A strain of Microbacterium tumefaciens H17 and its application

Abstract

This invention relates to a strain of *Microbacterium argentea* H17 and its application in the control of plant diseases. The *Microbacterium argentea* strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20242129. This *Microbacterium argentea* strain effectively inhibits the plant pathogen *Ralstonia solanacearum*, significantly reducing the incidence of tomato wilt disease. It effectively reduces the mortality rate caused by wilt disease (from 48% to 4.4%), achieving a biocontrol efficiency of 90%. Furthermore, it is harmless to humans and animals and causes no environmental pollution, showing broad application prospects in the control of related plant diseases.

Description

Technical Field

[0001] This invention relates to the fields of plant disease control and microbial technology, and to a strain of arborescent microbacterium H17 and its application, especially for the control of plant wilt caused by Ralstonia solanacearum. Background Technology

[0002] Plant diseases refer to abnormal plant growth and development or physiological dysfunction caused by biotic factors (such as pathogens, viruses, nematodes, etc.) or abiotic factors (such as environmental stress, nutrient imbalance, etc.). They are one of the main threats to crop growth, leading to reduced crop yield, decreased crop quality, and in severe cases, crop death, often causing serious losses to my country's national economy and agricultural production. Among these, diseases caused by pathogens are particularly widespread and harmful. Various pathogens invade plants through different pathways, utilizing plant nutrients for rapid growth and reproduction, while simultaneously producing toxins that damage plant cell structure and metabolic functions, ultimately leading to various disease symptoms such as yellowing and wilting leaves, and fruit rot.

[0003] Ralstonia solanacearum causes bacterial wilt in various crops, particularly damaging peppers, tomatoes, and eggplants belonging to the Solanaceae family. It belongs to the kingdom Procaryotae, phylum Proteobacteria, class Betaproteobacteria, order Burkholderiales, family Burkholderiaceae, and genus Ralstonia, and is one of the most damaging soil-borne pathogens worldwide. Fusarium wilt caused by Ralstonia solanacearum is widespread in the Yangtze River basin and southern regions of my country, severely impacting the yield and quality of economic crops. Ralstonia solanacearum has a wide host range and geographical distribution, and can survive in soil for extended periods without a host. Once an outbreak occurs, Ralstonia solanacearum wilt is difficult to control, causing widespread wilting and death of crops, and even total crop failure.

[0004] Existing control measures such as chemical control, grafting and crop rotation, soil improvement, and the cultivation of disease-resistant varieties have not been very effective and pose many risks, including environmental pollution and the development of pathogen resistance. In contrast, biological control, represented by antagonistic microorganisms, is environmentally friendly, safe for humans and animals, less likely to induce resistance, and can effectively reduce disease numbers, achieve significant control effects, and is inexpensive, offering good economic and social benefits. Therefore, it is currently a key focus and hot topic in plant disease control research and development. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a new Microbacterium arborescens H17, which can effectively prevent and control plant wilt disease, especially showing good antagonistic effect against the pathogen Ralstonia solanacearum, thus achieving the purpose of disease control.

[0006] The present invention is achieved through the following technical solution: a strain of Microbacterium arborescens H17, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 20242129.

[0007] Compared to existing technologies, this invention provides a novel *Microbacterium arborescens* H17, which effectively inhibits *Ralstonia solanacearum*, the pathogen of Fusarium wilt, with an inhibition zone diameter of 19 mm in 10 μL of fermentation supernatant. When used in tomato cultivation, it effectively reduces the mortality rate of Fusarium wilt (from 48% to 4.4%), achieving a biocontrol efficiency of 90% and increasing tomato yield. This *Microbacterium arborescens* H17 has broad application prospects in the control of plant diseases.

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

[0009] This invention also provides the application of the above-mentioned *Microbacterium arborescens* H17 in the prevention and control of plant diseases.

[0010] The present invention also provides the application of the above-mentioned *Microbacterium arborescens* H17 in the preparation of biological agents for the prevention and control of plant diseases.

[0011] Furthermore, the plant diseases mentioned include wilt caused by Ralstonia solanacearum.

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

[0013] The present invention also provides the application of the above-mentioned *Microbacterium arborescens* H17 in the preparation of antagonistic *Ralstonia solanacearum*.

[0014] The present invention also provides a method for the prevention and control of plant wilt disease, comprising the following steps: when planting the plants, apply 20-30 mL of Bacillus argentis H17 bacterial solution to each plant; during the plant growth period, apply 10-20 mL of Bacillus argentis bacterial solution to each plant every 7-10 days, for a total of 3-5 applications.

[0015] Furthermore, the concentration of *Microbacterium arborescens* H17 in the bacterial solution is 1.0 × 10⁻⁶. 5cfu / mL~1.0×10 7 cfu / mL.

[0016] The present invention also provides a method for preparing the above-mentioned *Microbacterium argentea* H17 bacterial suspension, comprising the following steps: inoculating the *Microbacterium argentea* H17 of claim 1 into a liquid culture medium, fermenting and culturing at 30℃~37℃ for 18~30 hours to obtain *Microbacterium argentea* H17 bacterial suspension.

[0017] Furthermore, the *Microbacterium arborescens* H17 described in this invention was deposited on September 30, 2024, at the China Center for Type Culture Collection (CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province, China), with accession number CCTCC NO: M20242129.

[0018] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of the *Bacillus arborescens* H17 cultured on NA medium for 24 hours according to the present invention.

[0020] Figure 2 This is an agarose gel electrophoresis image of the 16S rDNA sequence of *Microbacterium arboresense* H17 described in this invention, amplified by PCR.

[0021] Figure 3 A phylogenetic tree constructed from the 16S rDNA sequences of *Microbacterium arboresense* H17 and similar species described in this invention;

[0022] Figure 4 This is an agarose gel electrophoresis image of the gyrB gene sequence of *Microbacterium arboresense* H17 described in this invention, amplified by PCR.

[0023] Figure 5 A phylogenetic tree constructed from the gyrB gene sequences of *Microbacterium arboresense* H17 and similar species described in this invention;

[0024] Figure 6 The figures show the antibacterial effect of *Ralstonia solanacearum* H17 against *Ralstonia solanacearum*. Figure A shows *Ralstonia solanacearum* fermentation supernatant with 10 μL of *Ralstonia solanacearum* H17; Figure B shows *Ralstonia solanacearum* with 10 μL of 5 mg / mL ampicillin (positive control); Figure C shows *Ralstonia solanacearum* with 10 μL of sterile water (blank control).

[0025] Figure 7The diagram shows the control effect of the *Microbacterium argentea* H17 bacterial solution on tomato wilt disease as described in this invention; the experimental group was treated with *Microbacterium argentea* H17, and the control group was a blank control group treated with water. Detailed Implementation

[0026] In the fields of green and healthy crop cultivation and disease control, biocontrol utilizing the antagonistic effects of beneficial microorganisms or their metabolites against pathogens has become a new approach to controlling plant diseases. Antagonistic microorganisms, including bacteria and fungi, provide protection to plants through multiple mechanisms, such as competing with pathogens for ecological niches and nutrients, producing secondary metabolites that inhibit pathogens, secreting lysozymes, suppressing pathogen virulence, and inducing plant resistance. The elucidation of relationships between different microbial kingdoms provides a macro-ecological perspective for plant protection.

[0027] Furthermore, mangroves are woody plant communities located at the boundary between tropical and subtropical zones, serving as a transitional area between marine and terrestrial ecosystems. Their unique geographical environment results in a rich diversity of microorganisms with varied functions, making them a valuable resource for exploring various functional microorganisms, including antagonistic microorganisms. Therefore, this invention aims to discover antagonistic microbial agents from mangrove ecosystems, including those targeting pathogens such as *Ralstonia solanacearum*, to effectively control the occurrence of various plant wilt diseases and address the serious problem of plant disease transmission.

[0028] By exploring the mangrove ecosystem, this invention provides a novel antagonistic strain of *Ralstonia solanacearum* and a biological control method for controlling *Ralstonia solanacearum* and the plant wilt disease it causes. Specifically, this invention uses fermentation supernatants of different strains to conduct antibacterial experiments against *Ralstonia solanacearum*. The results showed that the fermentation supernatant of *Microbacterium argentea* H17 has a good antibacterial effect against *Ralstonia solanacearum*, and currently there are no reports on the antibacterial effect of *Microbacterium argentea* against *Ralstonia solanacearum*, either domestically or internationally. Based on the above research, this invention provides a new pollution-free and harmless biocontrol agent for the control of *Ralstonia solanacearum*.

[0029] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0030] The *Microbacterium arborescens* H17 described in this invention was deposited on September 30, 2024, at the China Center for Type Culture Collection (CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province, China), with accession number CCTCC NO: M 20242129.

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

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

[0033] The method for isolating and purifying the *Microbacterium arvense* H17 strain described in this invention comprises the following steps:

[0034] S101. Dilution of soil samples: Specifically, weigh 1g of soil sample, which is mangrove soil from the Hailing Island Mangrove National Wetland Park in Guangdong Province; add 10mL of sterile water to the soil sample, and then place it on a shaker and shake for 60min to evenly disperse the soil sample in the sterile water to form a soil suspension; after the soil is dispersed, take 100μL of the soil suspension into 900μL of sterile water to obtain a 10-fold dilution, and then dilute it 10 times in sequence to obtain a 10-fold dilution. 2 10 times dilution solution 3 10 times dilution solution 4 10 times dilution solution 5 10 times dilution and 10 6 The dilution solution was diluted 10 times, and the entire dilution process was carried out in a clean bench.

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

[0036] S103. Strain screening: After the culture is completed, single plaques of different shapes, colors and sizes are picked from appropriate dilution gradient plates according to the colony growth and streaked to isolate the *Bacillus arborescens* H17 described in this invention.

[0037] S104. Isolation and purification of *Microbacterium argentea* strain H17: The purified *Microbacterium argentea* strain H17 was picked from NA plates and inoculated into a 500 mL Erlenmeyer flask containing 200 mL of *Microbacterium argentea* H17 liquid culture medium. The flask was cultured at 37℃ and 220 rpm for 24 h to obtain a seed culture. The seed culture was then mixed with glycerol at a volume ratio of 1:4 to obtain a concentration of 1.0 × 10⁻⁶. 5 ~5.0×10 6 The glycerol seed culture at cfu / mL should be stored at -20℃ or for long-term storage at -80℃.

[0038] The morphological image of the *Microbacterium argentea* H17 strain isolated in this embodiment after 24 hours of culture on NA agar solid culture plates is shown below. Figure 1 As shown, the colonies are irregularly shaped and yellow, with light-colored and rough edges, and a soft and smooth center.

[0039] 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. Dissolve each component in sterile water by stirring. Add water to make up to 1L and adjust the pH to 7.2-7.4. Then dispense the solution into Erlenmeyer flasks. Seal the Erlenmeyer flasks with sterile paper and place them in an autoclave at 121℃ and 101kPa for 20min. When the temperature of the autoclave drops below 70℃ and the pressure returns to 0kPa, remove the flasks and pour them into petri dishes on a clean bench. Pour about 20mL of culture medium into each petri dish. After cooling and solidification, store the solid culture plates at 4℃.

[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. Dissolve each component in sterile water by stirring. Add water to make up to 1L and adjust the pH to 7.2-7.4. Then dispense the solution into Erlenmeyer flasks. Seal the Erlenmeyer flasks with sterile paper and place them in an autoclave at 121℃ and 101kPa for 20min. When the temperature of the autoclave drops below 70℃ and the pressure returns to 0kPa, remove the flasks and store them for later use.

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

[0042] This example describes the culture of strain *Microbacterium arborescens* H17 and the preparation of its fermentation supernatant. The specific steps are as follows:

[0043] S201: Using a sterile inoculation loop, streak *Microbacterium arborescens* H17 glycerol bacteria onto NA solid medium and incubate at 37°C for 24 hours. Observe the colony morphology.

[0044] S202: A single colony of *Microbacterium argentea* H17 was inoculated into 100 mL of NA liquid medium and cultured at 37℃ and 220 rpm for 24 h to obtain a seed culture. The seed culture was then inoculated into 250 mL of NA liquid medium at a ratio of 3% and cultured at 37℃ and 220 rpm for 24 h to obtain *Microbacterium argentea* H17 bacterial suspension. The bacterial suspension was centrifuged at 4℃ and 8000 rpm for 10 min, the precipitate was removed, and the supernatant was collected to obtain the fermentation supernatant of *Microbacterium argentea* H17.

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

[0046] This embodiment describes the PCR amplification and sequencing of the 16S rDNA sequence of *Microbacterium arboresense* H17, including the following steps:

[0047] S301: Extract genomic DNA from Microbacterium arborescens H17.

[0048] Specifically, genomic DNA was extracted using the Omega Bacterial DNA Kit (D3350-01): First, 2 mL of the seed culture described in Example 2 was placed in a sterile 2 mL centrifuge tube and 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 10 μL of lysozyme was added and mixed. 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 three vortexing cycles. 5 μL of RNase A was added, mixed, and allowed to stand at room temperature for 5 min. After centrifugation at 10000 rpm for 2 min, 200 μL of the supernatant was transferred to a new sterile 1.5 mL centrifuge tube. 200 μL of BTL Buffer was added. Mix the buffer thoroughly and incubate at 65°C for 10 min. Add 200 μL of anhydrous ethanol, vortex to mix, and transfer the entire sample to the adsorption column. Centrifuge at 10,000 rpm for 2 min, discard the supernatant and the adsorption column, and place the adsorption column into a new collection tube. Add 500 μL of HBC buffer to the adsorption column, centrifuge at 10,000 rpm for 2 min, and discard the supernatant. Add 700 μL of DNA Wash Buffer to the adsorption column, centrifuge at 10,000 rpm for 2 min, discard the supernatant, and repeat twice. Place the empty adsorption column back into the collection tube and centrifuge at 10,000 rpm for 2 min. Add 30–50 μL of Elution Buffer (preheated to 65°C) to the adsorption column to dissolve the DNA precipitate, thus obtaining genomic DNA. Store at -20°C for later use.

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

[0050] Specifically, using the genomic DNA obtained in step S301 as a template, PCR amplification was performed using Eubac27F and Eubac1492R primers. The PCR reaction system (50 μL) included: 0.5 μL TaKaRa LA Taq (5 U / μL), 5 μL 10×LA Taq Buffer II (Mg... 2+ The reagents included 8 μL of dNTPs Mixture (2.5 mM each), 2 μL of genomic DNA, 1 μL of upstream primer Eubac27F (10 μM), 1 μL of downstream primer Eubac1492R (10 μM), and 32.5 μL of 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 procedure was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, and repeated the denaturation, annealing and extension processes 30 times; extension at 72℃ for another 10 min, and storage of PCR amplification products at 4℃.

[0052] S303: Perform nucleic acid electrophoresis on the PCR products. After loading 5 μL of the PCR product obtained in step S302, perform nucleic acid electrophoresis at 120V for 25 min. The electrophoresis results are as follows: Figure 2 As shown, the 16S rDNA fragment amplified using the genomic DNA of *Microbacterium arborescens* H17 obtained in Example 1 of this invention as a template has a single and high-brightness band, and the 16S rDNA sequence length is approximately 1400 bp.

[0053] S304: Sequencing of the 16S rDNA sequence. After purification and recovery of the PCR product obtained in step S302, 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 described in this invention is 1390 bp in length, and the specific sequence is shown in SEQ ID NO: 3.

[0054] The 16S rDNA sequence of *Microbacterium argentea* H17 obtained from sequencing was entered into NCBI for BLAST alignment. A phylogenetic tree of *Microbacterium argentea* H17 was constructed using the neighbor-joining method in MEGA11, and the results are as follows. Figure 3As shown, based on BLAST homology analysis and phylogenetic tree, the *Microbacterium arborescens* H17 obtained in this invention shares 100% homology with *Microbacterium arborescens* strain N30 (MN555371.1). Therefore, the strain of this invention belongs to *Microbacterium arborescens*, and is named *Microbacterium arborescens* H17 in this invention.

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

[0056] This example demonstrates the gyrB gene sequence of Microbacterium arborescens H17 amplified by PCR and then sequenced.

[0057] S401: PCR amplification of the gyrB gene sequence. Using the genomic DNA obtained in step S201 of Example 3 as a template, PCR amplification was performed using degenerate primers UP-1 and UP-2r. 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+ The reagents included 8 μL of dNTPs Mixture (2.5 mM each), 2 μL of genomic DNA, 1 μL of upstream primer UP-1 (10 μM), 1 μL of downstream primer UP-2r (10 μM), and 32.5 μL of ddH2O. The sequence of the upstream primer UP-1 was 5'-gaagtcatcatgaccgttctgcaygcnggnggnaarttyga-3' (SEQ ID NO: 4), and the sequence of the downstream primer UP-2r was 5'-agcagggtacggatgtgcgagccrtcnacrtcngcrtcngtcat-3' (SEQ ID NO: 5).

[0058] The PCR reaction program was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min, and repeated the denaturation, annealing and extension processes 30 times; extension at 72℃ for another 10 min, and storage of PCR amplification products at 4℃.

[0059] S402: Nucleic acid electrophoresis of PCR products. After loading 5 μL of the PCR product obtained in step S302, perform nucleic acid electrophoresis at 120V for 25 min. The electrophoresis results are as follows: Figure 4 As shown, the gyrB gene fragment amplified using the genomic DNA of *Microbacterium arborescens* H17 as a template in Example 1 of this invention has a single and high-brightness band, and the gyrB sequence length is approximately 1700 bp.

[0060] S403: Sequencing of the gyrB gene sequence. After purification and recovery of the PCR product obtained in step S301, 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 described in this invention is 1774 bp in length, and the specific sequence is shown in SEQ ID NO: 6.

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

[0062] Example 5: Antibacterial activity of fermentation supernatant of *Microbacterium arboresense* H17 against *Ralstonia solanacearum*.

[0063] This embodiment studies the antibacterial activity of fermentation supernatant of *Microbacterium arvense* H17 against *Ralstonia solanacearum*, including the following steps:

[0064] A plate contrast experiment was conducted. 50 μL of *Ralstonia solanacearum* bacterial suspension was spread onto LB+ glucose solid medium. Filter paper discs were evenly placed on the plate, with 10 μL of *Microbacillus oryzae* H17 fermentation supernatant added to one disc. 10 μL of sterile water and 10 μL of 5 mg / mL ampicillin were used as blank and positive controls, respectively, with different filter paper discs added to each. The plates were incubated at 37℃ for 24 h, and the presence and diameter of inhibition zones were observed and measured. The experiment was repeated three times.

[0065] The results are as follows Figure 6As shown, *Microbacterium argentea* H17 exhibits significant inhibitory activity against *Ralstonia solanacearum*, with an average inhibition zone diameter of 19 mm. Its inhibitory capacity is approximately 90% of that of 5 mg / mL ampicillin (inhibition zone diameter 21 mm). This result demonstrates that *Microbacterium argentea* H17 possesses highly efficient inhibitory properties against *Ralstonia solanacearum*. Since *Ralstonia solanacearum* can cause wilt diseases in more than 200 plants, including important crops such as tomatoes, potatoes, peppers, and tobacco, *Microbacterium argentea* H17 can be used to control plant wilt diseases caused by *Ralstonia solanacearum* and can be used to prepare biological agents for the control of plant wilt diseases.

[0066] 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. Dissolve each component in sterile water by stirring. Add water to a final volume of 1L and adjust the pH to 7.2-7.4. Then, dispense the medium into Erlenmeyer flasks. Seal the Erlenmeyer flasks with sterile paper and sterilize them in an autoclave at 121℃ and 101kPa for 20 minutes. When the temperature of the autoclave drops below 70℃ and the pressure returns to 0kPa, remove the flasks and pour the medium into petri dishes on a clean bench, adding approximately 20mL of culture medium to each dish. After cooling and solidification, store the solid culture plates at 4℃. Example 6: Application effect of *Microbacterium arborescens* H17 in tomato cultivation affected by *Ralstonia solanacearum*.

[0067] This embodiment 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, the tomato wilt disease caused by Ralstonia solanacearum was severe in the greenhouse, with a mortality rate of over 53% in the previous season.

[0069] The experimental group consisted of 3 rows, with 100 tomato seedlings in each row. The specific experimental procedure was as follows: Tomato seedlings at the three-leaf stage were transplanted into a greenhouse. At transplanting, each seedling was watered with 20-30 mL of *Microbacillus argentea* H17 bacterial solution. During the plant growth period, each tomato seedling was drip-irrigated with 10-20 mL of *Microbacillus argentea* H17 bacterial solution every 10 days, for a total of 3 applications. The bacterial solution concentration was 1.0 × 10⁻⁶. 5 cfu / mL~1.0×10 7 CFU / mL. Calculate the number of surviving plants and the survival rate during the peak fruiting period of tomatoes.

[0070] The blank control group consisted of 3 rows, with 100 tomato seedlings in each row. Water was used instead of the bacterial culture of Microbacillus arborescens H17 in the blank control group. All other conditions were the same as those in the experimental group and were carried out in parallel with the experimental group.

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

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

[0073] The results are as follows Figure 7 As shown, in the experimental group, 287 out of 300 tomato plants survived, a survival rate of 95.6%, with a mortality rate of 4.4%. In the blank control group, 156 plants survived, a survival rate of 52%, with a mortality rate of 48%. The use of the *Microbacterium arvense* H17 bacterial suspension described in this invention significantly improved the survival rate of tomatoes and reduced their mortality rate, achieving a biocontrol efficiency of 90%.

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

[0075] (1) In this invention, *Microbacterium argentea* H17 has a good inhibitory effect on *Ralstonia solanacearum*. However, conventional chemical pesticides require the application of high concentrations of chemical reagents to achieve good control effects and are prone to causing environmental pollution. Therefore, *Microbacterium argentea* H17 has the potential to inhibit various plant diseases caused by *Ralstonia solanacearum* and can be used to prepare drugs for the prevention and control of various plant diseases caused by *Ralstonia solanacearum*.

[0076] (2) This invention first discovered that the fermentation supernatant of Microbacillus arborescens H17 has a good inhibitory effect on Ralstonia solanacearum, which causes Fusarium wilt of tomatoes. In the antibacterial activity experiment, 10 μL of Microbacillus arborescens H17 fermentation supernatant inhibited the Ralstonia solanacearum by 19 mm, which showed a good antibacterial effect. In the process of plant cultivation, long-term intermittent application of Microbacillus arborescens bacterial solution has a biocontrol efficiency of 90% against Fusarium wilt, which significantly inhibits the occurrence of the disease.

[0077] (3) The arborescent microbacterium H17 is environmentally friendly and harmless to humans and animals, which provides a new and safe biocontrol strain for the prevention and control of Ralstonia solanacearum and the plant diseases it causes.

[0078] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and the present invention also intends to include these modifications and variations.

Claims

1. A strain of *Microbacterium arborescens* H17, characterized in that... The described *Microbacterium arboresense* is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO:M 20242129.

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

6.

3. The application of the *Microbacterium arborescens* H17 as described in claim 1 in the prevention and control of plant diseases.

4. The application of the *Microbacterium arborescens* H17 as described in claim 1 in the preparation of biological agents for the prevention and control of plant diseases.

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

6. The application of the *Microbacterium arborescens* H17 as described in claim 1 in the preparation of a biological agent antagonizing *Ralstonia solanacearum*.

7. A biological agent, characterized in that, The active ingredient of the biological agent includes *Microbacterium arboresense* H17 as described in claim 1.

8. A method for controlling plant wilt disease, characterized in that, The procedure includes the following steps: When planting, apply 20-30 mL of *Bacillus argentea* H17 bacterial solution to each plant; during the plant growth period, apply 10-20 mL of *Bacillus argentea* bacterial solution to each plant every 7-10 days, for a total of 3-5 applications.

9. The prevention and control method as described in claim 8, characterized in that, The concentration of *Microbacterium arborescens* H17 in the bacterial culture was 1.0 × 10⁻⁶. 5 cfu / mL~1.0×10 7 cfu / mL.

10. The prevention and control method as described in claim 8, characterized in that, The preparation method of Microbacterium arborescens H17 bacterial suspension includes the following steps: inoculating Microbacterium arborescens H17 as described in claim 1 into a liquid culture medium, fermenting and culturing at 30℃~37℃ for 18~30 hours to obtain Microbacterium arborescens H17 bacterial suspension.

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