Abstract: 【Objective】To study the mechanism of resistance to tobacco gray mold induced by the active metabolite Roflamycoin and Men-myco-A（R&M）of Streptomyces roseoflavus Men-myco-93-63 under greenhouse conditions，so as to provide theoretical basis for the ecofriendly use of R&M to prevent and control tobacco gray mold.【Method】Eighty mg/L R&M was injected to 4-5 leaf stage of Nicotiana benthamiana to conduct hypersensitive response（HR）staining verification and 100 mg/L R&M was injected to 4-5 leaf stage of N.benthamiana to get expression of the disease resistance marker gene hsr203J respectively to verify the induced disease resistance of R&M.Changes of catalase（CAT） activity，lipoxygenase（LOX）activity and content of hydrogen peroxide（H₂O₂），a defense-related substance，in non-pharmaceutically treated tobacco leaves of N.benthamiana were detected by kits（treatments of 80 mg/L R&M spray and water treatment）.Quantitative real-time PCR（qRT-PCR）technology was used to detect changes of relative expression of lignin synthesis-related gene PAL and pathogenesis-related protein（PR）genes PR1 and PR5 after tobacco treatments（treatment 1，80 mg/L R&M solution for root treatment for 2 d，friction inoculation with Botrytis cinerea spore suspension on the upper third leaves；treatment 2，root irrigation with water for 2 d，friction inoculation with water on the upper third leaves）.【Result】Catalase（CAT）activity in tobacco leaves gradually increased after induction treatment with 80 mg/L R&M；while lipoxygenase（LOX）activity increased significantly after 3 d（P<0.05，the same below），and both reached the highest on 7 d.H₂O₂ content increased rapidly on the 1 d and then decreased.Relative expression of PAL gene significantly was up-regulated after 24 h，which was 25.37 folds of that of CK.Relative expression of PR genes，PR1 and PR5，reached the highest at 24 h and 72 h respectively，which were 11.23 times and 11.17 times of those of the CK.【Conclusion】R&M enhances disease resistance of tobacco plants through increasing the activity of related defense enzymes in tobacco leaves，coordinating the level of active oxygen and increasing the expression level of disease resistance-related genes.R&M，an active metabolite produced by S.roseoflavus Men-myco-93-63，is potential to be elicitor of disease of plant and expected to be applied in ecofriendly prevention and control of diseases such as gray mold.