Study On Resistance Of Tobacco To Tobacco Etch Virus By Hypersensive Protein

Tobacco etch virus(TEV),which is a member of Potato virus Y,was first discovered in 1930 in the United States Kentucky and then was founded inpart of Canadian tobacco areas,and now it becomes a worldwide virus disease.Tobacco etch viruses are mainly transmitted through non-persistent aphids,as while as seed propagation and mechanical transmission.Because of the differences of virus strain,species of host and growth state,the symptoms of the disease are not the same.Generally speaking,the initial onset of the plant is small spot,with the increase in the number of spots,here comes the linear necrotic tissue and mottled,along the veins development.When the disease is serious,the lesion is full of leaves,to the late onset of disease tissue will dry off,and only left the dry veins.Recent years,the tobacco etch virus has becomes the fourth largest virus,following the Cucumber mosaic virus(CMV),Tobacco common mosaic virus(TMV),and thePotato virus Y(PVY).Tobacco etch virus in China's major tobacco areas generally occur,in Shaanxiprovince the incidence rate was around 10% and increased year by year.Bacillus amyloliquefaciens,a kind ofBacillus genus,a high affinity with Bacillus subtilis,is a class of important beneficial microbial resources.In the previous laboratory work,a Bacillus amyloliquefaciensBa-168 strain resistant to virus disease was screened from the rhizosphere soil of Qinling plant.Synthesis of Bacillus from predecessors,the Bacillus amyloliquefaciens strain Ba-168 was used as the starting point,and the protein elicitors in the fermentation broth of the strain were induced to induce plant resistance,many method were used to separate and predicate the protein,such as filtration centrifugation,ammonium sulfate fractionation,anion exchange chromatography and so on.The molecular weight of the protein was found to be between 25 and 30 kDa by protein electrophoresis.In order to clarify the induced resistance of the hypersensitive protein JDF-d to the tobacco etch virus in the fermentation broth of Bacillus amyloliquefaciens,in this experiment,Sani tobacco and ordinary cigarette NC89 were used as experimental materials,the inactivation in vitro of the hypersensitive protein JDF-d was determined,as while as the preventive effect ofhypersensitive protein JDF-d.In addition to that,by determining the activity of tobacco-related defensive enzymes to clear the resistance of the hypersensitive protein JDF-d to tobacco and the effect of the protein on tobacco growth.In this study,we examined the effect of hypersensitive protein JDF-d crude protein extract on tobacco leaf erosion virus;It was found that different concentrations of hypersensitive protein JDF-d crude protein extract could improve the inhibitory effect of tobacco on tobacco etch virus.The inhibitory effect gradually increased with the increase of the concentration of crude protein extract,when it comes to300?g/mL,the inhibition rate remains at a steady level,so the 300?g/mL was chosen as the further experiments.The passivation test of tobacco etch virus was carried out using 300?g/mL of the hypersensitive protein JDF-d crude protein extract,the experimental results showed that the effect of hypersensitive protein on in vitro passivation of tobacco etch virus was not obvious.Indicating that the hypersensitive protein JDF-d itself does not have the inhibitory effect on the virus particles.And then the protective effect of 300?g/mL hypersensitive protein JDF-d crude protein extract on tobacco was determined,the experimental results showed thatthe protective effect of 48 hours and 24 hours before inoculation were 67.08% and 71.33% which were significantly higher than the control group.A variety of metabolites secreted by Bacillus amyloliquefaciens during fermentation or self-growth,which can be a variety of pathogens have a significant inhibitory effect,also promote the growth of plant,has a broad development and application prospects.But because of its fermentation broth in 5% sheep blood agar plate on the hemolysis phenomenon,which restrictions on the further development of Bacillus amyloliquefaciens,and the safety of the strain caused by hidden dangers.Mutagenesis of Bacillus amyloliquefaciensBa-168 was carried out by physical and chemical mutagenesis;six mutant strains with hemolytic deletions were screened out by mutant strains on blood agar plates.Through this test,Bacillus amyloliquefaciens was found to be more sensitive to UV mutagenesis,higher mortality and more successful.The lethal rate and the success rate of mutagenesis were lower,the highest lethal rate of nitrite was 54.09%,and the time was 6min.The mutant strain LXY series was detected by Colletetrichum orbicalare as an indicator bacterium.It was found that the hemolytic ring and the inhibition zone of the five mutant strains were significantly smaller than those of the original strain.However,the antimicrobial activity of a mutant strain LXY-6 obtained by UV mutagenesis was not affected.The mutant strain had a strong antibacterial activity without hemolysis.