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【目的】筛选对烟草花叶病毒(Tobacco mosaic virus,TMV)和马铃薯Y病毒(Potato virus Y,PVY)具有显著拮抗活性的菌株。【方法】从山东诸城等植烟区采集烟草病毒病高发区健康烟株中烟100(Nicotianatabacum var.Zhongyan 100)的根际土壤,分离抗TMV的活性菌株,通过生理生化测定和分子鉴定确定菌株。采用Real-time PCR技术定量检测该菌株对TMV和PVY的抑制作用进一步验证该菌株的抗病毒活性。田间试验进行3个处理,菌液与病毒混合,2 h后接种(I),喷菌液2 h后接种病毒汁液(II),接种病毒汁液2 h后喷施菌液(III),以8%宁南霉素和肥皂水分别与等体积病毒汁液混合为阳性对照,以LB培养基与病毒汁液混合为空白对照。利用菌株发酵液对剪刀上病毒的钝化作用进行验证试验。【结果】筛选到的菌株通过枯斑寄主半叶法显示其对TMV的抑制效果达98%以上,命名为4A1,分子鉴定结果显示该菌株16S rDNA序列与Pseudomonas monteilii的16s rDNA序列同源率达到99%;菌株4A1为革兰氏阴性,氧化酶、淀粉水解VP、吲哚等反应为阴性,明胶液化、葡萄糖反应为阳性,能运动,确定该菌株为蒙氏假单胞菌(P.monteilii)。Real-time PCR检测结果显示其发酵液对TMV、PVY具有显著的抑制活性。田间试验结果显示,处理I可有效降低大部分病毒侵染,TMV和PVY发病率分别为5.3%和7.7%,病情指数分别为0.8和2.9;处理II可显著减少TMV和PVY的侵染几率,发病率分别为33.3%和36.7%,病情指数分别为4.2和6.0;处理III烟株发病率为95.0%和92.0%,病情指数为24.5和20.1;CK组TMV、PVY发病率分别为99.3%和95.4%,病情指数为别为38.6和45.1。菌株对剪叶工具的处理,钝化病毒效果可达95%以上,防效显著高于其它处理。【结论】在烟叶生产上,该菌株可以作为生产工具的生物消毒剂和抗病毒剂。
【Objective】 The objective of this study is to screen for strains that have significant antagonistic activity against Tobacco mosaic virus (TMV) and Potato virus Y (PVY). 【Method】 The rhizosphere soil of Nicotiana tabacum var. Zhongyan 100, a tobacco strain highly susceptible to tobacco virus disease, was collected from the tobacco planting areas in Zhucheng, Shandong Province, and the active strains against TMV were isolated. The strains were identified by physiological and biochemical assays and molecular identification . Real-time PCR was used to quantitatively test the inhibition of TMV and PVY by this strain to further verify the antiviral activity of this strain. In the field experiment, three treatments were carried out. The bacteria and virus were mixed and inoculated after 2 h (I). The virus solution (II) was inoculated 2 h after inoculation and the virus solution was inoculated for 2 h. % Ningnanmycin and soapy water were mixed with the same volume of virus juice as a positive control, with LB medium and virus juice mixed as a blank control. The use of strain fermentation broth on the passivation of scissors virus validation test. 【Result】 The results showed that the isolates showed more than 98% inhibitory effect on TMV by semi-leaf blotch of hemoblast, named as 4A1. The molecular identification showed that 16S rDNA sequence of this strain was homologous to 16s rDNA sequence of Pseudomonas monteilii 99%; strain 4A1 was negative for Gram, oxidase, starch hydrolysis VP, indole and other reactions were negative, liquefaction of gelatin, glucose reaction was positive, able to exercise, to determine the strain of Pseudomonas aeruginosa (P.monteilii ). Real-time PCR results showed that the fermentation broth of TMV, PVY significant inhibitory activity. The results of field trials showed that treatment I can effectively reduce the majority of virus infections, the incidence rates of TMV and PVY were 5.3% and 7.7% respectively, and the disease indices were 0.8 and 2.9 respectively. Treatment II could significantly reduce the infection rates of TMV and PVY, The incidences were 33.3% and 36.7% respectively, and the disease indices were 4.2 and 6.0 respectively. The incidences of tobacco plants in treatment III were 95.0% and 92.0% respectively, and the disease indices were 24.5 and 20.1 respectively. The incidences of TMV and PVY in CK group were 99.3% and 95.4%, disease index was 38.6 and 45.1 respectively. Strains of leaf cutting tools, passivation virus effect of up to 95%, control effect was significantly higher than the other treatments. 【Conclusion】 The strain can be used as biological disinfectant and antiviral in the production of tobacco leaves.