting rice and causing Bacterial Leaf Blight (BLB) and rice streak (BLS) in rice. These two diseases seriously compromise the yield and quality of rice, and occur more severely in tropical and subtropical asian regions.

The bacterial leaf blight of Rice is one of three diseases of Rice, and occurs in various Rice regions of the world, in China, the bacterial leaf blight of Rice is also called as smoldering or plague, and the yield of Rice can be reduced by 20-30% after the Rice is infected with the disease, and can reach 50-60% in severe cases (Ou, S, H., Rice diseases, Commonwelth Agricultural Bureau, Kew Surrey [ J ]. 1985). The rice streak disease mostly occurs in rice planting areas of Asia, Africa and the like, in China, the disease is widely prevalent mainly in a two-season rice area in south China and a single-double mixed planting rice area in China, and the disease becomes a main disease of rice in China (Zhangrong Sheng, Chengxiang Ying, Liu Yong Feng, research progress of the rice bacterial streak disease [ J ]. Jiangsu agricultural science report, 2014,30(04):901 and 908.). The yield loss after rice affection is related to weather, the disease is reduced by about 20% when the weather is proper, the disease is more serious under storm conditions, and the yield reduction can reach more than 30% (Zou, L, F., Wang, X, P., Xiaong, Y., etc., the reduction of the hrp regulators of Xanthomonas oryzae pv. oryzae, which is the same as that of the allergic reaction in negative bacteria and phosphorus sensitivity in reactive phosphorus microorganism [ J ]. Appl Environ Microbiol,2006,72(9), pp 6212-24.).

The rice bacterial leaf blight and rice streak disease are not only strong in outbreak and high in epidemic speed, but also difficult to control, and the main control method at present is the combination of chemical and biological control, the combination of improvement of cultivation technology and breeding of disease-resistant varieties. At present, no medicine capable of radically treating bacterial leaf blight of rice is found in production, and copper preparations and zinc preparations are mostly used, but the ecological environment is greatly damaged in the past (alpine, zhugong. genetic research on bacterial leaf blight resistance [ J ]. proceedings of Nanjing agriculture university, 1982,5(1): 22-35.). At present, at least 30 bacterial leaf blight resistance related genes have been found in Rice (White F, Yang B, Hawes M, etc. host and pathogens controlling the Rice-Xanthomonas oryzae interaction [ J ] Plant Physiology,2009,150(4):1677 1686.), more Xa13(Suh J P, Jeung J U, Noh T H, et al. development lines with a nutrient resistant genes which are resistant to bacterial strain-specific blue and the bacterium organism resistant in Rice Plant breeding [ J ] 2013,6(1): Xa 5, Xa 32 (Xa. land) nutrient resistant strain, Fagnong resistance No. 3621, Chengyuan Plant resistance of Chongnan J52. Pan Fagnomon K.K.: Chuannan K.K. K.K.K. K.K. Sakyo-resistant to bacterial strain of wheat grass K.K.K.K.K.K. Sativ et al., Zhang J.K.K.K.K.K.K. Shih. No. Shih No. K., tengfei Qin, Xiaooping Zhang, Kaijun Zhao, high-resolution genetic mapping of a rice bacterial light resistance gene Xa23[ J ]. Molecular Genetics and Genomics,2014,289 (5)). However, little is known about the genes related to rice leaf streak resistance, and more than 30 rice varieties are reported, and pathogenic bacteria also show high genetic changes. Therefore, the method also has certain limitation on the control of the two diseases.

In recent years, biological control has become a research focus of scientists, and on one hand, the biological control has mild influence on the environment, and on the other hand, the biological control has certain specificity on pathogenic bacteria and is not easy to cause resistance to the pathogenic bacteria. Therefore, the method is a work with great significance for screening biological resources with antagonistic action on diseases from natural environment in vigorous response to national advocates and calls for popularization of biological control measures.

Disclosure of Invention

The invention aims to provide a biocontrol bacillus beleisi SF259 and application thereof.

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

the first aspect of the present invention is: a strain of Bacillus is provided, is separated from cherry tree soil in a garden of a Lodite district in Qinghai province, China and is named as Bacillus velezensis SF259(Bacillus velezensis SF259), and the Bacillus is preserved in China type culture Collection (CCTCC) in 2019, 9 and 20 months with the preservation number of CCTCC M2019735.

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 bacillus belgii is a gram-positive bacterium, can secrete gelatinase and cannot hydrolyze beta-galactosidase, arginine double hydrolase, lysine decarboxylase, ornithine decarboxylase and urease; can utilize 3-hydroxy butanone to produce acetyl methyl methanol, can not produce indole; is incapable of oxidizing glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose. Can utilize 19 carbon sources such as glycerol, L-arabinose, ribose, D-xylose, glucose, etc.

Phylogenetic trees are respectively constructed through 16S rRNA genes and gyrA genes, genetic relationship comparative analysis is carried out, and physiological and biochemical characteristics of the strain are combined, so that the strain is proved to be 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 second aspect of the present invention is: applications of the bacillus are provided, which comprise the following applications:

in one embodiment of the present invention, the Bacillus belgii has an antagonistic effect on Xanthomonas oryzae. The Xanthomonas oryzae includes two pathogenic varieties of Xanthomonas oryzae (Xanthomonas oryzae pv. oryzae, Xoo) and Xanthomonas oryzae (Xanthomonas oryzae pv. oryzae, Xoc).

In one embodiment of the present invention, the Bacillus belgii SF259 has a significant antagonistic effect against Padina bacterial blight.

In one embodiment of the present invention, the rice bacterial blight comprises PXO99^A、AH1、YNB01-4、XZ35、JL3、JC1、YC11、LYG46、XC18。

In one embodiment of the present invention, the Bacillus belgii SF259 has an antagonistic effect against rice streak disease.

In one embodiment of the invention, the rice bacterial leaf streak pathogens comprise HANB12-26, RS85, HNB07-3, ZJB01-25, RS105, JSB1-39, AHB3-7, YNB01-3 and HNB 3-17.

In a specific embodiment of the present invention, the Bacillus belgii has an antagonistic effect against Padina bacterial white leaf spot and Padina streak.

In one embodiment of the invention, the Bacillus belgii SF259 is antagonistic to a wide variety of Xanthomonas pathogens. The plant pathogenic xanthomonas includes banana bacterial wilt pathogen (X.campestris pv. muraearum), cowpea wilt pathogen (X.axonopodis pv. vignicola), cotton bacterial angular leaf spot pathogen (X.campholris pv. malvacearum), pepper spot pathogen (X.campestris pv. vesicaria), bean wilt pathogen (X.campestris pv. phaseoli), sugarcane gummosis (X.axonopodis pv. vascurulum), soybean scab pathogen (X.axonopodis pv. glycerinum).

In a particular embodiment of the invention, said bacillus belgii SF259 has an antagonistic effect on phytopathogenic fungi. The plant pathogenic fungi include Botrytis cinerea (Botrytis cinerea), Magnaporthe oryzae (Magnaporthe oryzae), Fusarium graminearum (Fusarium graminearum), Phytophthora capsici (Phytophthora capsici), Fusarium oxysporum (Fusarium oxysporum), and the like.

Compared with the prior art, the Bacillus beiLeisi SF259 provided by the invention has obvious antagonism to rice bacterial blight and rice streak germ, has antagonism effect to common pathogenic bacteria of multiple xanthomonas at present, has antagonism activity to important plant pathogenic fungi, and provides new resources for biological control of multiple plant bacterial diseases in agricultural production at present.

Drawings

FIG. 1 is a photograph of Bacillus belgii SF259 observed with a microscope (1000X) and shows the colony morphology.

FIG. 2A phylogenetic tree constructed from Bacillus belgii SF259 based on the 16S rRNA gene.

FIG. 3A phylogenetic tree constructed by Bacillus beleisi SF259 based on 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 is a graph showing the antagonistic effect of bacillus beijerinckii SF259 on 9 strains of fusarium oxysporum f.oryzae pv. oryzae, Xoo, a: PXO99^A；B：AH1；C:YNB01-4；D：XZ35；E：JL3；F：JC1；G：YC11；H：LYG46；I：XC18。

Fig. 6 is a graph showing the antagonistic effect of bacillus belgii SF259 on 9 strains of rice leaf streak germ (Xanthomonas oryzae pv. oryzicola, Xoc), a: HANB 12-26; b: RS 85; c: HNB 07-3; d: ZJB 01-25; e: RS 105; f: JSB 1-39; g: AHB 3-7; h: YNB 01-3; i: HNB 3-17.

FIG. 7 antagonistic effect of B.beijerinckii SF259 on the other 9 plant pathogenic xanthomonas, A: banana bacterial wilt (x. campestris pv. musaceae); b: fusarium wilt of cowpea (x. axonopodis pv. vignicola); c: cotton bacterial angular leaf spot (x. campestis pv. malvacearum); d: pepper spot pathogen (x. campestis pv. vesicatoria); e: bean wilt pathogen (x. campestris pv. phaseoli); f: gummosis illustrative disease of sugarcane (X.axonopodis pv. vascullurum); g: scab (x. axonopodis pv. glycines); h: bacterial leaf blight of onion (x. axonopodis pv. allii); i: walnut bacterial alternaria nigra (Xanthomonas campestris pv. jugladis).

FIG. 8 is a graph showing the antagonistic effect of Bacillus belgii SF259 on Botrytis cinerea, in which A: botrytis cinerea; b: sterile water control; c: cultivation effect of Bacillus belgii SF259 and Botrytis cinerea.

FIG. 9 is a graph showing the antagonistic effect of Bacillus belgii SF259 on Pyricularia oryzae (Magnaporthe oryzae), in which A: rice blast germs; b: sterile water control; c: the effect of Bacillus belgii SF259 on the growth of rice blast fungus was shown.

FIG. 10 is a graph showing the antagonistic effect of Bacillus belgii SF259 on Fusarium graminearum (Fusarium graminearum), in which A: fusarium graminearum; b: sterile water control; c: cultivation effect of Bacillus belgii SF259 and Fusarium graminearum.

FIG. 11 is a graph showing the antagonistic effect of Bacillus belgii SF259 against Phytophthora (Phytophthora capsici), wherein A: phytophthora; b: sterile water control; c: cultivation effect of Bacillus belgii SF259 and Phytophthora.

FIG. 12 is a graph showing the antagonistic effect of Bacillus belgii SF259 on Fusarium oxysporum (Fusarium oxysporum), in which A: fusarium oxysporum; b: sterile water control; c: the effect of Bacillus beijerinckii SF259 on the culture of Fusarium oxysporum in pairs.

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).

PDA solid medium (g/L): 200g of potato, 20g of glucose and 15g of agar are dissolved in water, and finally the volume is determined to be 1000mL, the pH value is 7.0-7.2, and the mixture is sterilized by high pressure (121 ℃,20 min).

Example 1 preparation of Bacillus belgii SF259

1. Source of soil

Soil for cherry trees in orchard lands of garden of orchards in the areas of orchestra of Qinghai province of China

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 20min, standing at room temperature for 10min, and preparing a soil bacterium suspension stock solution. The soil bacterium suspension stock solution is subjected to gradient dilution to respectively obtain 10⁰、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. Placing into a biochemical incubator at 28 deg.C, culturing for 24 hr, 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 an NA liquid culture medium, culturing for 12h in a shaking table at 28 ℃ and 180rpm, sucking 200 mu L of bacterial suspension, fully and uniformly mixing with an NA solid culture medium, turning the plate, then placing oxford cups with the diameter of 6mm in the center of an NA flat plate, internally connecting 50 mu L of bacillus liquid to be tested (OD600 is about 2.0) in each oxford cup, repeating for 2-3 times each strain of pathogenic bacteria, placing the strain in a biochemical incubator at 28 ℃ for culturing for 24h, observing whether a bacteriostatic circle exists, recording the serial number of the strain, measuring the size of the bacteriostatic circle, and finishing and photographing.

Example 2 identification of 16S rRNA Gene of Bacillus beilesiensis SF259

Genomic DNA of strain SF259 was extracted, using primers:

27F 5'-AGAGTTTGATCCTGGCTCAG-3', and

1492R 5'-TACGGCTACCTTGTTACGACTT-3', using the extracted DNA as a template, and performing PCR amplification 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 DNA Star and BLAST comparison is carried out on NCBI website to determine the species of the related strains.

The results show that: the 16S rRNA gene of the strain SF259 has 99% 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 beilesiensis SF259

Genomic DNA of strain SF259 was extracted, using 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 is finished, the PCR products are checked by 1% agarose gel electrophoresis, and the results are detected and recorded by a gel imager. The PCR stock was sent to Shitanhua Biotech (Shanghai) Co., Ltd for sequencing. The sequencing result is analyzed by using DNA Star and BLAST comparison is carried out on NCBI website to determine the species of the related strains.

The results show that: the similarity of the gyrA gene of the strain SF259 and the gyrA gene sequence of Bacillus velezensis model bacteria reaches 99 percent, and the similarity of the gyrA gene sequence of the strain SF259 and the gyrA gene sequence of Bacillus amyloliquefaciens model bacteria is only 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 beilesiensis SF259

The physiological and biochemical characteristics of the bacillus beleisi SF259 are as follows: can secrete gelatinase, and can not hydrolyze beta-galactosidase, arginine double hydrolase, lysine decarboxylase, ornithine decarboxylase and urease; can utilize 3-hydroxy butanone to produce acetyl methyl methanol, can not produce indole; is incapable of oxidizing glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose. Can utilize 19 carbon sources such as glycerol, L-arabinose, ribose, D-xylose, glucose, etc., as shown in tables 3 and 4.

TABLE 3 physiological and biochemical characteristics of strain SF 259-enzyme activity, carbon source oxidation

+: positive reaction; -: negative reaction;

TABLE 4 physiological and biochemical characteristics of Strain SF259 acid production Using carbon sources

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

Example 5 determination of antagonistic spectra of Bacillus belgii SF259

1) Determination of antagonistic activity of Bacillus beleisi SF259 on 9 kinds of rice bacterial blight

Respectively inoculating 9 different kinds of rice bacterial blight and test Bacillus beilesiensis SF259 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 6mm in the center of an NA flat plate, internally connecting 50 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.

TABLE 5 bacteriostatic effect of Bacillus beilesiensis SF259 on different rice bacterial blight germs

2) Determination of antagonistic activity of Bacillus beleisi SF259 on 9 rice leaf streak pathogens

Respectively inoculating 9 different rice streak pathogens and test Bacillus belgii SF259 into NA liquid culture medium, culturing at 28 deg.C in a shaking table at 180rpm for 12 hr, and unifying OD₆₀₀Are all 2.0; respectively sucking 200 mu L of corresponding pathogenic bacteria liquid and NA solid culture medium, fully mixing, pouring, placing an Oxford cup with the diameter of 6mm in the center of an NA flat plate, internally connecting 50 mu L of test bacteria in each Oxford cup, repeating each pathogenic bacteria for 2-3 times, placing in a biochemical incubator at 28 ℃, observing the existence of a bacteriostatic zone after culturing for 24 hours, and recording the bacteriostatic zoneAnd (5) sorting and photographing the size of the fungus ring.

TABLE 6 bacteriostatic effect of Bacillus belgii SF259 on different rice streak pathogens

3) Determination of antagonistic Activity of Bacillus beiLeisi SF259 against 9 plant pathogenic Xanthomonas

Respectively inoculating 9 plant pathogenic xanthomonas such as banana bacterial wilt pathogen, cowpea wilt pathogen, cotton bacterial angular leaf spot pathogen, etc. and tested Bacillus beilaisi SF259 in 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 6mm in the center of an NA flat plate, internally connecting 50 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.

TABLE 7 bacteriostatic effect of Bacillus beilesiensis SF259 on 9 plant pathogenic xanthomonas

4) Determination of antagonistic Activity of Bacillus belgii SF259 against 5 plant pathogenic fungi

Respectively culturing rice blast, fusarium graminearum, botrytis cinerea, fusarium oxysporum and phytophthora on a PDA culture medium by adopting a plate confronting culture method, after the pathogenic bacteria grow on a plate, punching a bacterial block on the edge of the plate for later use by using a puncher, taking one of the bacterial cakes, inoculating the bacterial cake with the hypha facing downwards to the center of a new PDA plate, placing aseptic oxford cups at equal distance (20mm) from left to right of the bacterial cake, inoculating 50 mu L of test-ready beilaisi bacillus SF259 in each oxford cup, repeating each treatment for 3 times, and meanwhile, using the plate which is not connected with the beilaisi bacillus SF259 and is only connected with pathogenic fungi as a contrast. Culturing in 25 deg.C incubator, and observing and recording bacteriostasis after 5 days, wherein the bacteriostasis is shown in figure 8-figure 12. The result shows that the Bacillus belgii SF259 has obvious inhibition effect on botrytis cinerea, and the inhibition rate is 95.39%; the bacteriostasis rates of the bactericidal composition on rice blast, fusarium graminearum, phytophthora and fusarium oxysporum are respectively 79.25%, 75.84%, 58.46% and 41.10%.

Therefore, the Bacillus beilisi SF259 provided by the invention has obvious antagonism on rice bacterial blight Xoo, broad-spectrum bacteriostasis on rice stripe blotch Xoc, and antagonism on pathogenic bacteria of various xanthomonas and important plant pathogenic fungi, and provides new resources 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

<110> Shanghai university of transportation

<120> biocontrol bacillus beiLeisi 259 and application thereof

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gggcggggtg ctatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 60

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120

accggggcta ataccggatg gttgtctgaa ccgcatggtt cagacataaa aggtggcttc 180

ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240

aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420

caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720

gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780

gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 840

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960

ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg 1020

gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080

acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1140

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tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1320

cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380

cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tatggagcca gccgccgaag 1440

tgacaga 1447

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acagacggat tttgtacgca atgaatgatt taggcatgac cagtgacaaa ccatataaaa 120

aatctgcccg tatcgtcggt gaagttatcg gtaagtacca cccgcacggt gactcagcgg 180

tttacgaatc aatggtcaga atggcgcagg attttaacta ccgctacatg cttgttgacg 240

gacacggcaa cttcggttcg gttgacggcg actcagcggc cgcgatgcgt tacacagaag 300

cgagaatgtc aaaaatcgca atggaaattc tgcgtgacat tacgaaagac acgattgact 360

atcaagataa ctatgacggt tcagaaagag agcctgccgt catgccttcg agatttccga 420

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atcagcttgg ggaagtcatt gaaggcgtgc ttgccgtaag tgagaatcct gagattacaa 540

accaggagct gatggaatac atcccgggcc cggattttcc gactgcaggt cagattttgg 600

gccggagcgg catccgcaag gcatatgaat ccggacgggg atcaatcacg atccgggcta 660

aggctgaaat cgaagagact tcatcgggaa aagaaagaat tattgtcacg gaacttcctt 720

atcaggtgaa caaagcgaga ttaattgaaa aaatcgcgga tcttgtccgg gacaaaaaaa 780

tcgaaggaat taccgatctg cgagacgaat ccgaccgtaa cggaatgaga atcgtcattg 840

agatccgccg tgacgccaat gctcacgtca ttttgaataa cctgtacaaa caaacggccc 900

tgcagacgtc tttcggaatc aacctgctgg cgctcgtgac ggacagccga agacttggcc 960

atccg 965

Claims

1. A biocontrol Bacillus belgii strain is characterized in that the biocontrol Bacillus belgii strain is named as Bacillus belgii SF259(Bacillus belgii)Bacillus velezensisSF259), the Bacillus beleisi strain has been preserved in China center for type culture Collection (CCTCC M2019735) in 2019, 9, 20 months.

2. The biocontrol Bacillus belgii of claim 1, wherein the Bacillus belgii colonies are milky white, rough and irregular in edges, dry, rough in surface, and opaque; the thallus is short rod-shaped and can produce spores.

3. The biocontrol Bacillus belgii of claim 1, wherein said Bacillus belgii is a gram-positive, aerobic or facultative anaerobic bacterium; can secrete gelatinase, and can not secrete beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and urease; can utilize 3-hydroxy butanone to produce acetyl methyl methanol, can not produce indole; cannot oxidize glucose, mannitol, inositol, sorbitol, rhamnose, melibiose, amygdalin and arabinose, and can utilize glycerol, L-arabinose, ribose, D-xylose, glucose.

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

5. The biocontrol Bacillus belgii of claim 4, wherein said Xanthomonas oryzae comprises Xanthomonas oryzae (K.oryzae) (B.albilineans (K.oryzae)) (Xanthomonas oryzae pv. oryzae, Xoo) And bacterial leaf streak disease of rice: (Xanthomonas oryzae pv. oryzicola, Xoc) Two pathogenic varieties;

the rice bacterial leaf blight comprises PXO99^A、AH1、YNB01-4、XZ35、JL3、JC1、YC11、LYG46、XC18；

The rice bacterial leaf streak germ comprises HANB12-26, RS85, HNB07-3, ZJB01-25, RS105, JSB1-39, AHB3-7, YNB01-3 and HNB 3-17.

6. The biocontrol Bacillus belgii of claim 1, wherein the Bacillus belgii has an antagonistic effect against Paddy rice bacterial blight and rice streak disease.

7. The biocontrol Bacillus belgii of claim 1, wherein the Bacillus belgii has antagonistic activity against a plant pathogenic xanthomonas comprising banana bacterial wilt (Byssaceae: (Byssaceae))X. campestris pv. musacearum) Cowpea blight bacteria (A)X. axonopodis pv. vignicola) Bacterial angular leaf spot of cottonX. campestris pv. malvacearum) Pepper spot germs (1)X. campestris pv. vesicatoria) Bacterial wilt of kidney bean (A)X. campestris pv. phaseoli) Sugar cane gummosis bacteria (X. axonopodis pv. vasculorum) Bacteria of soybean scab (1)X. axonopodis pv. glycines）。

8. The biocontrol Bacillus belgii of claim 1, wherein the Bacillus belgii has antagonistic activity against phytopathogenic fungi, including Botrytis cinerea (Botrytis cinerea)Botrytis cinerea) Pyricularia oryzae (Pyricularia oryzae) (II)Magnaporthe oryzae) Fusarium graminearum（Fusarium graminearum) Phytophthora bacteria (A), (B), (C)Phytophthora capsici) Fusarium oxysporum (F.), (Fusarium oxysporium）。

9. The use of biocontrol bacillus belgii as claimed in claim 1, comprising the use of:

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

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

as banana bacterial wilt bacterium (X. campestris pv. musacearum) Antagonistic bacteria, Fusarium oxysporum (A) of cowpeaX. axonopodis pv. vignicola) Antagonistic bacteria, cotton bacterial angular leaf spot bacteria (X. campestris pv. malvacearum) Antagonistic bacteria, pepper spot bacteria: (X. campestris pv. vesicatoria) Antagonistic bacteria, Fusarium oxysporum (A)X. campestris pv. phaseoli) Antagonistic bacteria, gummosis of sugarcane: (X. axonopodis pv. vasculorum) Antagonistic bacteria, scab bacteria of soybean (X. axonopodis pv. glycines) The application of antagonistic bacteria;

the product can be used as plant pathogenic fungi Botrytis cinerea antagonistic bacteria, Pyricularia oryzae antagonistic bacteria, Fusarium graminearum antagonistic bacteria, Phytophthora bacteria antagonistic bacteria, and Fusarium oxysporum antagonistic bacteria.