Isolation And Preliminary Inhibitory Mechanism Against Potato Virus Y Of Active Metabolites From Strain JZ2-1-12

The disease caused by Potato virus Y which is widely-distributed is difficult to be controlled.It seriously threatened the quality and yield of crops and had caused huge economic losses to the society.The second metabolites from microbes have inhibitory activities against pathogens,weeds,and plant viruses,which have shown magnificent development valuable in agricultural biological control field.In this research,strain JZ2-1-12 isolated from the distiller's grain of highland barley wine was the research object.The strain was identified.The active substance with inhibitory activity against PVY was isolated and purified from its secondary metabolites.Then,the inhibitory mechanism of the active substance was preliminary explored.The inhibitory activity of different extracts from the fermentation broth of strain JZ2-1-12 against PVY was determined by half leaf method.The activity of ethyl acetate and n-butanol extracts at the concentration of 100 mg/m L against PVY is equivalent to that of the control agent ningnanmycin at the concentration of 100 mg/m L,with inhibition rates 66.89% and 71.46%,respectively.The ethyl acetate and n-butanol extracts was determined as active extracts of strain JZ2-1-12.The strain JZ2-1-12 was identified as Bacillus subtilis subsp.subtilis by morphology,physiological and biochemical characteristics,and molecular biology methods.The active extract was prepared from the fermentation broth of strain JZ2-1-12.Then,extract was isolated and purified by silica gel column chromatography,thin layer chromatography analysis,and high performance liquid chromatography techniques to obtain fraction E5-6-5.This fraction was analyzed by spectral analysis to obtain a total of 8 compounds: 1,2-Chrysenediol(1),3,4-Chrysene-diol(2),1,2-Dihydro-1,2-chrysenediol(3),5-Methyl-1,2-dihydro-1,2-chrysendiol(4),1,1 '-[1,1-Ethanediylbis(oxymethylene)]dibenzene(5),1,4-Diphenylbutane-2,3-diol(6),(11-Methyl-3,4-dihydro-3,4-chrysenediol(7),and Ferulic acid ethyl ester(8).The inhibitory activity of fraction E5-6-5 at different concentrations against PVY was determined by half leaf method.The inhibitory activity of fraction E5-6-5 against PVY at the concentration of 500.00 ?g/m L was higher than that of ningnanmycin at the concentration of 1000.00 ?g/m L with inhibition rate 73.44%.The EC50 value of the fraction E5-6-5 against PVY was 209.58 ?g/m L.The fraction E5-6-5 was determines as the active fraction of this strain.The effect of fraction E5-6-5 on the expression of PVY nucleic acid RNA was detected by q RT-PCR method.After 7 days of treatment of fraction E5-6-5 at the concentration of 500.00 ?g/m L,the expression of PVY nucleic acid RNA reached to the lowest level,with inhibition rate 77.41%.The results showed that the active fraction E5-6-5 significantly down-regulated the expression of PVY nucleic acid RNA,and inhibited the proliferation of PVY before the photosynthetic capacity and respiration rate of the plant significantly decreased.This study is expected to develop a new microbial energy from distiller's grain of highland barley wine,which could provide new bioactive fractions for the biological control of PVY.After that,it will provide a new way for development and application of microbes from special environments with antiviral acitivity.