Microbacterium arbor H17 and application thereof

Abstract

The invention relates to microbacterium arbor H17 and application thereof in preventing and treating plant diseases. The microbacterium arbor is preserved in the China Center for Type Culture Collection, and the preservation number of the microbacterium arbor is CCTCC NO: M 20242129. The microbacterium arbor can effectively inhibit plant pathogenic bacteria Ralstonia solanacearum, can significantly reduce the occurrence of tomato fusarium wilt, can effectively reduce the death rate (reduced from 48% to 4.4%) caused by the fusarium wilt, has biocontrol efficiency of 90%, is harmless to human and livestock, has no pollution to the environment, and can be widely applied to the field of biocontrol. Wide application prospects are realized in the aspect of treating related plant diseases.

Description

Technical Field

[0001] The invention relates to the field of plant disease prevention and control and microbial technology, and relates to a Microbacterium arborescens H17 and an application thereof, in particular to the use for preventing and controlling plant wilt disease caused by Ralstonia solanacearum. Background Art

[0002] Plant diseases refer to abnormal plant growth and development or physiological dysfunction caused by biological factors (such as pathogens, viruses, nematodes, etc.) or non-biological factors (such as environmental stress, nutritional imbalance, etc.). They are one of the main threats to crop growth, leading to reduced crop yields, reduced crop growth quality, and in severe cases, crop death, often causing serious losses to my country's national economy and agricultural production. Among them, diseases caused by pathogens are particularly common and serious. Various pathogens invade plants through different pathways, use plant nutrients to grow and reproduce rapidly, and produce toxins to destroy plant cell structures and metabolic functions, ultimately leading to various plant disease symptoms, such as yellowing and wilting of leaves, and fruit rot.

[0003] Ralstonia solanacearum can cause bacterial wilt in many crops, especially pepper, tomato and eggplant in the Solanaceae family. Ralstonia solanacearum belongs to the Procaryotae kingdom, Proteobacteria, Betaproteobacteria, Burkholderiales, Burkholderiaceae, and Ralstonia genus. It is one of the most harmful soil-borne pathogenic bacteria in the world. The wilt caused by Ralstonia solanacearum is common in the Yangtze River Basin and southern my country, seriously affecting the yield and quality of cash crops. Ralstonia solanacearum has a wide host range and a wide geographical distribution. The bacteria can survive in the soil for a long time without a host. Once the wilt caused by Ralstonia solanacearum breaks out, it is difficult to control, causing large-scale wilting and death of crops or even crop failure.

[0004] The existing prevention and control measures, such as chemical control, grafting and crop rotation, soil improvement and breeding of disease-resistant varieties, have unsatisfactory control effects and pose many risks such as environmental pollution and pathogen resistance. Biological control represented by antagonistic microorganisms is the focus and hotspot of plant disease control research and development because it is environmentally friendly, safe for humans and animals, and not easy to produce resistance. It can effectively reduce the number of diseases, has significant control effects, is inexpensive, and has good economic and social benefits. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a new Microbacterium arborescens H17, which can effectively prevent and control plant wilt disease, especially has a good antagonistic effect on the pathogenic bacteria Ralstonia solanacearum, so as to achieve the purpose of disease prevention and control.

[0006] The present invention is realized by the following technical scheme: a strain of Microbacterium arborense H17, wherein the Microbacterium arborense H17 is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20242129.

[0007] Compared with the prior art, the present invention provides a new Microbacterium arborense H17, which can effectively inhibit the wilt pathogen Lawrence solanacearum, and the diameter of the inhibition zone of 10 μL fermentation supernatant reaches 19 mm. When used in tomato planting, it can effectively reduce the mortality rate caused by wilt (from 48% to 4.4%), the biological control efficiency reaches 90%, and the yield of tomatoes is increased. The Microbacterium arborense H17 of the present invention has broad application prospects in preventing and controlling plant diseases.

[0008] Furthermore, the 16S rDNA nucleotide sequence of the Microbacterium arborescentis H17 contains the DNA molecule shown in SEQ ID NO: 3, and / or the nucleotide sequence of the gyrB gene of the Microbacterium arborescentis H17 is shown in SEQ ID NO: 6.

[0009] The present invention also provides the use of the Microbacterium arborescens H17 in preventing and controlling plant diseases.

[0010] The present invention also provides the use of the above-mentioned Microbacterium arborescentis H17 in preparing biological preparations for preventing and controlling plant diseases.

[0011] Furthermore, the plant disease includes wilt caused by Ralstonia solanacearum.

[0012] In some embodiments, the plant is tomato.

[0013] The present invention also provides the use of the Microbacterium arborescens H17 in the preparation of antagonistic Ralstonia solanacearum.

[0014] The present invention also provides a method for preventing and controlling plant wilt disease, comprising the following steps: when the plants are planted, 20 to 30 mL of Microbacterium arborense H17 bacterial solution is applied to each plant; during the growth period of the plants, 10 to 20 mL of Microbacterium arborense bacterial solution is applied to each plant every 7 to 10 days, and the application is continued for 3 to 5 times.

[0015] Furthermore, the concentration of Microbacterium arborescens H17 in the bacterial solution is 1.0×10 5cfu / mL~1.0×10 7 cfu / mL.

[0016] The present invention also provides a method for preparing the above-mentioned Microbacterium arborense H17 bacterial liquid, comprising the following steps: inoculating the Microbacterium arborense H17 described in claim 1 into a liquid culture medium, fermenting and culturing at 30°C to 37°C for 18 to 30 hours to obtain the Microbacterium arborense H17 bacterial liquid.

[0017] Furthermore, the Microbacterium arborescens H17 described in the present invention has been deposited in the China Center for Type Culture Collection (abbreviated as: CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province, China) on September 30, 2024, with a deposit date of September 30, 2024 and a deposit number of CCTCC NO: M20242129.

[0018] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology diagram of Microbacterium arborescentis H17 of the present invention cultured on NA medium for 24 hours;

[0020] Figure 2 The agarose gel electrophoresis diagram of the PCR amplification of the 16S rDNA sequence of Microbacterium arborescens H17 of the present invention;

[0021] Figure 3 A phylogenetic tree constructed from the 16S rDNA sequences of Microbacterium arborescentis H17 and similar species of bacteria;

[0022] Figure 4 This is an agarose gel electrophoresis diagram of the PCR amplification of the gyrB gene sequence of Microbacterium arborescentis H17 of the present invention;

[0023] Figure 5 A phylogenetic tree constructed from the gyrB gene sequences of Microbacterium arborescentis H17 and similar species of bacteria;

[0024] Figure 6 The diagram is a diagram of the antibacterial effect of Microbacterium arborescens H17 on Ralstonia solanacearum; wherein, Figure A: Ralstonia solanacearum + 10 μL of Microbacterium arborescens H17 fermentation supernatant; Figure B: Ralstonia solanacearum + 10 μL of 5 mg / mL ampicillin (positive control); Figure C: Ralstonia solanacearum + 10 μL of sterile water (blank control);

[0025] Figure 7The figure is a control effect diagram of the bacterial liquid of Microbacterium arborense H17 of the present invention on tomato wilt; wherein, the experimental group was applied with Microbacterium arborense H17, and the control group was a blank control group applied with water. DETAILED DESCRIPTION

[0026] In the field of green and healthy crop cultivation and disease prevention and control, biological control using beneficial microorganisms or their metabolites to antagonize pathogens has become a new way to control plant diseases. Antagonistic microorganisms include bacteria and fungi, and the protection they provide to plants includes a variety of mechanisms, including competing with pathogens for niches and nutrients, producing secondary metabolites that inhibit pathogens, secreting lysozymes, inhibiting pathogen virulence, and inducing plant resistance. The revelation of the relationship between different kingdoms of microorganisms provides a macroscopic perspective of microecology for plant protection.

[0027] Furthermore, mangroves are a woody plant community located at the junction of tropical and subtropical areas. They are the transitional area between the ocean and land ecosystems. Their unique geographical environment leads to a rich variety of microorganisms with diverse functions, and they are a valuable resource library for mining a variety of functional microorganisms including antagonistic microorganisms. Therefore, the present invention intends to mine antagonistic bacteria including pathogens such as Lawrence Livermore National Laboratory from mangrove ecosystems to effectively prevent and control the occurrence of various plant wilt diseases and solve the serious problem of plant disease transmission.

[0028] By exploring the mangrove ecosystem, the present invention provides a new antagonistic strain of Ralstonia solanacearum and a biological control method for preventing and controlling Ralstonia solanacearum and the plant wilt disease caused by it. Specifically, the present invention uses fermentation supernatants of different strains to conduct antibacterial experiments on Ralstonia solanacearum, and finally finds that the fermentation supernatant of Microbacterium arborescens H17 has a good antibacterial effect on Ralstonia solanacearum, and there is currently no report on the antibacterial effect of Microbacterium arborescens on Ralstonia solanacearum at home and abroad. Based on the above research, the present invention provides a new pollution-free and pollution-free biocontrol agent for the prevention and control of Ralstonia solanacearum.

[0029] The present invention will be further described in detail below in conjunction with specific examples, which are only used to illustrate the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0030] The Microbacterium arborescens H17 described in the present invention has been deposited in the China Center for Type Culture Collection (abbreviated as: CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province, China) on September 30, 2024, with a deposit date of September 30, 2024 and a deposit number of CCTCC NO: M 20242129.

[0031] The present invention is further described in detail below with reference to specific embodiments.

[0032] Example 1: Isolation and purification of Microbacterium arborescens H17

[0033] The separation and purification method of the Microbacterium arborescens H17 strain of the present invention comprises the following steps:

[0034] S101. Diluting the soil sample: Specifically, weighing 1 g of soil sample, the soil sample is from the mangrove soil of Hailing Island Mangrove National Wetland Park in Guangdong Province; adding 10 mL of sterile water to the soil sample, and then placing it on an oscillator to oscillate for 60 min, so that the soil sample is evenly dispersed in the sterile water to form a soil suspension; after the soil is dispersed, 100 μL of the soil suspension is drawn into 900 μL of sterile water to obtain a 10-fold dilution, and then diluting it 10 times in sequence to obtain 10 2 Dilution, 10 3 Dilution, 10 4 Dilution, 10 5 times dilution and 10 6 The whole dilution process was carried out in an ultra-clean workbench.

[0035] S102, strain culture: take 100 μL of 10-fold dilution, 10 2 Dilution, 10 3 Dilution, 10 4 Dilution, 10 5 times dilution and 10 6 The 100-fold dilutions were spread on NA agar solid culture plates, and then the culture plates were placed in an incubator at 37°C for 24 days, and bacterial plaques grew on the culture plates.

[0036] S103, strain screening: After the culture is completed, single plaques of different shapes, colors, sizes, etc. are picked one by one from appropriate dilution gradient plates according to the growth of the colonies, and the plates are streaked to isolate the Microbacterium arborescens H17 of the present invention.

[0037] S104, Isolation and purification of Microbacterium arborense H17 strain: The purified and cultured Microbacterium arborense H17 strain was picked from the NA plate, inoculated into a 500 mL conical flask containing 200 mL of Microbacterium arborense H17 liquid culture medium, and cultured for 24 hours at 37°C and 220 rpm constant temperature shaking incubator to obtain seed solution. The seed solution and glycerol were placed in a tube at a volume ratio of 1:4 and mixed to obtain a concentration of 1.0×10 5 ~5.0×10 6 cfu / mL glycerol seed solution and store at -20°C or -80°C for long-term storage.

[0038] The morphology of the Microbacterium arborescens H17 strain isolated in this example after being cultured on a NA agar solid culture plate for 24 hours is shown in the figure below. Figure 1 As shown, the colonies are yellow and irregular in shape, with lighter and rougher edges, softer texture in the middle and a smooth surface.

[0039] Among them, the preparation method of NA solid culture medium is as follows: weigh 10g of peptone, 3g of beef extract, 5.0g of sodium chloride, and 15.0g of agar powder, stir and dissolve each component in sterile water, add water to make up to 1L and adjust the pH to 7.2-7.4, and then divide it into conical flasks, seal the conical flasks with sterilization paper, place them in a sterilizer for 121°C, 101kPa high-pressure steam sterilization for 20min, wait for the temperature of the sterilizer to drop below 70°C and the pressure to return to 0kPa when it is taken out, pour it into the culture dish on the clean bench, pour about 20mL of culture medium into each culture dish, and after cooling and solidification, store the solid culture plate at 4°C.

[0040] The preparation method of NA liquid culture medium is as follows: weigh 10g of peptone, 3g of beef extract, and 5.0g of sodium chloride, stir and dissolve each component in sterile water, add water to make up to 1L and adjust the pH to 7.2-7.4, then divide it into conical flasks, seal the conical flasks with sterilization paper, place them in an autoclave and sterilize them with high-pressure steam at 121°C and 101kPa for 20min, take them out when the temperature of the autoclave drops below 70°C and the pressure returns to 0kPa, and store them for later use.

[0041] Example 2: Cultivation method of Microbacterium arborescens H17 and preparation of fermentation supernatant

[0042] This example is about the cultivation of strain Microbacterium arborescens H17 and the preparation of fermentation supernatant, and the specific steps are as follows:

[0043] S201: Use a sterile inoculation loop to streak the Microbacterium arborescentis H17 glycerol strain on NA solid culture medium, culture at 37°C for 24 hours, and observe its colony morphology.

[0044] S202: Take a single colony of Microbacterium arborense H17 and inoculate it into 100 mL NA liquid culture medium, and culture it at 37°C and 220 rpm for 24 hours to obtain a seed solution; inoculate the seed solution into 250 mL NA liquid culture medium at a ratio of 3%, and culture it at 37°C and 220 rpm for 24 hours to obtain a bacterial solution of Microbacterium arborense H17. Centrifuge the bacterial solution at 4°C and 8000 rpm for 10 minutes, remove the precipitate and collect the supernatant to obtain a fermentation supernatant of Microbacterium arborense H17.

[0045] Example 3: PCR amplification and sequencing of 16S rDNA sequence of Microbacterium arborescens H17

[0046] This example is to PCR amplify the 16S rDNA sequence of Microbacterium arborescens H17 and sequence it, including the following steps:

[0047] S301: Extract the genomic DNA of Microbacterium arborescentis H17.

[0048] Specifically, the Omega Bacterial DNA Kit (D3350-01) was used to extract genomic DNA: first, 2 mL of the seed solution described in Example 2 was taken into a sterile 2 mL centrifuge tube, centrifuged at 12000 rpm for 2 min, the supernatant was discarded and the precipitate was retained; then, 100 μL of 1×TE Buffer was added to the precipitate, vortexed and mixed, 10 μL of lysozyme was added and mixed, and the mixture was incubated at 37° C. for 10 min; 100 μL of BTL Buffer and 20 μL of proteinase K were added, mixed, and incubated at 55° C. for 1 h, with oscillation and mixing three times in between; 5 μL of RNase A enzyme was added, mixed, and allowed to stand at room temperature for 5 min, then centrifuged at 10000 rpm for 2 min, and 200 μL of supernatant was transferred to a new sterile 1.5 mL centrifuge tube; 200 μL of BTL Buffer, mix well, and place in a 65℃ warm bath for 10min; add 200μL anhydrous ethanol, vortex mix well, transfer all the samples to the adsorption column, centrifuge at 10000rpm for 2min, discard the supernatant and the adsorption column, and place the adsorption column in a new collection tube; add 500μL HBC Buffer to the adsorption column, centrifuge at 10000rpm for 2min, and discard the supernatant; add 700μL DNA Wash Buffer to the adsorption column, centrifuge at 10000rpm for 2min, discard the supernatant, and repeat twice; put the empty adsorption column back into the collection tube, centrifuge at 10000rpm for 2min; add 30-50μL Elution Buffer (preheated at 65℃) to the adsorption column to dissolve the DNA precipitate, and obtain genomic DNA, which is stored at -20℃ for later use.

[0049] S302: PCR amplification of the 16S rDNA sequence of Microbacterium arborescentis H17.

[0050] Specifically, PCR amplification was performed using the genomic DNA obtained in step S301 as a template and Eubac27F and Eubac1492R as primers. The PCR reaction system (50 μL) included: 0.5 μL TaKaRa LA Taq (5 U / μL), 5 μL 10× LA TaqBuffer II (Mg 2+ Plus), 8 μL dNTPs Mixture (2.5 mM Each), 2 μL genomic DNA, 1 μL upstream primer Eubac27F (10 μM), 1 μL downstream primer Eubac1492R (10 μM) and 32.5 μL ddH2O. The sequence of the upstream primer Eubac27F is: 5'-agagtttgat cctggctcag-3' (SEQ ID NO: 1). The sequence of the downstream primer Eubac1492R is: 5'-ggttaccttg ttacgactt-3' (SEQ ID NO: 2).

[0051] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The process of denaturation, annealing, and extension was repeated 30 times; extension was performed at 72°C for another 10 min, and the PCR amplification product was stored at 4°C.

[0052] S303: Perform nucleic acid electrophoresis on the PCR product. Take 5 μL of the PCR product obtained in step S302 and spot it, then perform nucleic acid electrophoresis at 120V for 25 minutes. The electrophoresis results are as follows: Figure 2 As shown, the 16S rDNA fragment band amplified using the genomic DNA of Microbacterium arborescentis H17 obtained in Example 1 of the present invention as a template is single and has high brightness, and the length of the 16S rDNA sequence is about 1400 bp.

[0053] S304: 16S rDNA sequence sequencing. After the PCR product obtained in step S302 was purified and recovered, 30 μL of the purified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results showed that the 16S rDNA sequence of Microbacterium arborescens H17 of the present invention was 1390 bp in length, and the specific sequence was shown in SEQ ID NO: 3.

[0054] The 16S rDNA sequence of Microbacterium arborense H17 obtained by sequencing was input into NCBI for BLAST comparison. The phylogenetic tree of Microbacterium arborense H17 was constructed using the neighbor-joining method in MEGA11. The results are shown in Figure 2. Figure 3As shown, according to BLAST homology analysis and phylogenetic evolutionary tree, the homology between the Microbacterium arborescens H17 obtained by the present invention and the Microbacterium arborescens strain N30 (MN555371.1) reaches 100%. Therefore, it can be known that the strain of the present invention belongs to Microbacterium arborescens in classification, and the present invention names it Microbacterium arborescens H17.

[0055] Example 4: PCR amplification and sequencing of the gyrB gene of Microbacterium arborescentis H17

[0056] This example is to amplify the gyrB gene sequence of Microbacterium arborescentis H17 by PCR and sequence it.

[0057] S401: PCR amplification of gyrB gene sequence. The genomic DNA obtained in step S201 in Example 3 was used as a template, and the degenerate primers UP-1 and UP-2r were used as primers for PCR amplification. The PCR reaction system (50 μL) was as follows: 0.5 μL TaKaRa LATaq (5 U / μL), 5 μL 10×LA Taq Buffer II (Mg 2+ Plus), 8 μL dNTPs Mixture (2.5 mM Each), 2 μL genomic DNA, 1 μL upstream primer UP-1 (10 μM), 1 μL downstream primer UP-2r (10 μM) and 32.5 μL ddH2O. The sequence of the upstream primer UP-1 is: 5'-gaagtcatcatgaccgttctgcaygcnggnggnaarttyga-3' (SEQ ID NO: 4), and the sequence of the downstream primer UP-2r is: 5'-agcagggtacggatgtgcgagccrtcnacrtcngcrtcngtcat-3' (SEQ ID NO: 5).

[0058] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min. The process of denaturation, annealing, and extension was repeated 30 times; extension was performed at 72°C for another 10 min, and the PCR amplification product was stored at 4°C.

[0059] S402: Electrophoresis of PCR products. Take 5 μL of the PCR products obtained in step S302 and spot them and perform electrophoresis of nucleic acids at 120V for 25 minutes. The electrophoresis results are as follows: Figure 4 As shown, the gyrB gene fragment amplified using the genomic DNA of Microbacterium arborescentis H17 as a template in Example 1 of the present invention has a single band with high brightness, and the gyrB sequence length is about 1700 bp.

[0060] S403: Sequencing of gyrB gene sequence. After the PCR product obtained in step S301 was purified and recovered, 30 μL of the purified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results showed that the gyrB sequence of Microbacterium arborescens H17 of the present invention was 1774 bp in length, and the specific sequence was shown in SEQ ID NO: 6.

[0061] The sequence of the gyrB gene of Microbacterium arborescens H17 obtained by sequencing was input into NCBI for BLAST comparison. BLAST showed that the homology between its gyrB gene and the corresponding sequence of Microbacterium arborescens CP128474.1 was 90.80%. The phylogenetic tree of Microbacterium arborescens H17 was constructed using the neighbor-joining method in MEGA11. The results are as follows: Figure 5 As shown, according to the phylogenetic tree, the Microbacterium arborescens H17 obtained by the present invention is clustered with the Microbacterium arborescens strain (SSF12). Therefore, it can be known that the strain of the present invention belongs to Microbacterium arborescens in classification, but it is a new strain, so the present invention names it as Microbacterium arborescens H17.

[0062] Example 5: Antibacterial activity of the fermentation supernatant of Microbacterium arborescens H17 against Ralstonia solanacearum

[0063] This example studies the antibacterial activity of the fermentation supernatant of Microbacterium arborescens H17 against Ralstonia solanacearum, comprising the following steps:

[0064] Through the plate confrontation experiment, 50μL of Ralstonia solanacearum liquid was spread on LB+glucose solid medium, and filter paper was evenly placed on the plate. One of the filter paper was added with 10μL of arborescent Microbacterium H17 fermentation supernatant, and 10μL of sterile water and 10μL of 5mg / mL ampicillin were used as blank control and positive control, respectively, and different filter paper were added. The plate was placed in a 37℃ incubator for 24h to observe whether an inhibition zone was produced and measure the diameter of the inhibition zone. The experiment was repeated 3 times.

[0065] The results are as follows Figure 6As shown, Microbacterium arborense H17 has a significant antibacterial effect on Ralstonia solanacearum, with an average diameter of the inhibition zone of 19 mm, and its antibacterial ability is about 90% of that of 5 mg / mL ampicillin (inhibition zone diameter 21 mm). This result shows that Microbacterium arborense H17 has a highly effective function of inhibiting Ralstonia solanacearum. Since Ralstonia solanacearum can cause the occurrence of more than 200 plant wilt diseases including important crops such as tomatoes, potatoes, peppers and tobacco, Microbacterium arborense H17 can be used to prevent and control plant wilt diseases caused by Ralstonia solanacearum, and can be used to prepare biological agents for preventing and controlling plant wilt diseases.

[0066] Among them, the preparation method of LB+glucose solid culture medium is as follows: weigh 10g of tryptone, 5g of yeast powder, 7g of NaCl, 5g of glucose, and 15g of agar powder, stir and dissolve each component in sterile water, add water to make up to 1L and adjust the pH to 7.2-7.4, and then divide it into conical flasks, seal the conical flasks with sterilization paper, and place them in a sterilizer for 20 minutes at 121°C and 101kPa high-pressure steam sterilization. When the temperature of the sterilizer drops below 70°C and the pressure returns to 0kPa, take it out and pour it into a culture dish on the clean bench. Pour about 20mL of culture medium into each culture dish. After cooling and solidification, store the solid culture plate at 4°C. Example 6: The application effect of Microbacterium arborescens H17 in tomato planting affected by Ralstonia solanacearum

[0067] This example studies the application effect of Microbacterium arborescens H17 in tomato cultivation affected by Ralstonia solanacearum.

[0068] The experiment was conducted in a tomato greenhouse at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. Due to continuous cropping in the greenhouse, tomato wilt caused by Ralstonia solanacearum was seriously ill, and the death rate of tomatoes due to wilt was more than 53% last season.

[0069] The experimental group included 3 rows, with 100 tomato seedlings in each row. The specific experimental process was as follows: the three-leaf stage tomato seedlings were transplanted into the greenhouse for planting. Each seedling was irrigated with 20-30 mL of Microbacterium arborescens H17 solution during planting. During the growth period of the plants, each tomato seedling was drip-irrigated with 10-20 mL of Microbacterium arborescens H17 solution every 10 days for a total of 3 times, with a solution concentration of 1.0×10 5 cfu / mL~1.0×10 7 cfu / mL. The number of surviving tomatoes was counted during the fruit-bearing period, and the survival rate was calculated.

[0070] The blank control group included 3 rows, with 100 tomato seedlings in each row. The blank control group used water instead of the bacterial solution of Microbacterium arborescens H17. The other conditions were the same as those of the experimental group and were carried out in parallel with the experimental group.

[0071] Among them, the mortality rate = the number of dead plants / the total number of plants × 100%, and the survival rate = the number of surviving plants / the total number of plants × 100%.

[0072] Biological control efficiency: (number of dead plants in the control group - number of dead plants in the experimental group) / number of dead plants in the control group × 100%.

[0073] The results are as follows Figure 7 As shown, 287 of the 300 tomatoes in the experimental group finally survived, with a survival rate of 95.6% and a death rate of 4.4%. 156 tomatoes in the blank control group survived, with a survival rate of 52% and a death rate of 48%. The use of the Microbacterium arborescens H17 bacterial solution of the present invention significantly increased the survival rate of tomatoes and reduced the mortality rate of tomatoes, and its biological control efficiency reached 90%.

[0074] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0075] (1) Microbacterium arborense H17 in the present invention has a good antibacterial effect on Ralstonia solanacearum, while in the use of conventional chemical pesticides, high concentrations of chemical reagents need to be applied to achieve good prevention effects and are prone to environmental pollution and other problems. Therefore, Microbacterium arborense H17 has the potential to inhibit various plant diseases caused by Ralstonia solanacearum and can be used to prepare drugs for preventing and controlling various plant diseases caused by Ralstonia solanacearum.

[0076] (2) The present invention first discovered that the fermentation supernatant of Microbacterium arborense H17 has a good inhibitory effect on Ralstonia solanacearum, which causes tomato wilt. In the antibacterial activity experiment, the antibacterial diameter of 10 μL of the fermentation supernatant of Microbacterium arborense H17 on Ralstonia solanacearum is 19 mm, and the antibacterial effect is good. During the plant cultivation process, the long-term interval application of Microbacterium arborense bacterial liquid has a biological control efficiency of 90% against wilt disease, which significantly inhibits the occurrence of the disease.

[0077] (3) Microbacterium arborescentis H17 is harmless to the environment and humans and animals, which provides a new type of safe biocontrol strain for the prevention and control of Ralstonia solanacearum and the plant diseases it causes.

[0078] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A strain of Microbacterium arborescens H17, characterized in that The Microbacterium arborescentis is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO:M 20242129.

2. The Microbacterium arborescentis H17 according to claim 1, characterized in that The 16S rDNA nucleotide sequence of the Microbacterium arborescentis H17 contains the DNA molecule shown in SEQ ID NO: 3, and / or the nucleotide sequence of the gyrB gene of the Microbacterium arborescentis H17 is shown in SEQ ID NO:

6.

3. Use of Microbacterium arborescentis H17 according to claim 1 in preventing and controlling plant diseases.

4. Use of Microbacterium arborescentis H17 as claimed in claim 1 in the preparation of biological agents for preventing and controlling plant diseases.

5. The use according to claim 3 or 4, characterized in that: The plant diseases include wilt caused by Ralstonia solanacearum.

6. Use of Microbacterium arborescens H17 as claimed in claim 1 in the preparation of biological preparations antagonizing Lawrella solanacearum.

7. A biological agent, characterized in that: The active ingredient of the biological preparation includes the Microbacterium arborescentis H17 as claimed in claim 1.

8. A method for preventing and treating plant wilt disease, characterized in that: The method comprises the following steps: when the plants are planted, 20 to 30 mL of Microbacterium arborescens H17 bacterial solution is applied to each plant; during the growth period of the plants, 10 to 20 mL of Microbacterium arborescens bacterial solution is applied to each plant every 7 to 10 days, and the application is continued for 3 to 5 times.

9. The control method according to claim 8, characterized in that: The concentration of Microbacterium arborescens H17 in the bacterial solution was 1.0×10 5 cfu / mL~1.0×10 7 cfu / mL.

10. The method for preparing the Microbacterium arborescentis H17 bacterial solution according to claim 8, characterized in that: The method comprises the following steps: inoculating the Microbacterium arborense H17 described in claim 1 into a liquid culture medium, and fermenting and culturing at 30° C. to 37° C. for 18 to 30 hours to obtain a bacterial liquid of Microbacterium arborense H17.

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