Actinomyces rarus and application thereof in prevention and treatment of tobacco bacterial wilt

Abstract

This invention relates to a rare actinomycete and its application in controlling tobacco bacterial wilt, wherein the strain is *Nivea pseudoamycetes* (…). Amycolatopsis mediterranei YPL-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 18, 2023, with accession number CGMCC NO.28656, significantly inhibits the growth of *Ralstonia solanacearum*, the pathogen of tobacco bacterial wilt. The inhibition zone diameter against the pathogen reaches 55.6 mm, preventing its colonization in the tobacco rhizosphere and reducing the amount of *Ralstonia solanacearum* in the soil. Furthermore, this strain, isolated from tobacco rhizosphere soil, exhibits strong rhizosphere colonization ability and is easily cultured, making it highly valuable for the biological control of tobacco bacterial wilt.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a rare actinomycete, *Nivea pseudoamycetes*, and its application in the prevention and control of tobacco bacterial wilt. Background Technology

[0002] Tobacco bacterial wilt is caused by Ralstonia solanacearum (also known as the solanaceous bacterium). Ralstonia solanacearum Bacterial wilt is a soil-borne plant disease that is prevalent in major tobacco-growing areas of my country. Because it requires high temperatures and abundant rainfall, it primarily occurs in southern tobacco-growing regions. However, in recent years, with the frequent occurrence of abnormal weather and the adaptive variations of the bacterium *Ralstonia solanacearum*, the disease has shown a trend of spreading to northern tobacco-growing areas, with reports of damage in provinces such as Henan, Shandong, Shaanxi, and Liaoning. The disease incidence rate in the field is generally 10%–30%, but in severe cases it can reach over 50%, even leading to total crop failure. Therefore, this disease has become a devastating threat to tobacco yield and quality. In nature, *Raulella solanaceae* has a wide range of hosts, infecting economic crops such as tobacco, tomatoes, and potatoes. Some variants can also infect other non-solanaceae crops such as bananas and mulberries. The infection mainly occurs through wounds in the plant roots, secondary roots, or natural openings. During infection, *Raulella solanaceae* colonizes the xylem of tobacco and proliferates rapidly. The extracellular polysaccharides produced by *Raulella solanaceae* block the vascular bundles of the host, hindering nutrient supply and transport. The tobacco plant cannot obtain nutrients and water to ensure the normal growth of the above-ground parts, and the host will exhibit symptoms such as wilting leaves that spread to the death of the entire plant, accompanied by the gradual blackening, rotting, and hollowing of the vascular bundles in the roots and stems.

[0003] Currently, control measures for tobacco bacterial wilt mainly include chemical control, breeding of resistant varieties, crop rotation, soil improvement, and biological control. In production, cultivating disease-resistant varieties is the simplest and most effective propagation method. However, the breeding cycle for tobacco varieties resistant to bacterial wilt is too long, and due to the diversity and variation of physiological races of Ralstonia solanacearum, resistance in resistant tobacco varieties may degenerate or even disappear after prolonged consecutive cropping in the same plot. Crop rotation is one of the effective measures for controlling the disease, but the rotation period is too long, and the per capita arable land area in tobacco-producing areas is relatively small. Due to farmers' economic needs, this method cannot be widely adopted. While chemical control is effective... While fast, this method is not only costly, but long-term use can also lead to the development of drug-resistant pathogens in the soil, and pesticide residues and environmental pollution can persist in tobacco products and soil for extended periods. Soil improvement is also an effective method for controlling tobacco bacterial wilt. In production, alkaline fertilizers such as quicklime and calcium silicate fertilizer are often used to improve the soil in fields severely affected by tobacco bacterial wilt. This effectively reduces the occurrence of tobacco bacterial wilt in the short term, but long-term and excessive application of quicklime and other substances can damage the soil structure, causing soil compaction and hindering production. Therefore, biological control, as a safe, efficient, and environmentally friendly control method, is of great significance to tobacco production.

[0004] Biological control refers to the use of beneficial microorganisms to inhibit pathogens, reduce their vitality and reproductive capacity, thereby mitigating crop diseases. Common mechanisms of biological control of plant diseases include: improving soil physical and chemical properties to promote plant growth and enhance the host plant's disease resistance; utilizing the antagonistic, parasitic, or predatory effects of biocontrol microorganisms on pathogens, as well as their competition with pathogens for nutrients and ecological niches to inhibit and eliminate pathogens; and inducing host plants to develop systemic resistance to pathogens.

[0005] pseudoamycin ( Amycolatopsis *Amycium pseudomycoides* is a unique genus of Nocardia-type actinomycetes and is a rare actinomycete. Currently, 82 species of *Amycium pseudomycoides* have been published, most of which were isolated from soil and marine sediments. As a rare genus of actinomycetes, the applications of *Amycium pseudomycoides* have mainly focused on high-yield antibiotics and the degradation of chemical substances. It was once considered the most effective source of antibacterial, antifungal, or antiviral drugs. For example, Wang Huihui et al.'s patent (CN202211341535.6) confirmed a method for producing vancomycin using *Amycium orientalis*; Zhao Haibing et al.'s patent (CN201710609367.7) confirmed a culture medium and method for producing rifamycin using *Amycium thaliana* fermentation. To date, *Amycium pseudomycoides* still has great potential for drug development.

[0006] There have been many studies on the production of antibiotics using Amylopectin bacterium, but research on the direct use of Amylopectin bacterium for biological control, especially for the control of bacterial diseases, is still very rare. Therefore, exploring the use of Amylopectin bacterium to control tobacco bacterial wilt omits the step of extracting metabolites after large-scale cultivation of Amylopectin bacterium, simplifies the production process, and reduces production costs. At the same time, Amylopectin bacterium applied to the soil can also change the soil microenvironment in the long term, which will broaden the application prospects of Amylopectin bacterium in agricultural production and is of great significance.

[0007] Based on the above analysis, the shortcomings and unresolved problems of existing technologies are as follows: among the existing methods for controlling tobacco bacterial wilt, resistant varieties cannot achieve good disease control effects for a long period of time; large-scale, long-term crop rotation is not feasible; the application of quicklime and chemical pesticides can cause soil compaction, excessive pesticide residues, and the development of pesticide resistance in pathogens, which does not meet the requirements for healthy and sustainable agricultural development. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a Nivea pseudo-amycin bacterium. Amycolatopsis nivea YPL-1 and its application in the control of tobacco bacterial wilt. This strain was isolated from the rhizosphere soil of healthy tobacco plants in a tobacco bacterial wilt field. As a rare actinomycete isolated from the rhizosphere soil of healthy tobacco, this strain can colonize well in the tobacco rhizosphere and does not have a negative impact on tobacco growth, showing good application prospects.

[0009] The present invention is implemented using the following technical solution.

[0010] A rare actinomycete, the strain described in this invention is the rare actinomycete *Nivea pseudomycorrhizal*. Amycolatopsis nivea YPL-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 18, 2023, with accession number CGMCC NO.28656.

[0011] The application of the rare actinomycetes described above in this invention, wherein the application is the use of the rare actinomycete *Nivea pseudomycorrhizal*. Amycolatopsis nivea YPL-1 was prepared as a biocontrol agent to prevent and control tobacco bacterial wilt.

[0012] The biocontrol agent described in this invention is derived from Nivea pseudomycolic acid bacteria. Amycolatopsis nivea Aqueous formulation made from YPL-1 fermentation.

[0013] The preparation method of the aqueous agent of the present invention includes the following steps: Step 1) Preparation of seed solution: Nivea pseudonephritobacterium was inoculated into NA liquid culture medium. Amycolatopsis niveaYPL-1 was cultured at 28℃ and 180 r / min for 2 days to obtain seed culture. Step 2) Fermentation culture: The seed culture was inoculated into a fermentation medium, which was NA liquid medium. The fermentation conditions were as follows: 500 mL of medium was added to a 1000 mL Erlenmeyer flask; the initial pH of the fermentation medium was 7.0; the temperature was 28℃; the fermentation speed was 180 rpm; and the fermentation time was 3 days. (The text then mentions *Deniveryl amylopectin*, but the connection to the previous sentence is unclear.) Amycolatopsis nivea YPL-1 fermentation broth is used as a biocontrol agent.

[0014] The NA liquid culture medium in steps 1) and 2) of this invention has the following formula by weight: 10 parts glucose, 5 parts peptone, 3 parts beef extract, 1 part yeast extract, and 1000 parts distilled water.

[0015] In step 2) of this invention, the ratio of seed liquid to fermentation medium is 5:95 by volume.

[0016] The biocontrol agent of this invention contains Nivea pseudomycolic acid bacteria. Amycolatopsis nivea The concentration of the YPL-1 fermentation broth is 1×10⁻⁶. 8 CFU / mL.

[0017] The method of preventing and controlling tobacco bacterial wilt described in this invention is root irrigation.

[0018] The root irrigation method described in this invention specifically involves irrigating each plant with 50 ml of biocontrol agent, repeating the application 2-3 times consecutively, with an interval of 7-10 days between each application.

[0019] The beneficial effects of this invention are that the expected benefits and commercial value of the technical solution after its transformation are significant, particularly for Nivea's pseudomycolic acid bacteria. Amycolatopsis nivea YPL-1 has simple culture conditions and is easy to industrialize. Isolated from tobacco rhizosphere soil, this strain exhibits good ecological compatibility with tobacco rhizosphere soil and demonstrates excellent colonization characteristics, which is beneficial for the control of tobacco bacterial wilt. Therefore, Nivea's *Amylopectinus* YPL-1 has great potential for exploration and development.

[0020] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0021] Figure 1 Image of Nivea pseudoelastomer isolated from the rhizosphere soil of healthy tobacco; Figure 2 For Nivea's pseudomycolic acid bacteria Amycolatopsis nivea The plate inhibition effect of YPL-1 on tobacco bacterial wilt pathogen; Figure 3 For Nivea's pseudomycolic acid bacteria Amycolatopsis niveaPhylogenetic tree of YPL-1. Detailed Implementation

[0022] The following embodiments are only a part of the technical solutions of the present invention and are not intended to limit all the technical solutions of the present invention. The embodiments of the present invention are provided to further explain and illustrate the details of the technical solutions of the present invention.

[0023] To address the problems existing in the prior art, the present invention provides a Nivea pseudomycolic acid bacterium strain and its application. The present invention will be described in detail below with reference to the accompanying drawings.

[0024] To enable those skilled in the art to fully understand how the present invention is specifically implemented, this section provides an explanatory description of the embodiments that expand upon the technical solutions of the claims.

[0025] Example 1: Isolation of pseudomycolic acid bacteria 1. Culture medium formulation NA medium: 10.0g glucose, 5.0g peptone, 3.0g beef extract, 1g yeast extract, 18.0g agar powder, 1000mL distilled water, pH adjusted to 7.0, autoclaved at 121℃ for 30min.

[0026] 2. Isolation and purification of strains The present invention Nivea pseudo-amycin ( Amycolatopsis nivea YPL-1 bacteria were isolated and purified from tobacco rhizosphere soil using the dilution plating method and the streak plating method. The isolation method was as follows: 10g of rhizosphere soil was placed in a 250mL Erlenmeyer flask, 90mL of sterile water was added, and the mixture was shaken at 180rpm and 28℃ for 30min to prepare 10... -1 Diluent. Take 100 μL of 10 -1 The diluent was diluted in 900 μL of sterile water to obtain 10 -2 Diluent, diluted sequentially in this manner, to obtain 10% of the soil solution. -3 and 10 -4 Diluent. Take 100 μL of each of the following solutions: -3 10 -4 The diluted solution was spread evenly onto NA solid medium plates using a sterile spreader. After incubation at 30°C for 48 hours, colonies of different morphologies from the NA solid medium were picked up with an inoculation loop and streaked onto fresh NA solid medium plates. The strains were then purified using the streak plating method, numbered, stored, and screened for antagonistic bacteria (see...). Figure 1 ).

[0027] Example 2: Plate inhibition assay of isolated strains against *Ralstonia solanacearum*, the pathogen of tobacco bacterial wilt. 1. Culture medium formulation NA medium: 10.0g glucose, 5.0g peptone, 3.0g beef extract, 1g yeast extract, 18.0g agar powder, 1000mL distilled water, pH adjusted to 7.0, autoclaved at 121℃ for 30min.

[0028] CPG medium: 1.0 g casein hydrolysate, 10.0 g peptone, 5.0 g glucose, 17.0 g agar powder, 1000 mL distilled water, pH adjusted to 7.0, autoclaved at 121°C for 30 min.

[0029] 2. Activation of the isolated strains and *Ralstonia solanacearum* (bacterial wilt pathogen of tobacco). Take out the cryopreservation tubes of antagonistic strains stored at -20℃, place them on ice to thaw slowly, and take out the cryopreservation tubes of Ralstonia solanacearum stored in sterile water at room temperature.

[0030] The antagonistic bacterial strains that showed good initial screening results were activated on NA medium.

[0031] Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum Activation was performed on CPG medium.

[0032] 3. Plate inhibition assays of various isolated strains against *Ralstonia solanacearum*. Colonies of *Ralstonia solanacearum*, the causal agent of bacterial wilt of tobacco, cultured on CPG medium for 2 days were scraped off and placed in 50 mL centrifuge tubes containing sterile water. The suspension was vortexed to form a homogeneous bacterial suspension. The OD600 of the bacterial suspension was adjusted to approximately 0.5 with sterile water. 100 μL of the *Ralstonia solanacearum* bacterial suspension was evenly spread onto NA solid medium. A single colony of the previously activated antagonistic bacterial strain was picked and inoculated into the center of the medium. NA medium coated only with *Ralstonia solanacearum* served as a control. The medium was incubated at 28°C for 2 days, and the size of the inhibition zone was measured. Each biocontrol strain was tested in triplicate. The results showed that strain YPL-1 had a good inhibitory effect on *Ralstonia solanacearum*, with an inhibition zone diameter of up to 48 mm (see...). Figure 2 ).

[0033] Example 3: Identification of strain YPL-1 1. Observation of morphological characteristics Strain YPL-1 was inoculated onto Gao's No. 1 medium and NA medium, and after incubation at 28℃ for 6 days, the morphological characteristics of the colonies were observed. The results showed that on Gao's No. 1 medium, strain YPL-1 colonies were small, off-white, with a rough and dry surface, tightly bound to the medium, with irregular edges, and difficult to pick up; when scraped, they were pulpy. After prolonged incubation, no pigment was produced. On NA medium, strain YPL-1 colonies were larger, grayish-white, with a rough and dry surface, not tightly bound to the medium, with irregular edges, and easily scraped off. After prolonged incubation, pink pigment was produced and released onto the medium, while the colony itself remained unchanged in color.

[0034] 2. 16S rDNA sequence analysis DNA was extracted from strain YPL-1 using the genome extraction kit from Tiangen Biotech Co., Ltd., and then PCR amplification was performed using the 16S universal primer 27F / 1492R.

[0035] The primer sequences are: 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′.

[0036] 1492R:5′-TACGGCTACCTTGTTACGACTT-3′ After the recovered PCR amplification products were verified by gel electrophoresis, the positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain nucleotide sequence information.

[0037] 16S rDNA sequence of biocontrol bacterium YPL-1: The sequenced sequences were submitted to the GenBank database for BLAST analysis and comparison. It was found that strain CSH S0261 showed 99.45% homology with YPL-1. Strain CSH S026 is... Amycolatopsis nivea It belongs to the rare actinomycete genus *Pseudomonas*, and no domestic literature has been found on it. Amycolatopsis nivea The Chinese translation is therefore Nivea pseudoechinococcus. A 16S rDNA sequence with high homology was selected as a reference, and a phylogenetic tree was constructed using the Neighbour-joining method (see...). Figure 3 The results showed that strain YPL-1 was... Amycolatopsis nivea The strain YPL-1 is Nivea pseudomycolic acid bacterium, which was deposited on October 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28656.

[0038] Example 4: Plate inhibition assay of strain YPL-1 against other pathogens The culture medium used is as follows: YEM medium and NA medium.

[0039] Tested pathogens: Including Carotene soft rot pectinobacter ( Pectobacterium carotovorum Blueberry crown gall pathogen ( Agrobacterium tumefaciens Rice streak pathogen ( Xanthomonas oryzae pv. oryzicola), black rot fungus of cabbage ( Xanthomonas campestris The pathogens mentioned above, such as pv. campestris, were all provided by the Bacteriology Research Laboratory of Yunnan Agricultural University.

[0040] Experimental method: Bacterial activation: *Carrot soft rot pectinobacter* T bacteria (… Pectobacterium carotovorum Blueberry crown gall pathogen ( Agrobacterium tumefaciens Rice streak pathogen ( Xanthomonas oryzae pv. oryzicola), black rot fungus of cabbage ( Xanthomonas campestris Cryopreservation tubes of *pv. campestris* were removed from a -80°C freezer and slowly thawed on ice. 80 μL of bacterial culture was then aspirated from the thawed tubes and inoculated into NA medium. All tested pathogenic bacteria were incubated upside down at 28°C for 48 hours. After bacterial activation, a small number of colonies were picked up with an inoculation loop and streaked three times consecutively to amplify the colonies, incubating them upside down at 28°C for 48 hours each time to allow single colonies to grow for use in antibacterial experiments.

[0041] Methods for determining antibacterial effect: Measurement of the inhibition diameter of pathogenic bacteria: A single colony of pathogenic bacteria was picked and inoculated into 100 mL of NA liquid medium and cultured at 28℃ and 180 rpm to prepare a bacterial suspension with OD600≈0.5. The bacterial suspension was mixed with NA solid medium thawed at 50℃ at a ratio of 5:95 to prepare a bacterial substrate. In a clean bench, the biocontrol bacteria YPL-1 was spot-inoculated into the center of the substrate using a pipette tip. The substrate was incubated upside down at 28℃ for 3 days, and the diameter of the inhibition zone was measured.

[0042] Experimental Results and Analysis: The experimental results are shown in Table 1.

[0043] Table 1. Determination of the inhibitory effect of YPL-1 on the growth of different pathogens. Table 1 shows that Nivea pseudoelastomer ( Amycolatopsis nivea YPL-1 showed strong antagonistic effects against all four tested pathogenic bacteria, including Nivea mycoides (Nivea mycoides). Amycolatopsis nivea YPL-1 showed the strongest inhibitory effect on the soft rot pathogen of Chinese cabbage, indicating that the metabolites of YPL-1 have a broad-spectrum inhibitory effect on pathogenic bacteria.

[0044] Example 5: Nivea pseudomycolic acid bacteria Amycolatopsis nivea Greenhouse control experiment of YPL-1 against tobacco bacterial wilt.

[0045] The tested crop was the tobacco variety Yunyan 87, which showed moderate resistance to tobacco bacterial wilt.

[0046] Test media: NA medium, CPG medium.

[0047] Experimental location: Constant temperature greenhouse, Engineering Center, Yunnan Agricultural University Preparation of biocontrol agents and pathogenic bacteria fermentation broth: Nivea pseudonephrolepis growing on NA solid medium Amycolatopsis nivea A single colony of YPL-1 was picked and inoculated into an Erlenmeyer flask containing 50 mL of NA liquid medium. The flask was incubated on a shaker at 28°C and 180 rpm for 2 days to obtain the YPL-1 seed culture. This seed culture was then inoculated into fermentation medium at a volume ratio of seed culture to fermentation broth of 5:95. The fermentation conditions were: 500 mL / 1000 mL flask volume, initial pH of fermentation medium of 7.0, 28°C, 180 rpm, and fermentation time of 3 days, yielding the fermentation broth of strain YPL-2 with a concentration of approximately 5–8 × 10⁻⁶. 9 CFU / ml, the bacterial suspension concentration was adjusted to approximately 1×10⁻⁶ CFU / ml with sterile water. 8 CFU / ml, used as a biocontrol agent.

[0048] Single colonies of *Ralstonia solanacearum*, the pathogen of *Ralstonia solanacearum*, grown on CPG solid medium were picked and inoculated into Erlenmeyer flasks containing 500 mL of CPG liquid medium. The flasks were incubated on a shaker at 28°C and 180 rpm for 1 day. The bacterial suspension concentration was adjusted to approximately 1 × 10⁻⁶ with sterile water. 8 CFU / ml.

[0049] Experimental methods: The experiment was designed with three treatments: biocontrol agent and control agent. Amycolatopsis nivea YPL-1 fermentation broth treatment group, water control group, and pesticide control group were selected. Tobacco seedlings at the 6-7 leaf stage were transplanted into flowerpots. Five days after transplanting, each tobacco plant was irrigated with 50 mL of Nivea pseudoemodinum bacteria. Amycolatopsis nivea YPL-1 strain fermentation broth (approximately 1×10⁻⁶) 8 CFU / ml), and 24 hours later, drench the roots with 30 mL of tobacco wilt bacteria fermentation broth (approximately 1×10⁻⁶ CFU / ml). 8 (CFU / ml). After inoculation with *Ralstonia solanacearum*, two applications of biocontrol fermentation liquid were applied, 7 days apart. A 500-fold dilution of 20% thiabendazole copper suspension was used as the pesticide. Twenty tobacco plants were placed in a constant-temperature, high-light greenhouse at the Engineering Center of Yunnan Agricultural University, with 16 hours of light per day, a temperature of 30℃, and 80% humidity. Disease surveys were conducted 21 days after the first tobacco plant in the CK experimental group (water control) showed symptoms. Tobacco disease classification and survey methods followed the national standard GB / T23222-2008.

[0050] The disease severity grading criteria for tobacco bacterial wilt are as follows: Grade 0: The entire plant is disease-free; Grade 1: Occasionally, there are chlorotic spots on the stem, or the leaves on the diseased side below 1 / 2 wither; Grade 3: There are black streaks on the stem, but they do not exceed 1 / 2 of the stem height, or the leaves on the diseased side from 1 / 2 to 2 / 3 wither; Grade 5: The black streaks on the stem exceed 1 / 2 of the stem height, but do not reach the top of the stem, or the leaves on the diseased side above 2 / 3 wither; Grade 7: The black streaks on the stem reach the top of the stem, or all the leaves of the diseased plant wither; Grade 9: The diseased plant is basically dead.

[0051] Disease index (%) = [∑(Disease grade number × Number of plants at that disease grade) / (Highest disease grade number × Total number of plants)] × 100% Prevention and control efficacy (%) = [(Disease index of blank control - Disease index of treatment) / Disease index of blank control] × 100% The experimental results are shown in Table 2: Table 2 Nivea pseudoemodinum Amycolatopsis nivea The effect of YPL-1 fermentation broth on the prevention and control of tobacco bacterial wilt The results showed that Nivea pseudomycolic acid bacteria Amycolatopsis niveaYPL-1 fermentation broth significantly reduced the incidence and disease index of tobacco bacterial wilt. Most tobacco plants treated with this broth were at disease grade 1, achieving a relative control effect of 68.3%, slightly higher than a 500-fold dilution of 20% thiamethoxam suspension. This indicates that Nivea's *Amylopectinus* var. *mairei* is effective in controlling bacterial wilt. Amycolatopsis nivea YPL-1 effectively controls tobacco bacterial wilt, and the experiment was reproducible; therefore, this *Nivea* strain... Amycolatopsis nivea YPL-1 has the basic conditions for large-scale production and promotion.

[0052] The above descriptions are merely some specific embodiments of the present invention. Commonly known details or common knowledge in the solutions are not described in detail here (including but not limited to abbreviations, acronyms, and units conventionally used in the art). It should be noted that the above embodiments do not limit the present invention in any way. For those skilled in the art, any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A rare actinomycete, characterized in that, The rare actinomycete strain is Nivea pseudoamycin (Nivea pseudoamycin). Amycolatopsis nivea YPL-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 18, 2023, with accession number CGMCC NO.28656.

2. The application of the rare actinomycetes according to claim 1, characterized in that, The application involves preparing Nivea pseudoelastomer YPL-1 into a biocontrol agent to prevent and control tobacco bacterial wilt.

3. The application according to claim 2, characterized in that, The biocontrol agent is an aqueous solution produced by fermentation of Nivea pseudomycolic acid bacteria YPL-1.

4. The application according to claim 3, characterized in that, The preparation method of the aqueous agent includes the following steps: Step 1) Preparation of seed solution: Nivea pseudoelastomer YPL-1 was inoculated in NA liquid medium at 28℃ and 180 r / min for 2 days to obtain seed culture. Step 2) Fermentation culture: The seed culture was inoculated into a fermentation medium, which was NA liquid medium. The fermentation conditions were as follows: 500 mL of medium was added to a 1000 mL Erlenmeyer flask; the initial pH of the fermentation medium was 7.0; the temperature was 28℃; the fermentation speed was 180 r / min; and the fermentation time was 3 days. The fermentation broth of Nivea pseudomycolic acid bacterium YPL-1 was obtained and used as a biocontrol agent.

5. The application according to claim 4, characterized in that, The NA liquid culture medium in steps 1) and 2) has the following formulation: by weight, it comprises: 10 parts glucose, 5 parts peptone, 3 parts beef extract, 1 part yeast extract, and 1000 parts distilled water.

6. The application according to claim 4, characterized in that, In step 2), the ratio of seed liquid to fermentation medium is 5:95 by volume.

7. The application according to claim 4, characterized in that, The concentration of the fermentation liquor of N. nevirae YPL-1 in the biocontrol agent is 1 x 10 8 CFU / mL.

8. The application according to claim 2, characterized in that, The method of application for preventing and controlling tobacco bacterial wilt is root irrigation.

9. The application according to claim 8, characterized in that, The root irrigation method specifically involves applying 50ml of biocontrol agent to each plant, repeating the application 2-3 times consecutively, with an interval of 7-10 days between each application.

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