CN110438049B

CN110438049B - Tobacco common mosaic virus biocontrol alternaria alternata strain and application thereof

Info

Publication number: CN110438049B
Application number: CN201910792441.2A
Authority: CN
Inventors: 杨金广; 李莹; 申莉莉; 王杰; 王凤龙; 周志成; 张超群; 刘天波
Original assignee: Jiangxi tobacco science research institute; TOBACCO AGRICULTURAL EXPERIMENT STATION OF CENTRAL-SOUTH CHINA; Tobacco Research Institute of CAAS
Current assignee: Jiangxi tobacco science research institute; TOBACCO AGRICULTURAL EXPERIMENT STATION OF CENTRAL-SOUTH CHINA; Tobacco Research Institute of CAAS
Priority date: 2019-08-26
Filing date: 2019-08-26
Publication date: 2021-05-25
Anticipated expiration: 2039-08-26
Also published as: CN110438049A

Abstract

The invention discloses a tobacco common mosaic virus biocontrol alternaria alternata (Pseudomonas issachhenkonii) strain, the strain number is H12, the strain is preserved in the China general microbiological culture Collection center in 2016, 10, 14 days, and the strain preservation number is CGMCC No. 13113. The strain can inhibit infection of Tobacco Mosaic Virus (TMV), and biocontrol experiments show that the strain can prevent and treat the TMV. Meanwhile, the invention also provides a method for obtaining the active substance of the strain, which has higher application value in the aspect of preventing and treating tobacco mosaic virus.

Description

Tobacco common mosaic virus biocontrol alternaria alternata strain and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a tobacco common mosaic virus biocontrol alternanthera stutzeri strain, wherein the strain and active substances separated from the strain are applied to the control aspect of the tobacco common mosaic virus.

Background

Tobacco Mosaic Virus (TMV) is a plant virus disease caused by TMV and is one of the most serious diseases damaging Tobacco. The TMV has strong stress resistance, simple propagation path and wide host range. Research reports that TMV can infect more than 350 host plants such as potatoes, tomatoes, tobaccos and the like, infected leaves are still high in infection activity after being treated at high temperature of 120 ℃ for 10min, and the infected leaves can survive for 52 years in dry tobacco leaves. If the plants are infected with TMV in the seedling stage, the yield and the quality are more influenced, so that the grade of tobacco leaves is reduced, and the requirements of the cigarette industry on high-quality cigarette raw materials cannot be met. According to statistics, the flue-cured tobacco yield loss caused by TMV in 2009 of 16 tobacco-planting provinces in China reaches 168.27 ten thousand kg, and the yield loss is 1806.81 ten thousand yuan.

Once the plants are infected with TMV, contact among diseased plants and field farming operation are easy to cause re-infection, the disease propagation speed is high, and the prevention and the control are harder along with the occurrence of compound infection. For a long time, the main control of TMV in production is chemical control, but the wide use of chemical pesticides causes a series of worrying problems of food safety, ecological environment and society. In 1925, researches show that the plant virus extract polluted by bacteria loses the infection capacity, which opens up a new direction for preventing and treating plant virus diseases by using microorganisms and metabolites thereof. The biological agent is safe and harmless, and the application of the biological agent in preventing and treating TMV can prevent pesticide residue and environmental pollution. Along with the increasing attention and concern of people on the health of smoking, green pollution-free biological agents are increasingly paid attention and become the core of comprehensive prevention and treatment, so that research and development of some green and effective biological agents for resisting tobacco mosaic disease are required.

Domestic researches on biological control of tobacco mosaic virus have been carried out, but most of the researches are limited to the stages of biological control bacteria screening and potted plant experiment, and the researches are rarely carried out on the aspects of separation of antibacterial active substances and the like. The invention explores from the aspect of biological prevention and control, screens antagonistic strains with excellent prevention and growth promotion effects, separates antagonistic active substances of the antagonistic strains, and utilizes the antagonistic active substances to prevent and control tobacco mosaic virus diseases.

Disclosure of Invention

The invention aims to provide a tobacco common mosaic virus biocontrol pseudoalteromonas strain, and the strain and active substances separated from the strain are applied to the aspect of tobacco common mosaic virus prevention and control, so as to provide a new microbial resource for the prevention and control of tobacco common mosaic virus.

The aim of the invention is realized by separating the strain from the ocean sandy soil sample, identifying the strain as a Pseudoalteromonas (Pseudoalteromonas sp) strain with the strain code of H12, and storing the strain in the China general microbiological culture Collection center at 10-14 months in 2016 with the strain preservation number of CGMCC No. 13113.

The method for preparing fermentation liquid and TMV-resistant active substance from pseudoalteromonas comprises inoculating activated H12 strain in LB culture medium, performing shaking culture at 28 deg.C and 140rpm for 48H, and diluting with sterile water to 1 × 10⁸cfu/mL as a fermentation broth for standby; centrifuging the fermentation liquor at 4 ℃ and 12000rpm for 10min, removing thallus, and preparing fermentation supernatant of extracellular metabolite to obtain the active substance for resisting TMV.

The biocontrol pseudoalteromonas H12 strain for antagonizing tobacco mosaic virus can be applied to prevention and control of the tobacco mosaic virus, the inhibition rate of fermentation liquor on TMV is determined to be 97.22% by using a local spot method, and the prevention effect of fermentation supernatant of the H12 strain on TMV is up to 84.72%. Meanwhile, the growth promoting effect of the H12 strain fermentation liquor is good, TMV infection and proliferation can be inhibited, and the strain fermentation liquor has certain practical application value in production.

Drawings

FIG. 1 is a graph showing the effect of antagonistic activity of a strain on TMV;

FIG. 2 is a graph of TMV inhibition by strain fermentation supernatants;

FIG. 3 is a graph showing the inhibitory effect of different treatments on TMV (a: H12 fermentation supernatant; b: 1000-fold liquid of Altailing; c: LB liquid medium);

FIG. 4 is a graph showing the growth promoting effect of H12 fermentation broth on Nicotiana benthamiana root systems (a: H12 fermentation broth; b: sterile water);

FIG. 5 is a graph showing the growth promoting and anti-TMV effects of H12 fermentation broth on Nicotiana benthamiana (a: sterile water; b: H12 fermentation broth);

FIG. 6H12 strain plate line graph;

FIG. 7 is a cell morphology of H12 strain under a transmission electron microscope.

Detailed Description

The invention is further described with reference to the accompanying drawings, but the invention is not limited in any way, and any variations or modifications based on the teachings of the invention are within the scope of the invention.

Example one

The inhibitory effect of the obtained strain on TMV was determined by the local focal spot method. Weighing 1g of fresh TMV diseased leaves, adding 40mL of sterilized phosphate buffer solution, grinding into homogenate, filtering by using sterilized gauze, and taking supernatant as inoculation liquid; inoculating the strain into LB liquid culture medium at a ratio of 1:100, performing large-scale fermentation culture, and diluting with sterile water to 1 × 10⁸cfu/mL fermentation liquor is used as anti-TMV bacterial liquid for standby. Mixing the anti-TMV bacterial liquid (fermentation liquid) and the TMV inoculation liquid in equal amount, mixing the LB liquid culture medium and the TMV inoculation liquid in equal amount, standing for 10min at room temperature, friction inoculating the Sansheng-NN tobacco, and spraying a proper amount of carborundum on the surface of the leaf before inoculation. The mixed solution of the diluted solution of the left half-leaf inoculation bacterial liquid and the TMV inoculation liquid, the mixed solution of the right half-leaf inoculation LB liquid culture medium and the TMV inoculation liquid are used as a reference, and 200 mu L of the mixed solution is inoculated to each half leaf. After inoculation, the leaves were washed with clear water and repeated 3 times. The observation results after 3d inoculation are shown in FIG. 1, and the inhibition rate is calculated by counting the number of dead spots. The result shows that the strain fermentation liquor has good effect of inhibiting TMV, the inhibition rate of the scorched spots is as high as 97.22 percent (Table 1), and the biocontrol strain generates a resistant substance for inhibiting the activity of the TMV in the growth and metabolism process.

The inhibition ratio (%) [ 1- (treatment average number of scorched spots/control average number of scorched spots) ] × 100.

TABLE 1 inhibition of TMV by different treatments

Example two

In order to compare the disease-resistant effect of the H12 fermentation supernatant and other antiviral agents on TMV, the inventor inoculates the H12 strain into an LB liquid culture medium by using an inoculating loop, cultures the strain in a shaking way at 28 ℃ and 140rpm for 48H, centrifuges the strain at 4 ℃ and 12000rpm for 10min, removes thallus to prepare the fermentation supernatant of extracellular metabolites, and simultaneously selects 1000-fold liquid of the Altailing and the LB liquid culture medium to compare the disease-resistant effect.

Transplanting the three-generation-NN tobacco seedlings in the 4-5 leaf stage into slow seedlings, and respectively spraying H12 fermentation supernatant, a 1000-fold liquid of Tailing, an LB liquid culture medium and sterile water, wherein 10mL of each plant is obtained, 4 plants are treated, and 3 groups are repeated. TMV was inoculated after 48h, and the growth of each treated tobacco strain after 3d was shown in FIGS. 2 and 3. The tobacco plant sprayed with 1000 times of liquid of the Altailing and LB liquid culture medium has relatively serious disease incidence, the prevention effect on the TMV is 31.00 percent and 5.35 percent respectively, while the tobacco plant sprayed with H12 fermentation supernatant has dark green leaf color, lighter disease incidence and less withered spots, and the prevention effect on the TMV is as high as 84.72 percent.

Example three

To verify the growth promoting effect of the H12 fermentation liquid on the Nicotiana benthamiana root system, the inventor transplants the Nicotiana benthamiana seedlings in the 4-5 leaf stage into test tubes, and cultures the seedlings with the H12 fermentation liquid and sterile water respectively. Repeat in 3 groups per treatment 7 strains. The growth condition of the Nicotiana benthamiana after 10 days is shown in figure 4, and compared with the Nicotiana benthamiana cultured in sterile water, the Nicotiana benthamiana cultured in the H12 fermentation liquid is longer in root system, good in growth condition and remarkable in growth promoting effect.

In order to further verify the growth promotion and anti-TMV effects of the H12 fermentation liquor on Nicotiana benthamiana, the inventor transplants the Nicotiana benthamiana seedlings in the 4-5 leaf stage for seedling rejuvenation, inoculates TMV, irrigates roots with the H12 fermentation liquor and sterile water after 6 hours, wherein 10mL of each strain is irrigated with roots for 3 times, 3d intervals are provided for each time, and 3 groups are repeated after 7 strains are treated. The growth condition of the Nicotiana benthamiana after 10 days is shown in figure 5, and the tobacco seedlings treated by sterile water contrast are short, partial tobacco leaves wither and the disease symptoms are obvious, while the tobacco seedlings treated by H12 fermentation liquor root irrigation are high in strain, the withering condition of the tobacco leaves is light, and the growth promoting effect of the H12 fermentation liquor is good, and the tobacco seedlings have a certain TMV (Tetramethylbenzidine) resistance effect.

Example four

The inventor refers to the kit specification to carry out DNA extraction and PCR amplification on the H12 strain, uses a bacterial universal primer as an amplification primer, and hands the PCR amplification result to Beijing Huada Dazhongsheng Biotech limited company to determine the 16s rDNA sequence. The determined sequence was compared with the sequence in GenBank by the BLAST program on the NCBI website, and as a result, it was found that the sequence determined by the H12 strain was identical to a partial sequence of the 16s rDNA of Alternaria alternata and had high homology, and thus the H12 strain was identified as Alternaria alternata.

The activated H12 strain is dipped in the inoculating loop and streaked on an LB solid culture medium, and the colony morphology is observed after the culture is carried out for 24H at the temperature of 28 ℃. As a result, H12 colonies were found to be orange, round, smooth in surface, wet, viscous, and flat (FIG. 6). After the H12 fermentation supernatant was subjected to JEM-100CX transmission electron microscopy, the shape of the cells was short rod-like and mobile as shown in FIG. 7.

Claims

1. A tobacco common mosaic virus (TMV) biocontrol alternaria alternata strain, alternaria alternata (A) and (B)Pseudoalteromonas issachenkonii) The strain has the code number of H12, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2016, 10 months and 14 days, and has the strain preservation number of CGMCC number 13113.

2. Use of the alteromonas reinhardtii strain of claim 1 for the control of tobacco mosaic virus.

3. A method for preparing a TMV-resistant bacterial liquid by using the Pseudomonas ehrlichi strain according to claim 1, wherein the TMV-resistant bacterial liquid comprises: inoculating the strain, fermenting, culturing, diluting with sterile water to 1 × 10⁸cfu/mL strain fermentation liquor to obtain the anti-TMV bacterial liquid.

4. The method for preparing a TMV-resistant bacterial liquid from the alternaria alternata strain according to claim 3, wherein the TMV-resistant bacterial liquid is prepared from the alternaria alternata strain: the activated H12 strain was inoculated in LB medium, cultured with shaking at 140rpm at 28 ℃ for 48 hours, and then diluted to 1X 10 with sterile water⁸cfu/mL, and centrifuging at 12000rpm at 4 deg.C for 10min to remove thallus and obtain fermentation supernatant of extracellular metabolite.

5. The TMV-resistant bacterial liquid prepared by the method for preparing TMV-resistant bacterial liquid by using the alternaria pseudomona stutzeri strain according to claim 4.

6. The application of the TMV-resistant bacterial liquid as defined in claim 5 in prevention and treatment of tobacco mosaic virus.