Bacillus aerobacter A3-5 with efficacy of preventing and treating tobacco mosaic disease, application of bacillus aerobacter A3-5 and tobacco planting method

Abstract

The invention discloses bacillus aerius A3-5 with a tobacco mosaic disease prevention and treatment effect and application thereof, and relates to the technical field of microbial prevention and treatment, the bacillus aerius A3-5 is preserved in the China Center for Type Culture Collection on July 18, 2024, the preservation number is CCTCC NO: M 20241594, and the bacillus aerius A3-5 is preserved in the China Center for Type Culture Collection on July 18, 2024, the preservation number is CCTCC NO: M 20241594, the preservation number is CCTCC NO: M 20241594, and the preservation number is CCTCC NO: M 20241594. The invention relates to application of bacillus aerosus A3-5 in prevention and treatment of tobacco mosaic disease. The effective component of the preparation for preventing and treating the tobacco mosaic disease comprises the aerobacillus A3-5. The strain has the advantages of being efficient in TMV resistance, green, free of pollution and the like, enriches a TMV-resistant biocontrol bacterium strain resource library, provides powerful support for diversification and sustainable development of plant virus prevention and treatment strategies, can induce tobacco to generate disease resistance, achieves a good antiviral effect and has good application prospects. The method can be applied to development of anti-mosaic virus biopesticides.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, relates to a microbial control technology for tobacco mosaic disease, and specifically relates to an airborne bacillus strain capable of inhibiting tobacco mosaic virus and a tobacco planting method using the strain. Background Art

[0002] Tobaco mosaic virus (TMV) is one of the main pathogens in the tobacco industry. It has a simple transmission route and a wide host range. It can infect 885 species of plants in more than 65 families, including tobacco. At present, measures to prevent and control TMV include agricultural management, chemical control and biological control. With the increasing public concern about health and the environment, the excessive use of chemical pesticides has caused problems such as environmental pesticide residues, increased resistance of pests and diseases, and recurrence of pests and diseases. In this context, biopesticides, as a new type of pesticide, have become the focus of the modern pesticide industry because of their low toxicity, low residue and low resistance.

[0003] Microbial pesticides, as a key branch of the field of biological pesticides, cover two major categories: living microbial preparations and agricultural antibiotics. These preparations are derived from biological resources in nature. After careful separation and purification, they can not only directly prevent and control pests and diseases, but also generate compounds with potential pesticide value through their metabolic processes. Among them, Bacillus ( Bacillus cereus ) has become the most widely used biocontrol bacteria resource due to its simple nutritional requirements, rapid reproduction, and rich metabolites. Bacillus velezensis ) SG11, for example, was effective against Botrytis cinerea ( Botrytis cinerea ) caused by tomato gray mold with an inhibitory effect of up to 79.81%. In addition, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) The suspension of F11 was effective against Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ) reached 100%. Air Bacillus ( Bacillus aerius )PAPM-14 strain against Rhizoctonia solani( Rhizoctonia solani ) also achieved an inhibition effect of 81.60%. Although biological pesticides cannot replace chemical pesticides for a certain period of time, with the recognition of the hazards of chemical pesticides and the restrictions on the conditions of use, Bacillus strains will become a type of microbial resource with a wide range of practical uses.

[0004] Since Mulvania first revealed the possibility of bacteria inhibiting plant virus activity in 1926, researchers have been committed to exploring TMV inhibitory active substances contained in microorganisms and their metabolites. At present, there have been many studies on the biological control of tobacco mosaic virus in China, and there have also been many screenings of biological control strains, most of which are bacteria and a few fungi. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art. In order to cope with the harm caused by excessive use of chemical pesticides and tap into local high-quality biocontrol bacteria resources, the present invention provides a strain of air Bacillus A3-5 with tobacco mosaic virus control effect. The strain has high anti-TMV efficiency, non-toxicity and non-pathogenicity, environmental friendliness, and antibacterial activity stability. The discovery of the strain provides a new and effective means for controlling tobacco mosaic virus and expands the biocontrol bacteria strain resource library.

[0006] The technical solution of the present invention is as follows: The first aspect of the present invention provides an air Bacillus A3-5 having the efficacy of preventing and treating tobacco mosaic disease, wherein the air Bacillus strain ( Bacillus aerius ) A3-5 was deposited in the China Center for Type Culture Collection on July 18, 2024, with the deposit number CCTCC NO: M 20241594. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] Preferably, the colony morphology of the aerobic Bacillus A3-5 is irregular, light yellow, easy to pick up, and has a smooth surface; under microscope observation, the bacterial body morphology is Gram-positive bacteria, rod-shaped.

[0008] The second aspect of the present invention provides the use of the aerobic Bacillus A3-5 in preventing and treating tobacco mosaic disease.

[0009] The third aspect of the present invention provides a method for preventing and controlling tobacco mosaic disease with the aerobic Bacillus A3-5, the method comprising the following steps: Inoculating aerobacillus sp. A3-5 into a liquid culture medium and culturing the liquid to obtain a fermentation liquid; The fermentation liquid is sprayed on tobacco leaves.

[0010] Preferably, the tobacco leaves are three-grown tobacco leaves.

[0011] Preferably, the liquid culture medium is beef extract peptone liquid culture medium.

[0012] Preferably, the culture conditions are: constant temperature shaking culture at 37°C and 200 r / min for 24 hours to obtain aerobic Bacillus A3-5 fermentation liquid, which is stored at 4°C for later use.

[0013] The fourth aspect of the present invention provides a biological agent for preventing and treating tobacco mosaic disease, wherein the active ingredient of the biological agent comprises the above-mentioned Bacillus aeruginosa A3-5.

[0014] A fifth aspect of the present invention provides a pharmaceutical composition for preventing and treating tobacco mosaic disease, wherein the active ingredient of the pharmaceutical composition comprises the above-mentioned aerobic Bacillus A3-5.

[0015] A tobacco planting method, comprising the steps of spraying the above-mentioned biological preparation or the above-mentioned pharmaceutical composition on tobacco leaves The present invention has at least one of the following beneficial effects: The air bacillus screened by the present invention ( Bacillus aerius ) A3-5 has the characteristics of high efficiency in resistance to TMV and being green and pollution-free. It not only enriches the resource library of biocontrol bacteria against TMV, but also provides strong support for the diversification and sustainable development of plant virus control strategies.

[0016] The fermentation liquid of the air Bacillus subtilis A3-5 strain of the present invention has a significant inhibitory effect on tobacco common mosaic virus. First, in the three-skinned tobacco dead spot experiment, the inhibition rate of TMV is high, and the inhibition rate of A3-5 fermentation liquid on TMV passivation can reach 91.18%. Further exploration of the inhibitory effect of A3-5 fermentation liquid on TMV shows that A3-5 can induce tobacco to produce disease resistance from the changes in the virus expression amount and defense enzymes in K326 tobacco. After inoculation of TMV, the resistance generated in the body makes it impossible for the tobacco-infected virus to reproduce in large quantities in the body, thereby achieving a relatively good antiviral effect. The strain A3-5 is applied to the development of anti-mosaic virus biopesticides, and new, efficient and safe microbial preparations can be developed to control plant viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The culture morphological characteristics and Gram staining observation results of strain A3-5 in Example 1 of the present invention are shown; Figure 2 is the phylogenetic tree of strain A3-5 in Example 1 of the present invention; Figure 3 This is a photo of tobacco leaves treated with the A3-5 fermentation liquid treatment group and the CK control group in Example 2 of the present invention; Figure 4 This is a graph showing changes in TMV expression in the A3-5 fermentation broth treatment group and the CK clear water control group in Example 2 of the present invention; Figure 5 This is a graph showing the determination of POD, SOD, and PAL defense enzyme activities in the A3-5 fermentation broth treatment group and the CK control group in Example 2 of the present invention. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0020] Example 1 Air Bacillus ( Bacillus aerius )A3-5 screening 1. Isolation and purification of strains The strain A3-5 was isolated from the perennial rotation tobacco soil of the tobacco planting base in Tanxi Township, Lichuan County, Fuzhou City, Jiangxi Province by pure culture. The specific method is to use the soil collected from the tobacco planting base in Tanxi Township, Lichuan County, Fuzhou City, Jiangxi Province during the tobacco harvest period in July 2020 and June 2021 for tobacco cultivation. When the tobacco grows to 6-7 leaves, the root soil is taken for separation and purification of soil microorganisms. After dilution according to the conventional separation method, it is spread on beef extract peptone medium, potato agar medium (PDA), and Gao's No. 1 medium, and cultured in a constant temperature incubator for 2-5 days. Among them, Gao's No. 1 medium and PDA medium are cultured in a 28°C incubator, and beef extract peptone medium is cultured in a 37°C incubator. Pick a single colony for plate streaking purification, and the purified colonies are used for TMV passivation test and plant disease resistance induction test, and the effective bacteria are sent for testing and preservation.

[0021] Results: The strain A3-5, which had a high inactivation effect on TMV and could induce disease resistance in plants, was successfully isolated from beef extract peptone medium.

[0022] Formula of beef extract peptone solid culture medium: peptone 10g, sodium chloride 5g, beef extract 3g, agar 15-20g. Dissolve in 400ml water first, then add water to 1000ml, adjust pH to 7.2±0.3, and sterilize at 121℃ for 20min.

[0023] Gao's medium No. 1 and PDA medium are commercially available culture media.

[0024] 2. Strain Identification The strain A3-5 was identified by morphological characteristics and 16SrDNA sequence analysis, as follows: 2.1. Culture morphological characteristics of strains Morphological observation and culture characteristics: The strain was streaked onto beef extract peptone medium and cultured at 37°C for 3-5 days. The morphological characteristics such as colony size, color, and morphology were observed under a microscope, and then a single colony was picked for staining and observation.

[0025] Gram staining: First, fix the smear, stain it with crystal violet for 2 minutes, wash it with sterile water, stain it with iodine solution for 1 minute, wash it with sterile water, decolorize it with 95% ethanol, rinse it and stain it with safranin solution for 2 minutes. Finally, check the staining results with an oil microscope. If the staining result is purple, it is Gram-positive bacteria, and if it is red, it is Gram-negative bacteria.

[0026] The results are as follows Figure 1 As shown, the colony morphology of strain A3-5 is irregular, light yellow, easy to pick up, and smooth; microscopic observation of the bacterial morphology is Gram-positive bacteria, rod-shaped.

[0027] 2.2. 16S rDNA sequence analysis and identification The DNA of the strain was extracted using the Novozymes bacterial DNA extraction kit. After extracting the total genomic DNA of the strain, universal primers for the 16 S rDNA gene were designed. The sequences of the amplification primers were: Forward 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; Reverse 1492R: 5′-GGTTACCTTGTTACGACTT-3′; The length of the amplified fragment was approximately 1445 bp.

[0028] The extracted total DNA was used as a PCR template, and the PCR products were analyzed by electrophoresis using 1% agarose gel, and the corresponding results were observed using a gel imager. The SanPrep column DNA gel recovery kit from Qingke Biotech Co., Ltd. was used. The recovered PCR products were sent to Qingke Biotech Co., Ltd. for sequencing. The sequencing results were compared for homology in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the phylogenetic tree of strain A3-5 was constructed using MEGA11 software, as shown in the figure. Figure 2 shown.

[0029] Air Bacillus ( Bacillus aerius ) The 16S rDNA sequencing result of A3-5 is shown in SEQ ID NO: 1:

[0030] The strain was deposited in the China Center for Type Culture Collection on July 18, 2024, and its classification name is Bacillus aeruginosa ( Bacillus aerius )A3-5, the deposit number is CCTCC NO: M 20241594, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0031] Example 2 Detection of antiviral activity of aerobic Bacillus A3-5 fermentation broth 1. Preparation of A3-5 Fermentation Broth Inoculate Bacillus aerogenes A3-5 into beef extract peptone liquid culture medium (preparation method: 3g beef extract, 5g NaCl, 10g peptone, add water to 1000mL, adjust pH 7.0-7.2), culture at 37℃, 200 r / min for 24h to obtain A3-5 fermentation broth, and store at 4℃ for later use.

[0032] 2. The control effect of A3-5 fermentation liquid on tobacco against TMV by dead spot method Choose healthy 8-leaf Sansheng tobacco ( Nicotiana tabacum var samsun NN), select leaves of uniform size and with symmetrical midribs.

[0033] Take common tobacco leaves infected with TMV and showing severe symptoms of tobacco mosaic disease, grind and mix with inoculated PBS buffer (10mM phosphate buffer, pH7.4) at a ratio of 1:10 (w / v) to serve as TMV virus inoculation solution; A3-5 treatment group: The A3-5 fermentation liquid obtained above was mixed with the TMV virus inoculation liquid at a ratio of 1:1 (v / v), and the mixture was placed at 25°C, allowed to stand for 30 minutes, and then the right half of the leaf of the selected 8-leaf Sansheng tobacco was inoculated with the inoculation liquid; CK control group: Mix the above buffer solution with the virus inoculation solution at a ratio of 1:1 (v / v), and inoculate the left half of the leaf half an hour later as a control; inoculate 6 leaves for each treatment, and perform 3 repeated treatments; After obvious necrosis appeared, the number of necrosis was recorded, the average value was taken, and the inhibition rate was calculated according to the following formula: inhibition rate (%) = [1-(number of treated necrosis spots / number of control necrosis spots)] × 100%.

[0034] The number of treatment necrotic spots is the average number of necrotic spots in the A3-5 treatment group (right half of the leaf), and the number of control necrotic spots is the average number of necrotic spots in the CK control group (left half of the leaf).

[0035] 3. Mechanism of A3-5 fermentation broth against TMV in tobacco We selected healthy and uniformly growing common tobacco K326 at the 6-8 leaf stage and set different treatments: CK: TMV inoculation 24 h after spraying with clean water; Group A3-5: The TMV virus solution obtained above was inoculated 24 hours after spraying the fermentation liquid of A3-5 strain.

[0036] Leaves from the same part of tobacco in each group were taken at 1, 3, 7, and 9 days after inoculation, and 3 plants were randomly selected for each treatment, repeated 3 times. The leaves were collected and quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator to determine the activity of related defense enzymes and the expression of viruses by qRT-PCR.

[0037] 3.1. qRT-PCR measurement of viral expression The RNA of the leaves collected above was extracted using the Trizol method. 50-100 mg of fresh plant tissue was minced as much as possible, and then 1 mL of Total RNA Extraction Reagent was added to mix well. The homogenized sample was shaken vigorously and placed at room temperature for 5 minutes to completely dissociate the ribosomes. Centrifuge at 4°C, 12, 000 rpm for 10 min, and the supernatant was collected. 0.2 mL of chloroform was added to the above lysate. The centrifuge tube was tightly capped, and the tube was shaken vigorously for 15 sec. The tube was placed at room temperature for 2-3 min. Centrifuge at 4°C, 12,000 rpm for 10-15 min. The upper aqueous phase was carefully pipetted into a new centrifuge tube, and an equal volume of isopropanol was added. Inverted to mix well and placed at room temperature for 10 min. Centrifuge at 4°C, 12, 000 rpm for 10 min. The supernatant was carefully discarded and 1 mL of 75% ethanol was added. Vortex to wash thoroughly, and flick the bottom of the tube to suspend the precipitate. Centrifuge at 4°C, 12,000 rpm for 5 min, and discard the supernatant. Air dry at room temperature for 5-10 min. Add 30-100 μL of RNase-free water to dissolve RNA. After complete dissolution, take a small amount for testing, and store the remaining solution at -70°C. Use Nanodrop ultra-micro spectrophotometer to measure the nucleic acid concentration of the extracted total RNA, synthesize and amplify tobacco cDNA after the determination, and use tobacco cDNA for real-time fluorescence quantitative PCR to determine the TMV content in tobacco. β-Actin of common tobacco K326 samples treated at different time periods was used as the internal reference gene, and TMV-CP in tobacco treated with fermentation broth of each strain was used as the target gene content to evaluate the effect of fermentation broth on tobacco resistance. The relative expression level of TMV-CP gene under each treatment in different time periods was calculated according to 2-ΔΔCt.

[0038] 3.2. Determination of defense enzyme activity The peroxidase (POD) activity was determined by the guaiacol method. The reaction system was 2.9 mL 0.05 mol / L PBS buffer (pH 5.5), 1.0 mL 2% H2O2, 1.0 mL 0.05 mol / L guaiacol and 0.1 mL enzyme solution. The mixture was kept in a 37 ℃ water bath for 5 min. PBS was used as a blank control instead of enzyme solution. The timer was immediately started after mixing. The absorbance change was measured at 470 nm within 3 min. A change of 0.01 per minute was considered as one enzyme activity unit U / (g·min).

[0039] The activity of superoxide dismutase (SOD) was determined using a superoxide dismutase activity detection kit. 0.1 g of tobacco leaves were added with 1 mL of extract and homogenized in an ice bath. The mixture was centrifuged at 8,000 g and 4 °C for 10 min, and the supernatant was taken as the crude enzyme solution, which was placed on ice for testing. The crude enzyme solution was added to the reagent and mixed well. The premixed solution without the sample was the blank control group. After being incubated in a 37 °C water bath for 30 min, the solution was placed in a 1 mL glass cuvette and the absorbance at 560 nm was measured.

[0040] Phenylalanine ammonia lyase (PAL) activity was determined using a phenylalanine ammonia lyase detection kit. 0.1 g of tobacco leaves were added with 1 mL of extract and homogenized in an ice bath; centrifuged at 8,000 g, 4 °C for 10 min, and the supernatant was taken as the crude enzyme solution, which was placed on ice for testing. The crude enzyme solution was added to the reagent and mixed well. The premix without sample was used as the blank control group. After standing for 10 min, the absorbance of the measuring tube was recorded at 290 nm.

[0041] 4. Test Results 4.1 Results of the test on the control effect of Bacillus aeruginosa A3-5 fermentation liquid on tobacco by the dead spot method After calculation, the inhibition rate was 91.18%.

[0042] Figure 3 The picture shows the tobacco leaves treated with A3-5 fermentation liquid (Bacillus aerogenes A3-5 fermentation liquid and TMV virus inoculum 1:1 mixed) and CK control group (PBS buffer and TMV virus inoculum 1:1 mixed). Figure 3 It can be seen that there are only a few white necrotic spots on the treated leaves (right half of the leaves) of the treatment group, while a large number of white necrotic spots appear on the treated leaves of the control group (left half of the leaves), indicating that the treatment group has a control effect on TMV.

[0043] Therefore, the above test results show that the fermentation liquid of Bacillus aeruginosa A3-5 in this example has a good control effect on tobacco mosaic disease.

[0044] 4.2 Test results of the anti-TMV mechanism of Bacillus aeruginosa A3-5 fermentation broth on tobacco Figure 4 The figure shows the changes in TMV expression in the A3-5 fermentation liquid treatment group and the CK water control group. Figure 4 It can be seen that on days 1, 3, 7, and 9, the expression levels of TMV in the A3-5 fermentation broth treatment group were significantly lower than those in the CK group, indicating that the A3-5 treatment group could effectively inhibit the proliferation of TMV.

[0045] Figure 5 The results show the results of the determination of defense enzyme activity. The activities of POD, PAL, and SOD in tobacco treated with A3-5 fermentation liquid were improved to varying degrees compared with the CK group. The results showed that the activities of POD and SOD reached their peaks on the 7th day; the activity of PAL reached its peak on the 5th day. This shows that A3-5 fermentation liquid can increase the activity of defense enzymes in tobacco to resist the invasion of TMV.

[0046] In summary, it can be seen from the changes in virus expression and defense enzymes in K326 tobacco that A3-5 can induce disease resistance in tobacco. After inoculation with TMV, the resistance produced in the body prevents the virus infected by tobacco from multiplying in large quantities in the body, thereby achieving a better antiviral effect.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A strain of Bacillus aeruginosa A3-5 having the efficacy of preventing and treating tobacco mosaic disease, characterized in that: The aerobic Bacillus strain ( Bacillus aerius ) A3-5 was deposited in the China Center for Type Culture Collection on July 18, 2024, with the deposit number CCTCC NO: M 20241594. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The air bacillus A3-5 according to claim 1, characterized in that The colony morphology of the air bacillus A3-5 is irregular, light yellow, easy to pick up, and smooth on the surface; under microscope observation, the bacterial body morphology is Gram-positive bacteria, rod-shaped.

3. Use of the air bacillus A3-5 as described in any one of claims 1 to 2 in preventing and treating tobacco mosaic disease.

4. A method for preventing and treating tobacco mosaic disease using the air Bacillus A3-5 as described in any one of claims 1 to 2, characterized in that: The method comprises the following steps: Inoculating aerobacillus sp. A3-5 into a liquid culture medium and culturing the liquid to obtain a fermentation liquid; The fermentation liquid is sprayed on tobacco leaves.

5. The method for preventing and treating tobacco mosaic disease according to claim 4, characterized in that: The tobacco leaves are three-year-old tobacco leaves.

6. The method for preventing and controlling tobacco mosaic disease according to claim 4, characterized in that: The liquid culture medium is beef extract peptone liquid culture medium.

7. The method for preventing and controlling tobacco mosaic disease according to claim 4, characterized in that: The culture conditions are: The culture was cultured at 37°C and 200 r / min for 24 h to obtain the fermentation liquid of Bacillus aerogenes A3-5, which was stored at 4°C for later use.

8. A biological agent for preventing and treating tobacco mosaic disease, characterized in that: The active ingredient comprises the aerobic Bacillus A3-5 described in any one of claims 1 to 2.

9. A pharmaceutical composition for preventing and treating tobacco mosaic disease, characterized in that: The active ingredient comprises the aerobic Bacillus A3-5 described in any one of claims 1 to 2.

10. A tobacco planting method, characterized in that: The method comprises the step of spraying the biological agent according to claim 8 or the pharmaceutical composition according to claim 9 onto tobacco leaves.

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