CN114958640A - Bacillus velezensis EM-1 and application thereof - Google Patents

Abstract

The invention discloses a Bacillus belgii separated from tobacco rhizosphere soil, which is named as EM-1 and has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 11 months and 09 days 2020, the preservation address is the institute of microorganisms of China academy of sciences No. 3 of North West Lu 1 of the Chaoyang district in Beijing, and the preservation number is CGMCC No. 21131. The strain EM-1 has broad-spectrum antibacterial activity, the EM-1 and metabolites thereof can inhibit the growth of ralstonia solanacearum pathogenic bacteria, effectively prevent and treat bacterial wilt, and simultaneously can inhibit the growth of various pathogenic fungi, thereby laying a foundation for the development and research of rich natural resources of biological pesticides and microbial pesticides.

Description

Bacillus subtilis EM-1 and application thereof

The technical field is as follows:

the invention relates to a control technology of bacterial wilt, in particular to a biocontrol strain Bacillus velezensis EM-1 and application thereof.

Background art:

bacterial wilt is a destructive soil-borne disease, mainly caused by Ralstonia solanacearum. It is a plant bacterial pathogen widely distributed in tropical, subtropical and temperate regions. Its host, disease condition and pathogenicity are gradually diversified, and it has a tendency to extend to cold areas with high altitude and high latitude. It can harm many important commercial crops, such as tomatoes, tobacco, potatoes, bananas, peanuts, and the like. The tobacco bacterial wilt affects most seriously on tobacco production in subtropical and tropical regions, and in the last forty centuries, the tobacco bacterial wilt causes immeasurable loss to tobacco industries in Indonesia and America, and the occurrence range of the tobacco bacterial wilt in China is very wide. In China, the tobacco bacterial wilt is called as 'marginless crazy' and 'tobacco pest', especially in Yangtze river basin and tobacco planting areas in the south of the Yangtze river basin, the bacterial wilt is serious and common in morbidity, the serious tobacco planting areas even have the phenomenon that diseases are spread to the top of the dead, and China has countless direct losses caused by the integral reduction of the yield and the quality of tobacco leaves due to the harm of the bacterial wilt every year. In recent years, the tendency of the bacterial wilt to spread to the north poses a great threat to the healthy production of tobacco leaves in China.

The wide geographical distribution and wide host range of ralstonia solanacearum has raised a high interest in crop production worldwide. If the traditional chemical pesticide is used properly, the bacterial wilt can be effectively solved or relieved, but serious problems are brought, such as environmental pollution, pesticide residue or the increase of drug resistance and drug resistance of pathogenic bacteria, which causes the worry of the safety of agricultural products. In order to realize green and sustainable agricultural development, biological control of bacterial wilt attracts more and more attention of researchers at home and abroad. Compared with chemical control, biological control has no pollution, does not kill natural enemies, is not easy to generate drug resistance, is beneficial to human and animal safety and environmental protection, can simultaneously control diseases and meet the requirements of developing organic agriculture, and has wide application prospect. Biological control is therefore the best choice for controlling this disease. Biological control mainly utilizes organisms or metabolites of certain organisms to replace chemical substances to inhibit the growth and development of pathogens, but the invention provides a bacillus belgii strain, the thallus and the generated metabolites of the bacillus belgii strain can inhibit the growth of ralstonia solanacearum, and simultaneously EM-1 can also inhibit the growth of various pathogenic fungi, and the bacillus belgii strain is used as an effective antibacterial agent and provides a new idea for controlling ralstonia solanacearum and other fungal diseases.

The invention content is as follows:

the invention aims to provide a biocontrol strain Bacillus velezensis EM-1 and application thereof.

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

a strain of Bacillus belgii, Bacillus velezensis EM-1, has been deposited in China general microbiological culture Collection center at 11 months and 09 days 2020, the deposition address is the institute of microbiology of China academy of sciences No. 3, North West Lu No. 1, North Cheng Yang district, Beijing, and the deposition number is CGMCC No. 21131.

The application of the strain in bacterial wilt prevention and control.

The application of the strain in preventing and controlling fungal diseases caused by fungi.

The fungus is tobacco brown spot, grape gray mold, tomato gray mold or tobacco black shank.

A biocontrol agent comprising said Bacillus belgii.

The biocontrol agent contains one or more of the bacillus belgii, the strain culture, the strain metabolite, the separated supernatant of the strain culture and the crude extract of the strain fermentation liquor.

The culture is a strain culture obtained by activating the strain EM-1 and then performing fermentation culture in an LB culture medium; filtering the obtained strain culture to obtain a supernatant of the strain culture; further extracting the obtained strain culture to obtain a crude extract of the strain fermentation liquor.

The application of a biocontrol agent is characterized in that: the biocontrol agent is applied to inhibition of bacterial wilt, brown spot, grape gray mold, tomato gray mold or black shank.

The invention has the beneficial effects that:

the Bacillus belgii EM-1 separated from tobacco has a very good inhibition effect on Ralstonia solanacearum and other pathogenic fungi, and further discovers that volatile gas and supernatant generated by the Bacillus belgii EM-1 can effectively inhibit the growth of the Ralstonia solanacearum, so that thalli, fermentation liquor or crude extracts of the fermentation liquor can be directly used for preparing medicines for resisting the Ralstonia solanacearum and other pathogenic fungi, and plant bacterial wilt and other fungal diseases can be prevented and treated.

Description of the drawings:

FIG. 1 is a colony morphology of EM-1 on LB medium provided in the present invention; wherein, A: EM-1 morphology after 1 day of culture; b: EM-1 morphology after 3 days of culture;

FIG. 2 is a 16S rDNA phylogenetic tree of EM-1;

FIG. 3 is a graph showing the effect of EM-1 in inhibiting Ralstonia solanacearum;

FIG. 4 is a graph showing the effect of EM-1 in inhibiting pathogenic fungi;

FIG. 5 shows the inhibition rate of EM-1 to pathogenic fungi;

FIG. 6 is a graph showing the effect of supernatant of fermentation broth of different concentrations of EM-1 in inhibiting Ralstonia solanacearum;

FIG. 7 is a graph showing the effect of volatile gas produced by EM-1 on the inhibition of Ralstonia solanacearum;

FIG. 8 shows the effect of EM-1 in preventing and treating tobacco bacterial wilt; wherein, A: comparison; b: the effect of preventing and treating bacterial wilt by EM-1;

FIG. 9 shows the amount of Ralstonia solanacearum in tobacco roots after EM-1 treatment; wherein RS is the number of ralstonia solanacearum in each gram of root of the control; EM-RS is the number of ralstonia solanacearum per gram of root after treatment with the strain EM-1.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: screening, separating and identifying biocontrol bacteria EM-1

Separation: the Bacillus belgii EM-1 is obtained by separating from tobacco rhizosphere soil, and the specific separation method comprises the following steps: collecting tobacco plants, collecting rhizosphere soil, diluting the rhizosphere soil with sterile water 10 ^-2 、10 ^-3 、10 ^-4 And coated on an LB plate. Culturing at 28 deg.C, selecting single colonies with different colors and forms, and repeatedly streaking and separating until purification. The Ralstonia solanacearum is used as a screening strain, and a strain with inhibitory activity is screened for further research.

The obtained strain is EM-1 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 09.11.2020, the preservation address is the microorganism research institute of China academy of sciences No. 3, West Lu No. 1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 21131.

As can be seen from FIG. 1, the growth state of strain EM-1 was different at different cultivation times. The bacterial colony of the strain on an LB culture medium is milky round, and the fresh bacterial colony has a wet, smooth and sticky surface. With the extension of the culture time, the surface of the bacterial colony becomes rough, the middle part is inwards sunken, and the bacterial colony is matt and dry in texture.

And (3) identification: EM-1 single colony is picked to amplify 16S rDNA sequence (27F5 '-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3') and sequenced for identification. The length of the sequenced fragment was 1511bp, and the sequence was as follows. Obtaining 22 standard strain sequences from NCBI (GenBank) database, analyzing 16S rDNA sequences of the separated strain and the reference strain by MEGA-X, and constructing phylogenetic tree of the separated strain and the reference strain (figure 2); as can be seen from FIG. 2, the similarity between the Bacillus strain 16S rDNA gene sequence provided by the invention and the Bacillus velezensis 16S rDNA gene sequence is extremely high, so that the EM-1 is determined to belong to the Bacillus velezensis and is named as EM-1.

GGCTACCTTGTTACGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACACATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTTCGCTCGACTTGCGTGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCT

Example 2: antagonistic Effect of Strain EM-1 on pathogenic fungi

The test pathogens include tobacco brown spot (Alternaria alternate (Fries) keislar), tobacco black shank (Phytophthora nicotiana), tomato gray mold (Botrytis cinerea per ex Fr.) and grape gray mold (Botrytis cinerea).

Taking the pathogenic bacteria out of the strain tube, inoculating the pathogenic bacteria to a PDA culture medium, performing static culture at 28 ℃ for 7 days, placing a bacterial cake with the diameter of 5mm in the center of the PDA culture medium by using a puncher, dripping 4 mu L of activated EM-1 strain at two ends of the bacterial cake, and dripping 4 mu L of LB culture solution at two ends of the bacterial cake. Then, the plate was cultured at 28 ℃ for 10 days, the diameter of the fungus between the EM-1 bacteria solutions was measured, and the inhibition rate of each fungus was calculated. The experiment was set up in triplicate. (antagonistic effect see FIG. 4, inhibition rate see FIG. 5).

As can be seen from FIGS. 4 and 5, EM-1 has inhibition effects on all 4 fungi, but the inhibition degrees are different, and the inhibition effects on two kinds of gray mold (botrytis cinerea and botrytis cinerea) are strongest, namely 85.67% and 80.39%, respectively, the inhibition rate on gibberellin is 72%, and the inhibition rate on black shank is 65.1%.

Example 3: preparation of EM-1 strain fermentation liquor and detection method of bacteriostatic activity of fermentation liquor with different concentrations

(1) Preparation of EM-1 strain fermentation liquor: inoculating EM-1 single colony in an LB culture medium, and performing shake culture in a constant temperature shaking table at 28 ℃ and 180r/min for 24 hours to obtain seed liquid; inoculating the strain into a fresh LB culture medium with the inoculation amount of 1 percent, and performing shake culture in a constant-temperature shaking table at 28 ℃ and 180r/min for 72 hours. Centrifuging the fermentation liquid at 10000r/min for 5min to remove thallus, filtering the supernatant with 0.22 μm disposable microporous membrane to obtain filtrate completely free of thallus, and freeze drying to obtain powder for use.

(2) Preparing fermentation liquor with different concentrations: when preparing fermentation liquor with different concentrations, taking dry powder, and preparing into 50, 100 and 300mg/mL concentration solutions by using methanol solutions with different volumes.

(3) And (3) bacteriostatic test: the experiment was performed by a punch method. Inoculating Ralstonia solanacearum to NB culture solution, performing shake culture in a constant temperature shaking table at 28 deg.C and 180r/min for 24h for primary activation, inoculating to NB culture solution at 1% inoculum size, and performing shake culture in a constant temperature shaking table at 28 deg.C and 180r/min for 24h for secondary activation. And (3) sterilizing the NA culture medium, cooling to below 50 ℃, adding the secondarily activated Ralstonia solanacearum bacterial liquid into the culture medium according to the inoculation amount of 0.5%, quickly mixing uniformly, pouring into a flat plate, solidifying the culture medium, and uniformly punching for three times by using a sterilized puncher, wherein the diameter of the hole is 5 mm. 60 μ L of the above-mentioned fermentation broths of different concentrations were added to the wells, and methanol was set as a control, and three replicates were set for each experimental concentration. Putting into an incubator at 28 ℃ for upright culture for 48 h. The size of the zone of inhibition for Ralstonia solanacearum was observed (see FIG. 6).

As can be seen from FIG. 6, the fermentation supernatants (without thallus) with different concentrations have different bacteriostatic effects, and the higher the concentration is, the stronger the activity of inhibiting the ralstonia solanacearum is, and the more effective components are contained therein. The size of the inhibition zone generated by the fermentation supernatant of 300mg/mL is 2.63cm, the size of the inhibition zone generated by the fermentation supernatant of 300mg/mL is 2.25cm, and the size of the inhibition zone generated by the fermentation supernatant of 50mg/mL is 1.97 cm.

Example 4: detecting the ralstonia effect of the volatile gas generated by EM-1:

the anti-ralstonia activity of volatile organic compounds produced by the EM-1 strain was evaluated using two sealed plates. One plate contained 15mL NA and the other plate 15mL LA. After the secondary activation of Ralstonia solanacearum, the OD value was adjusted to 0.6 with sterile water at a lambda of 600nm absorbance, 100. mu.L of the activated strain was applied to an NA plate, and 100. mu.L of the secondarily activated strain EM-1 was applied to an LA plate. Then, the two substrates were sealed with a sealing film and cultured in an incubator at 28 ℃ for 4 days, 100. mu.L of LB medium was applied to LA plates as a blank, and the growth state of Ralstonia solanacearum was observed for three repetitions of the experimental setup.

As can be seen from FIG. 7, the ralstonia solanacearum in the control has good growth vigor, and the growth rate of the ralstonia solanacearum in the experimental group fumigated by EM-1 is obviously inhibited, which indicates that the bacterial strain EM-1 not only produces a fermented product with good bacteriostatic activity, but also produces volatile gas with good bacteriostatic effect.

Example 5: biocontrol strain EM-1 biocontrol potential analysis on tobacco bacterial wilt

(1) The tobacco pot plant of the tieback strain EM-1 can prevent and treat bacterial wilt: the tobacco seeds are uniformly scattered on the matrix soil and cultured in a greenhouse. The illumination period is 12 hours/12 hours, the day and night temperature is 30 ℃ and 28 ℃ respectively, and the relative humidity is 70-80%. And selecting the seedlings with consistent growth states and transferring the seedlings into a pot. Taking out the strain EM-1 from-80 ℃, streaking on an LB solid culture medium, culturing at 28 ℃ until a single colony grows out, selecting the single colony, inoculating into an LB liquid culture medium, and culturing at 28 ℃ and 180rpm overnight. The cells were suspended in distilled water until the OD lambda was about 600 at 0.6 after centrifugation at 6000rpm for 10min, inoculated into the soil of a pot in which tobacco seedlings were planted at a ratio of 1:5 (bacterial liquid (mL): soil (g)) for 3 times with 2-day intervals, and the control group was replaced with distilled water. After incubating the tobacco seedlings with the EM-1 bacterial liquid for 2 days, carrying out root injury treatment on the tobacco seedlings, inserting the tobacco seedlings into soil by using a sharp scalpel to quickly scratch the roots of the seedlings, then inoculating ralstonia solanacearum, wherein the ralstonia solanacearum activation method is similar to that of EM-1, the bacterial liquid is resuspended by using distilled water until the OD value lambda is about 0.7, the inoculation is carried out according to the proportion of 1:5 (bacterial liquid (mL): soil (g)), and the ralstonia solanacearum is inoculated by using the same inoculation method and inoculation amount in a control group. The experimental group and the control group of EM-1 were inoculated with Ralstonia solanacearum and then cultured in a greenhouse under the same culture conditions, and the onset of disease was observed (see FIG. 8).

Incidence (%) ═ bacterial wilt onset strain/test effective strain × 100

Disease index [ sigma (number of diseased plant at each stage × value of disease)/(total number of investigated plants × 9) ] × 100

Relative control effect (%) (control disease index-treatment disease index)/control disease index) x 100

TABLE 1 Classification Standard of disease conditions for tobacco bacterial wilt

As can be seen from FIG. 8, the plants in the control group A are withered to be serious, and the plants in the experimental group EM-1 are good in growth and not diseased, which shows that EM-1 has obvious control effect on bacterial wilt and is a biological agent with development potential. The plant disease was counted according to the agricultural industry standards (see table 2). The control effect of EM-1 reaches 88.7 percent after 5 days of inoculation, 72.5 percent after 7 days and 80.6 percent after 11 days.

TABLE 2 prevention and control of bacterial wilt of EM-1 tieback-grafted potted tobacco seedlings

(2) The method for quantifying the amount of the ralstonia solanacearum in the tobacco roots by using a fluorescent quantitative PCR method comprises the following steps: the sample treatment method is the same as before. The roots were sampled after the onset of disease in the control group. Gently shaking off the soil at the root of tobacco, shearing off the plant root with scissors, placing the plant root in sterile water, shaking to remove rhizosphere soil, and then washing with sterile water. Samples were stored at-80 ℃ until use. The experiment was set up in triplicate.

Root bacteria DNA extraction: 0.5g of root sample was taken and extracted using the FastDNA Spin Kit for Soil Kit according to the instructions.

A fluorescent quantitative PCR step: bacterial abundance was determined using Rsol _ fliCF/Rsol _ fliCR primers (Rsol _ fliCF 5'-GAACGCCAACGGTGCGAACT-3' and Rsol _ fliCR 5'-GGCGGCCTTCAGGGAGGTC-3'). Quantitative PCR (qPCR) analysis was performed using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, USA). The standard curve was generated with 10-fold serial dilutions of the plasmid (containing iC fragments of ralstonia solanacearum). The qPCR amplification of the standard and DNA samples included 2. mu.L template, 10. mu.L SYBR Green premix Ex Taq (2X), 0.4. mu.L ROX reference dye II, 0.4. mu.L of each primer and 6.8. mu.L distilled water. qPCR uses a two-step procedure with a program of 95 ℃ for 30s, followed by 40 cycles at 95 ℃ for 5s, 60 ℃ for 34s, and finally 95 ℃ for 15s, 60 ℃ for 1 min. Three replicates were set up for the experiment (see figure 9).

As can be seen from FIG. 9, the amount of ralstonia solanacearum in the tobacco roots of the EM-1 experimental group is obviously lower than that of the control group, and the amount of ralstonia solanacearum in the roots of the control group is 7 times of that of the ralstonia solanacearum of the EM-1 experimental group, which shows that the EM-1 can effectively prevent the ralstonia solanacearum from entering the plant roots and can play a role in biocontrol.

In conclusion, the Bacillus beijerinckii EM-1 separated from tobacco has a very good inhibition effect on Ralstonia solanacearum and other pathogenic fungi, the produced fermentation liquid or the produced volatile gas also has a very obvious inhibition effect on Ralstonia solanacearum, and a potting test further verifies the good biocontrol effect of the EM-1, so that the strain EM-1 is a biocontrol strain with development and application values.

Sequence listing

<110> tobacco institute of Chinese academy of agricultural sciences (Qingzhou tobacco institute of Chinese tobacco Co., Ltd.)

ZUNYI TOBACCO COMPANY OF GUIZHOU TOBACCO Corp.

<120> Bacillus velezensis EM-1 and application thereof

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<170> SIPOSequenceListing 1.0

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<211> 1511

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg attccagctt 180

cacgcagtcg agttgcagac tgcgatccga actgagaaca gatttgtggg attggcttaa 240

cctcgcggtt tcgctgccct ttgttctgtc cattgtagca cgtgtgtagc ccaggtcata 300

aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtcacct 360

tagagtgccc aactgaatgc tggcaactaa gatcaagggt tgcgctcgtt gcgggactta 420

acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac tctgcccccg 480

aaggggacgt cctatctcta ggattgtcag aggatgtcaa gacctggtaa ggttcttcgc 540

gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 600

gtttcagtct tgcgaccgta ctccccaggc ggagtgctta atgcgttagc tgcagcacta 660

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ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agttacagac cagagagtcg 780

ccttcgccac tggtgttcct ccacatctct acacatttca ccgctacacg tggaattcca 840

ctctcctctt ctgcactcaa gttccccagt ttccaatgac cctccccggt tgagccgggg 900

gctttcacat cagacttaag aaaccgcctg cgagcccttt acgcccaata attccggaca 960

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taggtaccgt caaggtgccg ccctatttga acggcacttg ttcttcccta acaacagagc 1080

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cccatctgtt cgctcgactt gcgtgtatta ggcacgccgc cagcgttcgt cctgagccag 1500

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Claims (8)

1. A strain of Bacillus belgii is characterized in that: bacillus velezensis (Bacillus velezensis) EM-1 is preserved in China general microbiological culture Collection center (CGMCC) at 11/09.2020, the preservation address is the institute of microbiology, China academy of sciences, No. 3, West Lu No. 1, North Chen, west, of the Chaoyang region, Beijing, and the preservation number is CGMCC No. 21131.

2. Use of a biocontrol strain according to claim 1, characterized in that: the application of the strain in bacterial wilt prevention and control.

3. Use of a biocontrol strain according to claim 1, characterized in that: the application of the strain in preventing and treating fungal diseases caused by fungi.

4. Use of a biocontrol strain according to claim 3, characterized in that: the fungus is tobacco brown spot, grape gray mold, tomato gray mold or tobacco black shank.

5. A biocontrol agent characterized by: a biocontrol agent comprising the Bacillus belgii of claim 1.

6. The biocontrol formulation of claim 5, wherein: the biocontrol agent contains one or more of Bacillus beiLeisi as described in claim 1, a culture of the strain, a metabolite of the strain, a supernatant separated from the culture of the strain, and a crude extract of a fermentation liquid of the strain.

7. The biocontrol formulation of claim 6, wherein: the culture is obtained by activating the strain EM-1 and then performing fermentation culture in an LB culture medium; filtering the obtained strain culture to obtain a supernatant of the strain culture; further extracting the obtained strain culture to obtain a crude extract of the strain fermentation liquor.

8. Use of the biocontrol agent of claim 5, characterized in that: the biocontrol agent is applied to inhibition of bacterial wilt, brown spot, grape gray mold, tomato gray mold or black shank.

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