A strain of Siamese Bacillus antagonistic to multiple plant pathogens and its application

Abstract

The present invention discloses a Siamese Bacillus antagonistic to multiple plant pathogens and its application. Bacillus siamensis )A‑2 is deposited with GDMCC NO.64228. The bacterium has a significant inhibitory effect on Ralstonia syringae subspecies Indonesia, Pseudomonas solanacearum, Fusarium solani, Phomopsis, Fusarium oxysporum, Colletotrichum spp., Colletotrichum spp., Fusarium fujikura and Fusarium spp. It contains gene clusters for synthesizing plipastatin, fengycin, surfactin, siderophore, bacteriocin, dificidin, buprofezin A / B and some terpene compounds; it has a good control effect on tomato bacterial wilt (relative control effect 58.14%); and has the ability to degrade tetracycline hydrochloride (3-day degradation rate 49.28%). Therefore, Siamese Bacillus A‑2 has a very high development and utilization value in agricultural production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a siam bacillus capable of antagonizing multiple plant pathogens and application thereof. Background Art

[0002] Bacterial wilt is a common bacterial disease of plants, mainly harming Solanaceae crops such as tomatoes, peppers, flue-cured tobacco, potatoes, and eggplants, causing serious impacts on crop yield and quality. Ralstonia solanacearum ), Pseudomonas solanacearum ( Pseudomonas solanacearum ) and Ralstonia syringae ( Ralstonia syzygii ) and other pathogenic bacteria infection.

[0003] At present, the prevention and control of bacterial wilt mainly focuses on breeding resistant varieties and chemical control. Although the occurrence of crop wilt has been reduced to a certain extent, there are still limitations. For example, crop varieties that are completely resistant to bacterial wilt have not yet been bred, and the breeding process of some crops is complicated, difficult, costly, and unstable. Although chemical agents are effective in preventing and controlling bacterial wilt, the long-term and large-scale use of chemical pesticides will lead to increased drug resistance of bacterial wilt bacteria and a series of food safety issues. Therefore, it is necessary to find new methods for the prevention and control of bacterial wilt that are more efficient, green and safe.

[0004] Biological control is considered to be one of the effective measures to reduce the occurrence of soil-borne diseases such as bacterial wilt due to its high efficiency, non-toxicity, no residue and low cost. Biological control mainly achieves the prevention and control of plant diseases such as bacterial wilt by screening and utilizing antagonistic microorganisms. Currently, antagonistic microorganisms include antagonistic bacteria, antagonistic endophytes, antagonistic actinomycetes and antagonistic fungi. These antagonistic microorganisms mainly bring broad-spectrum benefits to host plants by fixing nitrogen, dissolving phosphorus, dissolving potassium, secreting antimicrobial compounds, promoting plant growth and inducing plant immune resistance.

[0005] So far, although a series of antagonistic microbial resources have been explored, there are still few strains that can be used on a large scale to prevent and control bacterial wilt in the field. This is mainly because different antagonistic strains have different morphologies and functions, and the secondary metabolites they produce, as well as the types and effects of preventing and controlling crop diseases, are significantly different. Even antagonistic bacteria of the same genus and species may have significant differences in their antibacterial ability and antibacterial effects. Therefore, there is no predictability and revelation between strains. In addition, in actual production applications, the occurrence of bacterial wilt is often accompanied by the occurrence of other diseases. Antagonistic bacteria with a single function and a narrow antibacterial spectrum are difficult to achieve ideal prevention and control effects. Therefore, it is necessary to screen more antagonistic microbial resources with strong antibacterial / antibacterial ability, broad antibacterial / antibacterial spectrum, and rich secondary metabolites to prevent and control bacterial wilt.

[0006] Antibiotics are a class of chemical substances that have inhibitory and killing effects on pathogenic microorganisms. They play an important role in medical health, animal husbandry and agricultural production. However, the abuse of antibiotics has posed potential risks to the farmland soil environment. Therefore, screening strains that have the ability to degrade antibiotics is of great significance for further dealing with the problem of antibiotic residues in the farmland soil environment. Summary of the invention

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] The first object of the present invention is to provide a siam bacillus ( Bacillus siamensis )A-2, whose deposit number is GDMCC NO.64228.

[0009] Siamese Bacillus A-2 was isolated from diseased tomato tissue and deposited in Guangdong Microbial Culture Collection Center. Its 16S rDNA gene sequence is shown in SEQ ID NO.1. Based on 16S rDNA gene sequence comparison (EZBioCloud), this strain is similar to Siamese Bacillus KCTC13613 ( Bacillus siamensis ) sequence homology is the highest. The evolutionary tree results based on housekeeping genes ( Figure 2 ), which is related to Bacillus siamensis ( Bacillus siamensis ) are in the same branch, so strain A-2 was named Bacillus siamensis ( Bacillus siamensis )A-2.

[0010] Siamese Bacillus A-2 grows rapidly on nutrient broth solid medium, with raised, milky white colonies, dry wrinkles, opaque, and irregular colonies; Gram staining is positive, and the bacteria are oblong under the microscope. Nitric acid reaction and gelatin reaction are positive, and it can hydrolyze α-glucosidase, produce alkaline phosphatase, esterase (C4), lipase (C8) and phenol-AS-BI-phosphohydrolase.

[0011] The second object of the present invention is to provide the use of the above-mentioned siam bacillus A-2 in any of the following:

[0012] 1) Control of plant diseases caused by plant pathogenic bacteria and / or plant pathogenic fungi;

[0013] 2) Prevent and control tomato bacterial wilt;

[0014] 3) Degradation of tetracycline;

[0015] 4) Synthetic lipopeptide active substances, polyketide compounds and / or terpene compounds.

[0016] Preferably, the plant pathogenic bacteria is Ralstonia syringae subsp. indonesia Ralstonia syzygii subsp.indonesiensis and / or Pseudomonas solanacearum Pseudomonas solanacearum .

[0017] Preferably, the plant pathogenic fungus is Fusarium solani Fusarium solani Phomopsis Phomopsis sp., Fusarium oxysporum Fusarium oxysporum , banana anthracnose Colletotrichum musae , Colletotrichum Colletotrichum gloeosporioides Fusarium fujikura Fusarium fujikuroi Fusarium Fusarium circinatum One or more of .

[0018] Preferably, the control of plant diseases caused by plant pathogenic bacteria is by destroying the integrity of the pathogenic bacteria and lysing the pathogenic bacteria; the control of plant diseases caused by plant pathogenic fungi is by causing the pathogenic fungi hyphae to fold, twist, swell or dissolve to inhibit the growth of the pathogens.

[0019] Preferably, the tetracycline is tetracycline hydrochloride, and the concentration of tetracycline hydrochloride is 1-20 mg / L.

[0020] Preferably, the lipopeptide active substance is plipastatin, fengycin, surfactin or siderophore, and the polyketide compound is bacteriocin, dificitin or butyrosine A / B.

[0021] The third object of the present invention is to provide a biocontrol microbial agent, which comprises one or more of the above-mentioned Bacillus siamensis A-2 bacteria, its metabolites, its fermentation supernatant and its fermentation broth crude extract.

[0022] The fourth object of the present invention is to provide a method for preventing and controlling plant diseases, comprising the following steps: treating plant plants with the above-mentioned Bacillus siamensis A-2 or the above-mentioned biocontrol microbial agent.

[0023] Preferably, the treatment is to apply the reagent containing the above-mentioned Siamese Bacillus A-2 or the above-mentioned biocontrol microbial agent to the roots of the plants, wherein the Siamese Bacillus A-2 is OD 600 =1.0~2.0.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention successfully isolated a strain of Siamese Bacillus A-2 which has a significant antagonistic effect on a variety of plant pathogens. Through morphological observation, physiological and biochemical determination and molecular biological identification, it was determined that strain A-2 is Siamese Bacillus ( Bacillus siamensis). Plate confrontation test and pot test showed that the strain has broad-spectrum antibacterial properties, has a good control effect on tomato bacterial wilt (relative control effect 58.14%), and has the potential to secrete lipopeptide active substances, polyketide compounds and terpene compounds, and has a very high utilization value in the biological control of plant diseases.

[0026] The present invention discloses for the first time the antibacterial effect of siam bacillus on bacterial wilt pathogenic bacteria of Ralstonia syringae subspecies Indonesia, and provides available biocontrol microbial resources for the prevention and control of bacterial wilt caused by infection of Ralstonia syringae subspecies Indonesia.

[0027] The invention discloses for the first time that Siamese bacillus has the ability to degrade tetracycline hydrochloride. After culturing for 3 days, the degradation rate of tetracycline hydrochloride is 49.28%.

[0028] The Siamese Bacillus A-2 disclosed in the present invention has the potential to produce plipastatin, fengyogenin, surfactin, siderophore, bacteriocin, difisicin, buprenorphin A / B and terpene compounds; has obvious inhibitory effect on Ralstonia syringae subspecies Indonesia and / or Pseudomonas solanacearum and / or Fusarium solani and / or Phomopsis phytomyces and / or Fusarium oxysporum and / or Colletotrichum banana and / or Colletotrichum sporeans and / or Fusarium fujikura and / or Fusarium spp.; and the strain has the ability to degrade tetracycline hydrochloride. Therefore, Siamese Bacillus A-2 has extremely high development and utilization value in agricultural production.

[0029] Bacillus siamensis ( Bacillus siamensis )A-2, deposited on December 29, 2023 in Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is GDMCC NO.64228. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The colony morphology of Bacillus siame A-2 in the example of the present invention on the NA plate and the bacterial morphology after crystal violet staining.

[0031] Figure 2 This is the evolutionary tree of Bacillus siamensis A-2 based on the housekeeping gene in the examples of the present invention.

[0032] Figure 3 This is a diagram showing the inhibitory effect of Bacillus siamensis A-2 on plant pathogenic bacteria in an example of the present invention.

[0033] Figure 4 This is a diagram showing the inhibitory effect of Bacillus siamensis A-2 on plant pathogenic fungi in an example of the present invention.

[0034] Figure 5 This is a graph showing the control effect of Siamese Bacillus A-2 on tomato bacterial wilt in an example of the present invention.

[0035] Figure 6 The tetracycline standard curve (A) and the degradation effect diagram of Bacillus siamese A-2 on tetracycline hydrochloride (B) in the examples of the present invention are shown. DETAILED DESCRIPTION

[0036] In order to facilitate understanding by those skilled in the art, the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited thereto and is merely illustrative.

[0037] Example 1: Isolation, identification and preservation of Siamese Bacillus A-2

[0038] Isolation of Siamese Bacillus A-2: Strain A-2 was isolated from leaf tissues of tomatoes with severe bacterial wilt. The specific isolation steps are as follows: Take the leaf tissues with severe disease, rinse them with sterile water three times, and then disinfect the surface with 75% anhydrous ethanol for 3 minutes. After disinfection, rinse them with sterile water once, wipe the surface moisture of the leaves with absorbent paper, and cut them into pieces with sterile scissors. Weigh 1 g of sample in a sterile mortar, add 9 mL of sterile water, and crush the leaf tissue with a grinding rod. Take 1 mL of juice sample in 9 mL of sterile water, shake it with a vortex shaker for 3 minutes, and dilute the sample with sterile water to 10 -1 , 10 -2 , absorb the original solution, 10 -1 , 10 -2 100 μL of the sample was spread on a plate containing NA solid culture medium (formula: 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, 15 g / L agar powder, preparation method: take 33 g of the above culture medium, add 1000 mL deionized water, stir and heat until completely dissolved, adjust the solution pH to 7.2, divide into Erlenmeyer flasks, autoclave at 121℃ for 15 min, and pour into disposable culture dishes for later use). Each concentration gradient was repeated 3 times, and the cells were placed in an incubator at 30℃ for inversion and observation. After culturing for about 1 to 3 days, colonies with different morphology and color were picked for streak purification. Purification was performed 2 to 3 times to obtain pure strains.

[0039] Identification of Siamese Bacillus A-2: The strain was identified based on colony morphology, bacterial color, transparency, glossiness, etc. Figure 1As shown, Siamese Bacillus A-2 grows rapidly on NA solid medium, with raised colonies, milky white, dry wrinkles, opaque, and irregular colonies; Gram staining is positive, and the bacteria are oblong under the microscope. A series of physiological and biochemical tests were performed, and the results showed that the nitric acid reaction and gelatin reaction were positive, and it could hydrolyze α-glucosidase, produce alkaline phosphatase, esterase (C4), lipase (C8) and phenol-AS-BI-phosphohydrolase. 16S rDNA identification was performed on Siamese Bacillus A-2, and the obtained 16S rDNA sequence is shown in SEQ ID NO.1. Based on 16S rDNA gene sequence alignment (EZBioCloud), this strain is similar to Siamese Bacillus KCTC13613 ( Bacillus siamensis ) sequence homology is the highest. The evolutionary tree results based on housekeeping genes ( Figure 2 ), which is related to Bacillus siamensis ( Bacillus siamensis ) were in the same branch, so strain A-2 was named Bacillus siamensis ( Bacillus siamensis )A-2.

[0040] Housekeeping gene construction method: By comparing with the local database, based on 31 housekeeping genes ( dnaG , frr , infC , nusA , pgk , pyrG , rplA , rplB , rplC , rplD , rplE , rplF , rplK , rplL , rplM , rplN , rplP , rplS , rplT , rpmA , rpoB , rpsB , rpsC , rpsE , rpsI , rpsJ , rpsK , rpsM , rpsS , smpB , tsf ) The 19 strains closest to each other at the species level were selected, and the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA 6.0 software. The numbers of the 31 housekeeping genes are shown in Table 1.

[0041] Table 1 Numbers of 31 housekeeping genes

[0042]

[0043] Deposit of Siamese Bacillus A-2: Siamese Bacillus ( Bacillus siamensis )A-2, deposited on December 29, 2023 in Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is GDMCC NO.64228.

[0044] Example 2: Inhibitory effect of Bacillus siamese A-2 on plant pathogenic bacteria

[0045] Preparation of pathogenic bacteria liquid: Ralstonia syringae subsp. indonesia and Pseudomonas solanacearum were activated on NA plates and fermented with NB medium (NA medium without agar powder) at 30°C and 200 r / min for 24 h, and then the fermented bacterial liquid was diluted to OD 600 =1.0 stock solution, then diluted to 10 -2 Siamese Bacillus A-2 was streaked onto a NA solid culture medium (the culture medium preparation method was the same as in Example 1) plate and activated and cultured for 24 h before use.

[0046] Bacteriostatic test operation: Take 100 μL of pathogen dilution and evenly spread it on the plate containing NA solid culture medium. After the plate is dried, inoculate the activated Siamese Bacillus A-2 bacteria at 4 points above, below, left and right, 25 mm away from the center of the plate. Use the control without inoculation of Siamese Bacillus A-2 bacteria. Incubate at 30℃ for 1~2 days, observe whether there is an inhibition zone, and take pictures to record the results. At the same time, use a stereo microscope to magnify and observe the changes of pathogenic bacteria.

[0047] Results statistics: Figure 3 It can be seen that the Siamese Bacillus A-2 bacteria showed good inhibitory effects on Ralstonia syringae subspecies Indonesia and Pseudomonas solanacearum, and formed obvious inhibition zones on each antibacterial plate. The diameter of the inhibition zone was measured (Table 2). The inhibition diameter of Siamese Bacillus A-2 against the pathogen Ralstonia syringae subspecies Indonesia reached 27.92 mm; the inhibition diameter against Pseudomonas solanacearum reached 29 mm. Observation under a stereomicroscope revealed that ( Figure 3 ), Bacillus siamea A-2 mainly destroyed the integrity of the Ralstonia solanacearum cell, causing the substances contained in the cell to leak out and lysed the Ralstonia solanacearum cell.

[0048] Table 2 Inhibition of Bacillus siame A-2 on pathogenic bacteria

[0049]

[0050] Example 3: Inhibitory effect of Bacillus siamese A-2 on plant pathogenic fungi

[0051] Strain activation: Fusarium solani, Phomopsis spp., Fusarium oxysporum, Colletotrichum banana, Colletotrichum spp., Fusarium fujikura and Fusarium spp. were inoculated on plates containing PDA medium (formula: 300 g / L potato extract powder, 20 g / L glucose, 15 g / L agar powder, 0.1 g / L chloramphenicol, the preparation method is the same as NA solid medium), and placed in a 28°C incubator for culture. After the mycelium has grown all over the plate, a sterilized 1 mL blue pipette tip is used to evenly punch out a round bacterial cake with a diameter of 8 mm from the outer edge of the colony for use. Siamese Bacillus A-2 was streaked and inoculated on a NA solid medium (the medium preparation method is the same as in Example 1) plate for activation and culture for 24 h.

[0052] Antibacterial test operation: The activated pathogen cakes were inoculated in the center of the plate containing PDA culture medium, and the activated Siamese Bacillus A-2 bacteria were inoculated at 4 points above, below, left and right 25 mm from the center of the plate. The Siamese Bacillus A-2 bacteria were not inoculated as a control. (The inoculation method of Phomopsis: The activated Phomopsis cakes were inoculated in the center of the plate containing PDA culture medium, and the Siamese Bacillus A-2 bacteria were inoculated on the left and right sides 25 mm from the center of the plate by streaking. The Siamese Bacillus A-2 bacteria were not inoculated as a control.) Repeat 3 times and place in a 28℃ constant temperature incubator for culture. Fusarium solani and Colletotrichum banana were cultured for 2 days, Fusarium oxysporum was cultured for 3 days, Phomopsis and Fusarium were cultured for 5 days, and Discosporium spondylosporum and Fusarium fujikura were cultured for 7 days. After the culture is completed, take photos to record the growth of pathogens on the plate, and measure the growth radius of the pathogenic fungi from the edge of the bacterial cake to the center of Siamese Bacillus A-2, which is recorded as the growth radius of the treatment group; the growth radius of the pathogenic fungi in the control group is recorded as the growth radius of the control group. Finally, the inhibition rate is calculated according to the following formula. At the same time, a stereo microscope is used to magnify and observe the changes in the hyphae of the pathogens.

[0053] Inhibition rate (%) = (control growth radius - treatment growth radius) / control growth radius × 100%

[0054] The results show that: Figure 4 As shown in Table 3, Bacillus siamea A-2 has obvious inhibitory effects on Fusarium solani, Fusarium oxysporum, Colletotrichum spp., Fusarium fujikura, Fusarium spp. and Phomopsis spp. Among them, the inhibitory effect on Fusarium solani and Phomopsis spp. is the best, with an inhibition rate of more than 90%, followed by Fusarium oxysporum and Colletotrichum spp., with an inhibition rate of more than 80%, and the inhibition rate on Fusarium fujikura and Fusarium spp. is between 30% and 60%. Observation by stereo microscope ( Figure 4), and found that Bacillus siamese A-2 inhibited the growth of pathogenic fungi by causing their hyphae to fold, twist, swell and dissolve.

[0055] Table 3 Inhibition of Bacillus siame A-2 on pathogenic fungi

[0056]

[0057] Example 4: Analysis of Synthesis of Secondary Metabolites of Bacillus siamensis A-2

[0058] After culturing Bacillus siameensis A-2 in NB liquid medium (30°C 200 r / min) for 12 h, the cells were collected by centrifugation at 10,000 r / min, and the total genomic DNA was extracted. After the DNA purity test was qualified, it was commissioned to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing. The sequencing results were analyzed for secondary metabolite synthesis gene clusters using antiSMASH software.

[0059] The results showed that: as shown in Table 4, the antiSMASH software analysis showed that the genome of Bacillus siameensis A-2 contained 12 gene clusters related to the synthesis of secondary metabolites, including 4 lipopeptide active substance gene clusters synthesized by non-ribosomal synthetase (NRPS) (plipastatin, fengycin, surfactin and bacillibactin) and 3 polyketide compound gene clusters synthesized by polyketide synthase (transAT-PKS) (bacillaene, difficidin and Butirosin A / B). In addition, some terpene compounds with unknown functions were also included.

[0060] Table 4 Identification results of the secondary metabolite synthesis region of Bacillus siamensis A-2

[0061]

[0062] Example 5: Application of Siamese Bacillus A-2 in the prevention and treatment of tomato bacterial wilt

[0063] Preparation of bacterial suspension of Pseudomonas solanacearum and Bacillus siamense A-2: The activated Pseudomonas solanacearum and Bacillus siamense A-2 were inoculated into Erlenmeyer flasks containing NB liquid culture medium, cultured in a shaker at 30°C and 200 r / min for 24 h, and then centrifuged at 10,000 r / min for 8 min. The supernatant was discarded and the Pseudomonas solanacearum was resuspended in sterile water to an OD of 0. 600 = 1.56, and Bacillus siameensis A-2 was resuspended to OD 600 =1.27 spare.

[0064] Potted inoculation test: The potted test was carried out in a greenhouse. When the tomato seedlings grew to 3 leaves and 1 heart, they were transplanted into pots with 300 g of soil per pot (the soil was sieved through a 2 mm sieve, mixed with river sand in a volume ratio of 1:1, and then placed in pots with a diameter of 11 cm and a height of 9 cm). After transplanting, each pot was watered with 50 mL of water, and then 30 mL of water was watered every day. After the tomato seedlings survived (about 3 days), they were inoculated with Pseudomonas solanacearum and Bacillus siamensis A-2 bacterial suspensions. The inoculation method was used to inoculate the bacterial suspension into the soil at 20 mL / pot. After inoculation, the tomato seedlings continued to be cultured in the greenhouse, and the management was carried out according to the normal cultivation and management methods of tomatoes. The incidence of bacterial wilt was counted after one week. The specific treatments of the potted test are shown in Table 5:

[0065] Table 5 Treatments of potted plant experiments

[0066]

[0067] The grading standards for bacterial wilt are shown in Table 6:

[0068] Table 6 Grading standards for bacterial wilt

[0069]

[0070] Incidence rate = number of diseased plants / total number of plants investigated × 100%; disease index = [Σ (number of diseased plants at each level × representative value of each level)] / (total number of plants investigated × highest level number) × 100%; T2 relative treatment effect = (T1 disease index - T2 disease index) / T1 disease index × 100%; T3 relative preventive effect = (T1 disease index - T3 disease index) / T1 disease index × 100%.

[0071] The results showed that Figure 5 As shown in Table 7, Siamese Bacillus A-2 has good preventive and therapeutic effects on tomato bacterial wilt, especially the best preventive effect. When the tomato plants were inoculated with Pseudomonas solanacearum 24 hours later and then inoculated with sterile water (T1), the incidence of bacterial wilt in tomato plants reached 100% after 7 days, and the disease index reached 95.56. When the tomato plants were inoculated with Siamese Bacillus A-2 24 hours later (T2), the incidence of tomato bacterial wilt was 20% lower than that of T1, and the disease index decreased by 30.23%, and the relative treatment effect reached 30.23%. As for the preventive effect, when the tomato plants were inoculated with Siamese Bacillus A-2 and then with Pseudomonas solanacearum 24 hours later (T3), the incidence of tomato bacterial wilt was only 40%, which was 60% lower than that of T1, and the disease index was 40, which was 58.14% lower than that of T1, and the relative preventive effect reached 58.14%, which was significantly better than the relative treatment effect of 30.23%.

[0072] Table 7 Control effect of Siamese Bacillus A-2 on tomato bacterial wilt

[0073]

[0074] Example 6: Application of Siamese Bacillus A-2 in Degrading Tetracycline Hydrochloride

[0075] Pure Siamese Bacillus A-2 was picked and inoculated into MSM solid culture medium containing 10 mg / L tetracycline hydrochloride (formula: 1.5 g / L K2HPO4, 0.5 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 1.0 g / L NaCl, 10 g / L bacteriological peptone, 15 g / L agar powder, preparation method: take the above culture medium and add 1000 mL deionized water, stir and heat until completely dissolved, adjust the solution pH to 7.0, divide it into Erlenmeyer flasks, autoclave at 121℃ for 15 min, and after completion, add 100 μL 10 g / L tetracycline hydrochloride solution when the culture medium is cooled to 55℃, mix well and pour into a disposable culture dish for use), and culture at 30℃ in the dark for 3 days, then pick a single colony and inoculate it into LB liquid culture medium (formula: 10 g / L tryptone, 5 g / L yeast extract, 10.0 g / L NaCl, preparation method: take the above culture medium and add 1000 mL of deionized water, stir and heat until completely dissolved, adjust the solution pH to 7.0, divide into triangular bottles, sterilize at 121℃ for 15 min, and then set aside), place in a shaker at 30℃ and 200 r / min for 24 h, inoculate in MSM liquid culture medium containing 10 mg / L tetracycline hydrochloride as the only carbon source (MSM solid culture medium without agar powder) at a volume ratio of 10%, and inoculate the same volume of LB sterilized liquid culture medium as a control, and place in a shaker at 30℃ and 200 r / min to avoid light. After 3 days of cultivation, take a sample to determine the residual concentration of tetracycline hydrochloride in the bacterial liquid. The determination steps are: centrifuge the culture solution in a high-speed refrigerated centrifuge at 4℃ and 8000 r / min for 15 min, take the supernatant and filter it through a 0.22 μm microporous filter membrane into a 1.5 mL brown injection bottle for determination by high performance liquid chromatography (HPLC).

[0076] Liquid chromatography determination conditions: chromatographic column: C18 chromatographic column (5 μm, 4.6 mm×250 mm); mobile phase: phase A 0.01 mol / L oxalic acid, phase C: acetonitrile (in this embodiment, the injection tube is: phase A: 0.01 mol / L oxalic acid, phase B: methanol, phase C: acetonitrile, and only phase A and phase C are used in the liquid chromatography determination process). Gradient elution program: 0-15 min: 90% oxalic acid, 10% acetonitrile; 15-20 min: 75% oxalic acid, 25% acetonitrile; 20-30 min: 0% oxalic acid, 100% acetonitrile. Flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 355 nm, injection volume: 20 μL.

[0077] Method for drawing the standard curve: 100 mg / L standard stock solution is diluted with ultrapure water to a gradient of 5, 10, 20, 50, and 100 mg / L, filtered with a 0.22 μm microporous filter membrane into a 1.5 mL brown injection bottle and determined by high performance liquid chromatography (HPLC). The determination method is the same as above, and the standard curve is drawn with the obtained peak area as the ordinate and the concentration as the abscissa, and the corresponding regression equation is obtained. The concentration of tetracycline hydrochloride in the sample is calculated based on the regression equation.

[0078] The results show that ( Figure 6 ), after 3 days of cultivation, compared with the control group (CK) not inoculated with Siamese Bacillus A-2, the treatment group (A-2) inoculated with Siamese Bacillus A-2 had a significant degradation effect on tetracycline hydrochloride, with a degradation rate of 49.28%. This shows that Siamese Bacillus A-2 has the ability to degrade tetracycline hydrochloride.

[0079] SEQ ID NO.1 (16S rDNA gene sequence of Bacillus siamese A-2)

[0080]

Claims

1. A strain of Bacillus siamensis ( Bacillus siamensis ) A-2, characterized in that The deposit number is GDMCCNO.64228.

2. Use of the siam bacillus A-2 according to claim 1 in any of the following: 1) Control of plant diseases caused by plant pathogenic bacteria and / or plant pathogenic fungi; 2) Prevent and control tomato bacterial wilt; 3) Degradation of tetracycline hydrochloride; 4) Synthesize lipopeptide active substances or polyketide compounds; The plant pathogenic bacteria is Ralstonia syringae subsp. indonesia Ralstonia syzygii subsp. indonesiensis and / or Pseudomonas solanacearum Pseudomonas solanacearum ; The plant pathogenic fungus is Fusarium solani Fusarium solani Phomopsis Phomopsis sp., Fusarium oxysporum Fusarium oxysporum , banana anthracnose Colletotrichum musae , Colletotrichum Colletotrichum gloeosporioides Fusarium fujikura Fusarium fujikuroi Fusarium Fusarium circinatum One or more of; The lipopeptide active substance is plipastatin, fengycin, surfactin or siderophore, and the polyketide compound is bacteriocin, difhizoctonia or butyrosine A / B.

3. The use according to claim 2, characterized in that: The prevention and control of plant diseases caused by plant pathogenic bacteria is carried out by destroying the integrity of the pathogenic bacteria and lysing the pathogenic bacteria; the prevention and control of plant diseases caused by plant pathogenic fungi is carried out by causing the pathogenic fungi hyphae to fold, twist, swell or dissolve to inhibit the growth of pathogens.

4. A biocontrol microbial agent, characterized in that: The biocontrol microbial agent comprises the siam Bacillus A-2 bacteria body according to claim 1.

5. A method for preventing and controlling plant diseases caused by plant pathogenic bacteria or plant pathogenic fungi, characterized in that: The method comprises the following steps: treating plant plants with the siam bacillus A-2 according to claim 1 or the biocontrol microbial agent according to claim 4; The plant pathogenic bacteria is Ralstonia syringae subsp. indonesia Ralstonia syzygii subsp. indonesiensis and / or Pseudomonas solanacearum Pseudomonas solanacearum ; The plant pathogenic fungus is Fusarium solani Fusarium solani Phomopsis Phomopsis sp., Fusarium oxysporum Fusarium oxysporum , banana anthracnose Colletotrichum musae , Colletotrichum Colletotrichum gloeosporioides Fusarium fujikura Fusarium fujikuroi Fusarium Fusarium circinatum One or more of .

6. The method according to claim 5, characterized in that The treatment is to apply the agent containing the Siamese Bacillus A-2 of claim 1 or the biocontrol microbial agent of claim 4 to the root of the plant, wherein the Siamese Bacillus A-2 is OD 600 =1.0~2.0.

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