Application of Paenibacillus kriebensis space mutant ΔPS04-17 strain in controlling cucumber mosaic virus

Abstract

The present invention discloses the use of a space mutant of Paenibacillus cribensis, ΔPS04-17, in preventing and controlling cucumber mosaic virus. Studies have shown that the ΔPS04-17 strain inhibits CMV and can significantly reduce the incidence of CMV host plants. After treatment with ΔPS04-17, the relative efficacy against plant diseases caused by CMV reached 54.72%, reducing the incidence of tobacco cucumber mosaic virus disease. It can effectively inhibit the spread of CMV within the plant, delay the onset of disease, prevent the occurrence of cucumber mosaic virus disease, and prevent premature aging of the plant. Furthermore, the ΔPS04-17 strain can promote tobacco growth, root development, increase tobacco leaf area, enhance tobacco growth, and increase yield. The ΔPS04-17 strain is green and safe, not only reducing the use of pesticides but also promoting environmental safety. It can be used for the green prevention and control of CMV, and is of great significance for tobacco disease prevention and control and yield increase.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease prevention and control, and more specifically relates to the use of a space mutant of Paenibacillus kriebensis strain ΔPS04-17 in preventing and controlling cucumber mosaic virus. Background Art

[0002] Cucumber mosaic virus (CMV) is a serious viral disease with the widest host range and widest distribution. CMV can infect all parts of a plant, from the seedling stage to the mature plant stage. It is spread by aphids and friction. More than 60 aphid species can transmit the virus, primarily tobacco and cotton aphids. CMV infects approximately 124 plant species across 36 dicot families and four monocot families. CMV can cause yield and quality reductions in its host plants, severely impacting agricultural production. CMV is a major viral disease of tobacco, found and causing harm in all tobacco-growing regions worldwide, causing cucumber mosaic virus disease. CMV rapidly proliferates and metastasizes within tobacco plants, appearing in mesophyll cells within 6 hours of infection. It harms tobacco growth, reduces carbohydrate content, increases nitrogen content, and reduces quality in diseased leaves. In my country, the Huanghuai tobacco region is hardest hit, followed by the central and southern China tobacco regions. In severely affected areas, yields decrease by 25-30% in epidemic years, and in severe cases, yield reductions often reach around 50%. The disease caused by CMV poses risks to both the yield and quality of tobacco production in my country. Therefore, effective prevention and control measures are crucial to reducing the damage caused by cucumber mosaic virus.

[0003] Current methods for preventing and controlling CMV include selecting disease-resistant varieties, strengthening field management, and using pesticides. Furthermore, seed treatment, crop rotation, and grafting disinfection can also comprehensively control cucumber mosaic virus. However, the time required to select and breed disease-resistant varieties is long, and strengthening field management is time-consuming and labor-intensive. While the use of chemical agents, such as phytopathogens, ningnanmycin, and copper morpholino hydrochloride for spraying can improve control effectiveness, they increase the frequency of pesticide use, increase viral resistance, and negatively impact plant cultivation safety and the environment. Therefore, there is still a lack of products for preventing and controlling CMV that can inhibit CMV and thereby reduce its impact on plants, reduce the incidence of its diseases, and improve the yield and quality of its host plants. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of existing CMV prevention and treatment products and provide the application of a space mutant of Paenibacillus kriebensis △PS04-17 strain in preventing and treating cucumber mosaic virus.

[0005] The present invention aims to provide a space-induced mutant of Paenibacillus kribbensis, ΔPS04-17, for use in preventing and treating cucumber mosaic virus.

[0006] Another object of the present invention is to provide a method for preventing and controlling cucumber mosaic virus or reducing the incidence of cucumber mosaic virus disease.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a novel application of a space-induced mutant of Paenibacillus kribbensis, ΔPS04-17, in preventing and controlling cucumber mosaic virus. Studies have shown that the ΔPS04-17 strain can significantly reduce the incidence of CMV host plants, demonstrating its CMV-inhibiting effect. When the host plants are treated with the fermentation liquid of ΔPS04-17, the relative efficacy against the CMV-induced host plant disease, tobacco cucumber mosaic virus, reaches 54.72%. This effectively prevents the occurrence of tobacco cucumber mosaic virus, prevents premature aging of host plants, effectively inhibits the spread of CMV within the plant, and delays the onset of disease. Furthermore, a microbial agent prepared from the ΔPS04-17 strain can effectively promote tobacco plant growth, promote root development, increase leaf area, enhance tobacco growth, and increase tobacco yield. The space-induced mutant strain ΔPS04-17 of Paenibacillus kriebensis used in the present invention is green and safe. It can not only reduce the use of pesticides, but also contribute to the development of environmental safety. It can be used for the green prevention and control of CMV, and is of great significance to the prevention and control of tobacco diseases and the increase of production and efficiency.

[0009] Therefore, the present invention provides the use of the space mutant ΔPS04-17 strain of Paenibacillus kriebensis in preventing and controlling cucumber mosaic virus.

[0010] The present invention provides application of a space mutant ΔPS04-17 strain of Paenibacillus criebensis in preventing and treating plant diseases caused by cucumber mosaic virus.

[0011] In particular, the present invention demonstrates that the Paenibacillus kriebensis space mutant ΔPS04-17 strain has a moderate inhibitory effect against CMV and also exhibits significant efficacy against host plant diseases caused by CMV infection, significantly reducing the field morbidity of its host plants. Therefore, it is expected to have similar or similar efficacy against other plant diseases caused by CMV. Therefore, those skilled in the art can anticipate that the Paenibacillus kriebensis space mutant ΔPS04-17 strain will be useful against all plant diseases caused by CMV infection.

[0012] The present invention provides use of a space mutant ΔPS04-17 strain of Paenibacillus criebensis in preparing a cucumber mosaic virus prevention and control product.

[0013] The present invention provides application of a Paenibacillus cribenbergii aerospace mutant ΔPS04-17 strain in promoting tobacco growth.

[0014] The present invention provides the use of a space mutant ΔPS04-17 strain of Paenibacillus cribenbergii in preparing a growth-promoting agent.

[0015] The present invention provides application of a Paenibacillus cribenbergii aerospace mutant ΔPS04-17 strain in improving tobacco yield.

[0016] The invention provides a method for preventing and controlling cucumber mosaic virus or reducing the incidence of cucumber mosaic virus disease. Plants are treated with a space mutant of Paenibacillus criebensis △PS04-17 strain or its bacterial liquid.

[0017] Preferably, the bacterial liquid is a fermentation liquid.

[0018] More preferably, the concentration of the fermentation broth is not less than 1×10 9 CFU / mL.

[0019] Preferably, the fermentation liquid is used to perform foliar spraying on the plants.

[0020] More preferably, the fermentation broth needs to be diluted at least 50 times before use.

[0021] The present invention also provides a method for preparing a fermentation broth of the ΔPS04-17 strain: inoculating the ΔPS04-17 bacterial broth into a fermentation medium at an inoculation rate of 1-10%, and fermenting at 25-30° C. and 150-200 rpm for 5-9 days.

[0022] Preferably, the fermentation medium uses a modified Czapek liquid medium: 1.5 g / L sodium nitrate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate, and 30 g / L sucrose.

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides a novel application of a space-induced mutant strain of Paenibacillus cribensis, ΔPS04-17, in preventing and controlling cucumber mosaic virus. Studies have shown that the ΔPS04-17 strain can significantly reduce the incidence of CMV host plants, demonstrating its CMV-inhibiting effect. After treating host plants with the fermentation broth of ΔPS04-17, the relative efficacy against the tobacco cucumber mosaic virus disease caused by CMV reached 54.72%. This prevents the occurrence of tobacco cucumber mosaic virus disease, premature aging of tobacco plants, and effectively inhibits the spread of CMV within the plant, delaying the onset of disease. Furthermore, a microbial agent prepared from the ΔPS04-17 strain can effectively promote tobacco plant growth, root development, leaf area, and growth, significantly increasing yield and quality. This not only reduces pesticide use but also promotes environmental safety. The microbial agent can be used for the green prevention and control of CMV, and is of great significance for tobacco disease prevention and control, as well as increasing yield and efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a comparison of tobacco plants in the control group and the treatment group 30 days after inoculation with CMV.

[0026] Figure 2 This is a comparison of tobacco plants in the control group and the treatment group.

[0027] Figure 3 This is a comparison of the fresh weight of the aboveground part of tobacco in the control group and the treatment group.

[0028] Figure 4 This is a comparison of the fresh weight of the underground part of tobacco in the control group and the treatment group.

[0029] Figure 5 This is a comparison of the maximum leaf area of ​​tobacco in the control group and the treatment group. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0032] Example 1 Preparation of microbial agent

[0033] The space-induced mutant strain ΔPS04-17 of Paenibacillus kribbensis used in this embodiment is the research result of the applicant's research team. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 17, 2022, with the deposit number: GDMCC NO: 62233, and is recorded in the prior art: CN 114480222 A.

[0034] Preparation of microbial inoculum: ΔPS04-17 bacterial suspension was inoculated at a 1% inoculum into modified Czapek liquid medium (1.5 g / L sodium nitrate, 1 g / L potassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate, 30 g / L sucrose) and fermented at 25-30°C and 150 rpm for 7 days to prepare a fermentation broth containing the space-induced mutant strain ΔPS04-17 of Paenibacillus kriebensis. The fermentation broth concentration was 1×10 9 CFU / mL.

[0035] Example 2 Control of Cucumber Mosaic Virus

[0036] Since viruses cannot be cultured in vitro, they require a host to function. Cucumber mosaic virus (CMV) has a wide host range and can cause a variety of plant diseases, including tobacco cucumber mosaic virus disease in its host plant, tobacco. A potted plant experiment was conducted using the microbial agent containing the space-induced mutant strain ΔPS04-17 of Paenibacillus cribensis prepared in Example 1. The potted plant experiment was conducted at South China Agricultural University in Guangdong Province. The test plants in this example were tobacco, and the tobacco seeds were provided by the Plant Pathology Laboratory of South China Agricultural University.

[0037] After sowing tobacco seeds, place them in a constant temperature culture room for cultivation, water them regularly, and transplant them after germination. When the plants grow to 4 to 5 leaves, select plants with consistent growth conditions and no diseases or insect pests and divide them into two groups: a control group and a treatment group.

[0038] To prepare a CMV viral stock solution, first cut a fresh leaf containing CMV virus and mix it with PBS buffer (0.01 mol / L, pH 7.2) in a specific ratio (1 g:8 mL). Then, add a small amount of quartz sand and grind evenly to obtain a fine slurry. Then, filter through four layers of gauze to remove any remaining solid particles, and collect the filtered viral stock solution for later use.

[0039] The control group sprayed the tobacco plants with water, while the treatment group sprayed the tobacco plants with a 50-fold dilution of the microbial agent of Example 1. 48 hours later, the plants were inoculated with the prepared cucumber mosaic virus (CMV) stock solution by friction. Ten plants were treated in each group, with three replicates. Plant disease incidence was observed and recorded 20 and 30 days after inoculation with cucumber mosaic virus (CMV). A disease survey was conducted during the tobacco maturity stage to calculate the incidence of tobacco cucumber mosaic virus disease, a disease caused by cucumber mosaic virus (CMV). The disease index and relative control effect at different stages were calculated according to a grading standard.

[0040] According to the provisions on tobacco disease classification in the "Tobacco Industry Standard of the People's Republic of China YC / T 39-1996", especially the classification standards for tobacco mosaic disease, this standard adopts the method of using the whole plant as the evaluation unit.

[0041] Disease index = ∑(disease level × number of diseased plants) / (highest disease level × total number of plants in each treatment) × 100

[0042] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%

[0043] During the growth and fertilization period after sowing, the tobacco plants in the pot experiment were inoculated with cucumber mosaic virus (CMV) after microbial inoculants were applied. The phenotypic results of tobacco plants in different treatment groups were as follows: Figure 1 The statistical results of tobacco disease index and prevention and control effects are shown in Table 1 below.

[0044] Table 1 Statistics of the number of tobacco plants affected by diseases

[0045]

[0046] As shown in Table 1, at 20 days of tobacco growth, the incidence of cucumber mosaic virus in the control tobacco plant was approximately 65.63%, while that in the treatment group treated with the inoculant was approximately 34.38%, significantly reducing the incidence of CMV-induced diseases and demonstrating a CMV-inhibiting effect. At 30 days of tobacco growth, the incidence of cucumber mosaic virus in the control tobacco plant was approximately 93.75%, while that in the treatment group treated with the inoculant was approximately 56.25%. This indicates that the inoculant containing ΔPS04-17 has a significant control effect on cucumber mosaic virus in tobacco plants, reaching 54.72% against CMV-induced tobacco diseases in the field at 20 days of growth and 47.90% at 30 days of growth. This demonstrates that the space-induced mutant strain of Paenibacillus kriebensis ΔPS04-17 effectively prevents the occurrence of cucumber mosaic virus in tobacco plants, effectively inhibiting the spread of CMV within the plant, delaying the onset of disease, and preventing premature aging of the host plant.

[0047] Application of Experimental Example 3 in Tobacco Growth

[0048] After sowing, tobacco seeds are placed in a constant temperature culture environment for cultivation. During this period, regular irrigation is required to maintain appropriate moisture. After the tobacco seedlings successfully break through the soil, transplantation is carried out. The experiment is divided into two groups, with 7 tobacco plants in each group. The control group sprays clean water on the tobacco plant leaves, and the treatment group sprays the tobacco leaves with a 100-fold dilution of microbial agents. Both are sprayed once every 7 days, for a total of two sprayings. In addition to the difference in whether microbial agents are applied, the other treatments are the same. During the experiment, no other agents were used in each group to control pests and diseases. When the tobacco grows to the stage with 7 fully expanded leaves, the maximum leaf area, fresh weight of the aboveground part and the underground part of each group are measured.

[0049] The phenotypic results of tobacco plants in different treatment groups are as follows Figure 2 As shown in the figure, the average fresh weight of the aboveground part of each plant in the treatment group with microbial agents reached 3.5g, while the average fresh weight of each plant in the control group was 1.8g. The results showed a significant difference. Figure 3 As shown in the figure, the average fresh weight of the underground part of each plant in the treatment group reached 0.16g, while the average fresh weight of the underground part of each plant in the control group was 0.03g. The results showed significant differences, as shown in the statistics. Figure 4 As shown; the average maximum leaf area of ​​each plant in the treatment group reached 36cm 2 The maximum leaf area of ​​each plant in the control group was 22 cm 2 , the results are significantly different, as shown in the statistics Figure 5 The results showed that spraying a 100-fold diluted microbial agent of Paenibacillus kriebensis mutant △PS04-17 during tobacco growth promoted root development, enhanced tobacco growth, promoted tobacco growth, increased tobacco leaf area, and increased tobacco yield.

[0050] In summary, the present invention provides a new application of the space-induced mutant strain △PS04-17 of Paenibacillus cribensis in the prevention and treatment of cucumber mosaic virus. Studies have shown that the △PS04-17 strain can significantly reduce the incidence of CMV host plants and has the effect of inhibiting CMV. After the host plant is treated with the fermentation liquid of △PS04-17, the relative prevention effect against the tobacco disease tobacco cucumber mosaic virus caused by CMV reaches 54.72%. It can prevent the occurrence of tobacco cucumber mosaic virus disease, prevent premature aging of tobacco plants, effectively inhibit the spread of CMV in the plant body, and delay the onset of the disease. At the same time, the microbial agent prepared from the △PS04-17 strain can effectively promote the growth of tobacco plants, promote the development of tobacco roots, increase the area of ​​tobacco leaves, enhance the growth of tobacco, and significantly increase the yield and quality. It can not only reduce the use of pesticides, but also benefit the development of environmental safety. It can be used for green prevention and control of tobacco and is of great significance to improving the quality and efficiency of tobacco.

[0051] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Paenibacillus Cliburnii ( Paenibacillus kribbensis ) Application of the space mutant △PS04-17 strain in preventing and controlling cucumber mosaic virus, characterized in that: The Bacillus Cliburn space mutant △PS04-17 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

2. Use of the space mutant ΔPS04-17 strain of Paenibacillus kriebensis for preventing and controlling plant diseases caused by cucumber mosaic virus, characterized in that: The Bacillus Cliburn space mutant △PS04-17 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

3. Use of the space mutant ΔPS04-17 strain of Paenibacillus kriebensis in the preparation of a product for preventing and controlling cucumber mosaic virus, characterized in that: The Bacillus Cliburn space mutant △PS04-17 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

4. Use of the space mutant △PS04-17 strain of Paenibacillus kriebensis in promoting tobacco growth, characterized in that: The Bacillus Cliburn space mutant △PS04-17 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

5. Use of the space mutant △PS04-17 strain of Paenibacillus cribentii in the preparation of a growth-promoting agent, characterized in that: The Bacillus Cliburn space mutant △PS04-17 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

6. Use of the space mutant ΔPS04-17 strain of Paenibacillus kriebensis for increasing tobacco yield, characterized in that: The Bacillus Cliburn space mutant △PS04-17 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

7. A method for preventing and controlling cucumber mosaic virus or reducing the incidence of cucumber mosaic virus disease, characterized in that: The plants were treated with the space mutant △PS04-17 strain of Bacillus kriebensis or its bacterial liquid; the space mutant △PS04-17 strain of Bacillus kriebensis was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 10, 2022, with the deposit number: GDMCC NO: 62233.

8. The method according to claim 7, characterized in that: The bacterial liquid is a fermentation liquid.

9. The method according to claim 8, wherein The concentration of the fermentation liquid is not less than 1×10 9 CFU / mL.

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