Microbacterium strain nd023 and its application in resisting tobacco mosaic virus

Abstract

The present application relates to micrococcus, and provides a micrococcus strain ND023 and application of the micrococcus strain ND023 in resisting tobacco mosaic virus. The micrococcus strain ND023 has been preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on April 25, 2023, and the preservation number is CCTCC NO:M 2023617. The micrococcus strain ND023 can be used as a microbial pesticide to prevent and treat tobacco mosaic virus.

Description

Technical Field

[0001] This invention relates to microbes, and more specifically to microbes used for microbial control and their applications. Background Technology

[0002] Tobacco mosaic virus (TMV) is a pathogen that seriously harms tobacco. It is easy to spread and has a wide range of hosts, capable of infecting more than 885 plant species from over 65 families other than tobacco. When TMV infects tobacco plants, the most prominent symptom is the formation of mottled yellow-green leaves. The edges of diseased leaves sometimes curl backwards, severely impacting tobacco growth and causing a decline in both field yield and leaf quality.

[0003] Currently, methods for controlling TMV include agricultural control, chemical control, and biological control. Microbial pesticides are an important type of biological pesticide, including live microbial pesticides and agricultural antibiotics. Microbial pesticides are isolated and purified from natural products and can act directly on pests and diseases, or produce metabolites that can be used as new pesticides. Microorganisms themselves are abundant and widely distributed. Since Mulvania discovered in 1926 that bacteria could inhibit the biological activity of plant viruses, research has begun to search for TMV active substances from microorganisms and their secondary metabolites, but progress has been slow. Summary of the Invention

[0004] In view of this, the present invention provides a microbacterial strain ND023 and its application against tobacco mosaic virus, which at least solves one problem existing in the prior art.

[0005] In a first aspect, the present invention provides a strain of Microbacterium ND023 ( Microbacterium sp The microbacterial strain ND023 has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on April 25, 2023, with accession number CCTCC NO: M 2023617.

[0006] The 16S rDNA sequence of the microbacterial strain ND023 is shown in Sequence 1.

[0007] Secondly, the present invention provides the application of the aforementioned microbacterial strain ND023 in the prevention and control of tobacco mosaic disease.

[0008] In some preferred embodiments, the tobacco mosaic disease is common tobacco mosaic disease.

[0009] In some preferred embodiments, the application includes the step of spraying the fermentation broth of the microbacterial strain ND023 onto tobacco leaves.

[0010] In some preferred embodiments, the tobacco leaf is a terrestrial tobacco leaf.

[0011] Thirdly, the present invention provides a method for preventing and controlling tobacco mosaic disease, the method comprising the following steps: The microbacterial strain ND023 was inoculated into a liquid culture medium to obtain a fermentation broth; The fermentation liquid is sprayed onto tobacco leaves.

[0012] In some preferred embodiments, the liquid culture medium is Gao's No. 1 liquid culture medium.

[0013] Fourthly, the present invention provides a pharmaceutical composition for preventing and treating tobacco mosaic disease, the pharmaceutical composition comprising the microbacterial strain ND023.

[0014] Fifthly, the present invention provides a biological control agent for preventing and controlling tobacco mosaic disease, wherein the biological control agent is prepared using the Microbacterium strain ND023 as the strain.

[0015] Due to the adoption of the above technical solutions, the embodiments of the present invention have the following beneficial effects: The fermentation broth of *Microbacterium* strain ND023 has a significant inhibitory effect on tobacco mosaic virus (TMV). Firstly, it exhibits a high inhibition rate against TMV in the three-stage tobacco blight experiment, with the fermentation broth of *Microbacterium* strain ND023 showing an inhibition rate of up to 76% against TMV inactivation. Further investigation into the inhibitory effect of the fermentation broth of *Microbacterium* strain ND023 on TMV revealed changes in virus expression levels and defense enzymes in K326 tobacco plants. This indicates that *Microbacterium* strain ND023 can induce disease resistance in tobacco. After inoculation with TMV, the resistance produced within the tobacco prevents the virus from multiplying in large quantities, thus achieving a relatively good antiviral effect. Applying *Microbacterium* strain ND023 to the development of anti-mosaic virus biopesticides can lead to the development of novel, highly efficient, and safe microbial agents for controlling plant viral diseases. Attached Figure Description

[0016] Figure 1 The culture morphology characteristics and staining observation results of the microbacterial strain ND023 in the embodiments of the present invention are shown.

[0017] Figure 2 The phylogenetic tree of Microbacterium strain ND023 in an embodiment of the present invention is shown.

[0018] Figure 3 The images show the symptoms of leaf blight in the fermentation broth treatment group and the water control group of the Microbacterium strain ND023 in this invention.

[0019] Figure 4The changes in TMV expression levels in the fermentation broth treatment group and the water control group of Microbacterium strain ND023 in the embodiments of the present invention are shown.

[0020] Figure 5 The results of the determination of the activity of the defense enzyme in the embodiments of the present invention are shown. Detailed Implementation

[0021] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully illustrate the purpose, solution and effects of the present invention.

[0022] With the increasing severity of plant viral diseases and the long-term, extensive use of chemical pesticides, problems such as environmental pollution and pesticide residues have emerged. Developing new, efficient, and safe microbial agents to control plant viral diseases has become essential for agricultural safety and sustainable development. Therefore, the inventors explored the control of plant viral diseases from the perspective of tobacco roots, screening for the antagonistic strain ND023 (accession number: CCTCC NO: M 2023617) with good control effects, expanding the resources for combating plant viruses and providing a guarantee for further research and development of microbial antiviral agents. The following examples describe the isolation, identification, and anti-TMV activity detection of the microbial strain ND023.

[0023] Example 1 Isolation and purification of Microbacterium strain ND023: Soil collected from a tobacco planting base in Tanxi Township, Lichuan County, Fuzhou City, Jiangxi Province during the tobacco harvest in July 2020 was used to cultivate tobacco. When the tobacco plants grew to 6-7 leaves, soil samples were taken from the root system for the isolation and purification of soil microorganisms. Single colonies were isolated by plating on PDA medium, beef extract peptone medium, and Gao's No. 1 medium. After streak purification, the microbial strain ND023 was isolated.

[0024] The isolated and purified *Microbacterium* strain ND023 was inoculated onto three different culture media: PDA agar plates were incubated at 30°C, beef extract peptone agar plates at 37°C, and Gao's No. 1 agar plates at 28°C. The incubation period was 2-5 days. When distinct colonies were observed, their morphology was examined. Figure 1 As shown.

[0025] Example 2 Morphological characteristics analysis of Microbacterium strain ND023: Morphological observation and culture characteristics: Microbacterium strain ND023 was streaked onto Gao's No. 1 medium, and a coverslip was inserted into the medium at an angle. After incubation at 28°C for 3-5 days, the culture was observed under a microscope.

[0026] Lactophenol cotton blue staining: Place 1-2 drops of lactophenol cotton blue staining solution on a clean glass slide. Use an inoculation loop to take a small amount of the solution from the edge of the colony and place it in the staining solution. Then carefully cover it with a coverslip and observe it under a microscope.

[0027] Gram staining: First, fix the smear, then primary stain with crystal violet for 1 min. Wash with sterile water, mordant with iodine solution for 1 min, wash with sterile water, decolorize with 95% ethanol, rinse, and stain with safranin solution for 10 seconds. Finally, examine the staining results under an oil immersion microscope. Figure 1 As shown, the Gram staining result is purple, indicating that the strain is Gram-positive and has an irregular short rod-like morphology. Example 3 Identification of the 16S rDNA sequence of Microbacterium strain ND023: DNA was extracted from *Microbacterium* strain ND023 using a DNA extraction kit. After extracting the total genomic DNA of the strain, universal primers for the 16S rDNA gene were designed. The sequences of the primers for amplification are as follows: Forward 7F: 5'-CAGAGTTTGATCCTGGCT-3', Reverse 1540R: 5'-AGGAGGTGATCCAGCCGCA-3', The amplified fragment was approximately 1450 bp in length. Using the extracted total DNA as a PCR template, the PCR products were analyzed by electrophoresis on a 1% agarose gel, and the results were observed using a gel imaging system. The SanPrep column-based DNA gel extraction kit from Beijing Qingke Biotechnology Co., Ltd. was used. The recovered PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequencing results were analyzed using BLAST in the NCBI database for homology comparison. The results showed that strain ND023 had the highest similarity to *Microbacterium* (99.00%), and it was preliminarily identified as belonging to the *Microbacterium* genus.

[0028] The 16S rDNA sequencing results of Microbe strain ND023 are as follows:

[0029] Example 4 Antiviral activity assay of Microbacterium ND023 fermentation broth: (1) Preparation of fermentation broth of Microbacterium ND023 Microbacterium ND023 was inoculated into Gao's No. 1 liquid culture medium (preparation method: 20g soluble starch, 0.5g NaCl, 1g KNO3, 0.5g K2HPO4·3H2O, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 0.5g NaCl, add water to 1000 mL, and adjust pH to 7.4-7.6). It was cultured at 28℃ and 200 r / min for 3 days to obtain the fermentation broth of Microbacterium ND023, which was stored at 4℃.

[0030] (2) Detection of the effect of Microbe ND023 fermentation broth on TMV control on tobacco by the spot method Choose healthy 8-leaf stage Sansheng tobacco ( Nicotiana tabacum var samsun Select leaves of uniform size and with symmetrical midribs. Take common tobacco leaves with severe symptoms of tobacco mosaic virus caused by TMV (Tobacco Mosaic Virus) infection, grind and mix them with PBS buffer (10mM phosphate buffer, pH 7.4) at a ratio of 1:10 (w / v) to prepare TMV virus inoculation solution.

[0031] The above-obtained *Bacillus ND023* fermentation broth was mixed with TMV virus inoculation solution at a 1:1 ratio. After standing at 25℃ for 30 min, the mixture was used to rub and inoculate the left half of the selected 8-leaf stage *Nicotiana trifoliata* leaves. PBS buffer and TMV virus inoculation solution were mixed at a 1:1 ratio, and after half an hour, the mixture was rubbed and inoculated onto the right half of the leaves as a control. Six leaves were treated in each treatment, with three replicates. After obvious necrotic spots appeared, the number of necrotic spots was recorded, the average value was taken, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (number of treated necrotic spots / number of control necrotic spots)] × 100.

[0032] like Figure 3 As shown in the figure, the results indicate that the leaves of the control group treated with the fermentation broth of strain ND023 showed significantly lower TMV infection symptoms than the control group treated with water.

[0033] (3) Mechanism of resistance to TMV in tobacco by fermentation broth of Microbacterium ND023 Select healthy, uniformly growing common tobacco variety K326 at the 6-leaf stage, and set up different treatments for the experiment: CK: Inoculate with TMV 24 hours after spraying with water; T: Inoculate the obtained TMV virus solution 24 hours after spraying the microbacterium ND023 fermentation broth.

[0034] Leaves from the same part of the tobacco plants in each group were collected at 1, 3, 5, 7, and 9 days after inoculation. Three plants were randomly selected for each treatment, and the results were repeated three times. The leaves were collected, flash-frozen in liquid nitrogen, and stored at -80°C for later determination of relevant defense enzyme activities and qRT-PCR to determine virus expression.

[0035] (a) qRT-PCR to measure viral expression levels RNA was extracted from the leaves using the Trizol method. 50 mg of fresh plant tissue was minced and mixed with 1 mL of Total RNA Extraction Reagent. The homogenate was vigorously vortexed and incubated at room temperature for 5 minutes to allow complete ribosome dissociation. The sample was then centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. 0.2 mL of chloroform was added to the supernatant, the centrifuge tube was capped tightly, and the mixture was vigorously vortexed for 15 seconds, then incubated at room temperature for 2-3 minutes. The sample was then centrifuged at 12000 rpm for 10-15 minutes at 4°C. The upper aqueous phase was carefully transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The tube was inverted and mixed thoroughly, then incubated at room temperature for 10 minutes. The sample was then centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was carefully discarded, and 1 mL of 75% ethanol was added. The tube was vortexed thoroughly, and the bottom was gently tapped to resuspend the precipitate. The sample was then centrifuged at 12000 rpm for 5 minutes at 4°C, and the supernatant was discarded. After incubating at room temperature for 5 min, add 30 μL of RNase-free water to dissolve the RNA. Once completely dissolved, take a small amount for testing, and store the remaining solution at -70℃.

[0036] The total RNA extracted was analyzed using a Nanodrop micro-volume spectrophotometer to determine the nucleic acid concentration. Following the determination, tobacco cDNA was synthesized and amplified. Real-time quantitative PCR was then performed using the tobacco cDNA to determine the TMV content in the tobacco. K326 samples of ordinary tobacco were treated at different time points. β-Actin As an internal reference gene, it is used in tobacco plants treated with fermentation broths of various microbial strains. TMV-CP The target gene content was used to assess the effect of the fermentation broth on tobacco resistance, based on 2 -ΔΔCt Calculate the processing under different time periods TMV-CP The relative expression levels of genes. Results are as follows: Figure 4 As shown, there was no significant difference in the changes of TMV-CP content in tobacco from day 1 to day 7, and it increased after day 7. However, the TMV content of ND023 fermentation broth was significantly lower than that of the CK group, indicating that ND023 fermentation broth can inhibit the proliferation of TMV in tobacco and protect tobacco plants.

[0037] (b) Assay of defensive enzyme activity Peroxidase (POD) activity was determined using the guaiacol method: The reaction system consisted of 2.9 mL of 0.05 mol / L PBS buffer (pH 5.5), 1.0 mL of 2% H2O2, 1.0 mL of 0.05 mol / L guaiacol, and 0.1 mL of enzyme solution. The mixture was incubated in a water bath at 37°C for 5 min. PBS buffer served as a blank control instead of enzyme solution. Immediately after mixing, the absorbance was measured at 470 nm over 3 min. A change of 0.01 μL per minute was defined as one unit of enzyme activity, U / (g·min).

[0038] Superoxide dismutase (SOD) activity was detected using a superoxide dismutase activity assay kit: 0.1 g of tobacco leaves were added to 1 mL of extraction buffer and homogenized in an ice bath; the mixture was centrifuged at 8000 g and 4℃ for 10 min, and the supernatant was collected as the crude enzyme solution, which was then placed on ice for testing. The crude enzyme solution was added to the reagent and mixed thoroughly. The premixed solution without sample was used as a blank control group. After incubating in a 37℃ water bath for 30 min, the absorbance was measured at 560 nm in a 1 mL glass cuvette.

[0039] The activity of phenylalanine ammonia-lyase (PAL) was determined using a phenylalanine ammonia-lyase assay kit: 0.1 g of tobacco leaves were added to 1 mL of extraction solution and homogenized in an ice bath; the mixture was centrifuged at 8000 g and 4℃ for 10 min, and the supernatant was collected as the crude enzyme solution, which was then placed on ice for testing. The crude enzyme solution was added to the reagent and mixed thoroughly. The premixed solution without sample was used as a blank control group. After standing for 10 min, the absorbance of the test tube was recorded at 290 nm.

[0040] The results of the defense enzyme activity assay are as follows: Figure 5 As shown, the activities of POD, PAL, and SOD in tobacco treated with ND023 fermentation broth were increased to varying degrees compared with the CK group. The results showed that POD activity reached its peak on the third day, with an increase of 400%; PAL activity reached its peak on the ninth day, with an increase of 192.31%; and SOD activity reached its peak on the ninth day, with an increase of 278.50%.

[0041] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention using the same means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. A strain of microbacterium ( Microbacterium sp ND023, characterized in that: The microbacterial strain ND023 has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on April 25, 2023, with accession number CCTCC NO: M 2023617.

2. The application of the microbacterial strain ND023 according to claim 1 in the prevention and control of tobacco common mosaic disease.

3. The application of the microbacterial strain ND023 according to claim 2 in the prevention and control of tobacco mosaic virus, characterized in that: The application includes the step of spraying the fermentation broth of the microbacterial strain ND023 onto tobacco leaves.

4. The application of the microbacterial strain ND023 according to claim 3 in the prevention and control of tobacco mosaic virus, characterized in that: The tobacco leaves are Thomson's tobacco leaves.

5. A method for preventing and controlling tobacco mosaic virus, characterized in that, The method includes the following steps: The microbacterial strain ND023 described in claim 1 was inoculated into a liquid culture medium to obtain a fermentation broth; The fermentation liquid is sprayed onto tobacco leaves.

6. The method for controlling tobacco mosaic virus according to claim 5, characterized in that: The liquid culture medium is Gao's No. 1 liquid culture medium.

7. A pharmaceutical composition for preventing and controlling tobacco mosaic virus, characterized in that: The pharmaceutical composition comprises the microbacterial strain ND023 as described in claim 1.

8. A biological control agent for controlling tobacco mosaic virus, characterized in that: The biological control agent is prepared using the microbacterial strain ND023 described in claim 1 as the bacterial strain.

Images