The Screening And Investigation On Antagonistic Mechanism Of A Bacterium Strain With Antiviral Activity Against TMV

The production and character of Chinese crops was seriously threaten by tobacco mosaic virus（TMV）. Biological control bacteria had become an efficient way to control the tobacco virus disease. Anew bacterium strain named YNLP-2with great antiviral activity against TMV was screened usinghalf-leaf local necrosis lesion method from rhizosphere of healthy tobacco plants grown intobacco-growing area of Luoping of Yunnan province. And the identification, antiviral activity andmechanism were investigated in this study, and the small molecular components with antiviral activityagainst TMV were screened and identified in present study. The mainly results were as following:（1） The antiviral activity of YNLP-2bacterium strain was determined using half-leaf local necrosislesion method. It was showed significant antivirus activity against Tobacco mosaic virus （TMV） whichwas up to99%at the bacteria cell concentration of5.6×10⁸cfu/mL. The strain YNLP-2was determinedas Bacillus cereus through mycelia morphology, sequence analysis of16S rDNA, physiological andbiochemical test. The supernatant and the thallus of the strain YNLP-2displayed significant antivirusactivity against TMV, with high heat and acid stability by using half-leaf local lesion approach. YNLP-2suggested the control efficiency to TMV disease with incidence rate of45.65%.（2） Though half-leaf local necrosis lesion analysis, the TMV inhibitory activity of ethyl acetateextracted inorganic and organic phase was92.667%±1.528%and92.333%±2.517%respectively.Whereas the organic phase of8-2（absolute ethyl alcohol:purified water=8:2） extract and the inorganicphase from7-3（absolute ethyl alcohol:purified water=7:3） extract demonstrated the TMV inhibitoryactivity with92.000%and90.667%respectively. Together, these observations indicated the smallmolecular compound of YNLP-2possessing significant antivirus activity.12kinds of stable and smallmolecular components extracted from ethyl acetate were determined by GC-MS.（3） YNLP-2could disrupt the particles of TMV into small chunks at the observation oftransmission electron microscope. However, YNLP-2strain could not destroy the coat protein of TMVas the same width and the brilliance of protein strip with control in SDS-PAGE analysis. Duing to thewidth and the brilliance of protein strip decreased in SDS-PAGE analysis suggesting the inorganic phasefrom7-3extract could destroy the coat protein of TMV.（4） Detection of TMV by qPCR revealed the tobacco could be induced TMV resistance （IR） bystrain YNLP-2. After infection the disease resistant varieties of N. tabacum var. Samsun NN and thesusceptible varieties of N. tabacum var. NC89in10min by TMV, the content of Ca²⁺, K⁺and H₂O₂inSamsun NN was higher than NC89with non-significant variation of O₂. At the same time, the contentof K⁺and H₂O₂raised after infection NC89with strain YNLP-2. Hence we primary inferred themechanism of tobacco induced resistance by strain YNLP-2was accompany with the increase of K⁺andH₂O₂.A new bacterium YNLP-2was investigated in this study, and showed significant antivirus activityagainst TMV which was up to99%by half-leaf local necrosis lesion under5.6×10⁸cfu/mL. Themixture contained12kinds of small molecular components screened from ethyl butyl acetate were crude extract of YNLP-2fermentation broth determined in this study.