从烟草丛顶病病原复合物中分离烟草扭脉病毒的方法

Methods for the isolation of Tobacco vein distorting virus from tobacco bushy top disease virus complex

烟草丛顶病是由烟草丛顶病毒（TBTV）、烟草丛顶病毒类似卫星RNA（Sat-TBTV）、烟草扭脉病毒（TVDV）和烟草扭脉病毒相关RNA（TVDVaRNA）复合侵染引起的一类特殊病害,目前尚不清楚这些病原物各自在病害症状形成和病害传播中的作用。本文根据蚜虫传播烟草丛顶病复合病原物传毒特性的差异,探索了3种分离TVDV的生物学方法。方法A为将获得了烟草丛顶病复合病原物的蚜虫饥饿24h后,以单虫单苗的方式接到健康烟株上传毒,24h后灭虫。方法B为将无毒蚜虫饥饿2-4h后转接到感病烟株上饲毒48h,再以单虫单苗的方式接到健康烟株上传毒,24h后灭虫。方法C为将无毒蚜虫于感病烟株上饲毒48h后,以单虫单苗的方式接到健康烟株上传毒24h,此后每隔24h将蚜虫移到另一健康烟株上传毒,直到蚜虫死亡。其中方法 A和B均可将TVDV从烟草丛顶病病原复合体中分开,方法A操作简单、分离周期短且TVDV的分离效率高。单独TVDV侵染的烟株无明显的丛顶症状,叶片靠近主脉的地方会出现皱缩,生长后期叶片变得细长且顶端会发生扭曲。本试验可为研究TVDV与其他病原物、寄主植物和传播介体的相互作用提供很好的试验材料,也可为蚜虫传播的复合侵染病毒的生物学分离提供参考。

Tobacco bushy top disease（TBTD）is caused by a complex agents comprising Tobacco bushy top virus（TBTV）,Tobacco bushy top virus satellite-like RNA（Sat-TBTV）,Tobacco vein distorting virus（TVDV）and Tobacco vein distorting virus-associated RNA（TVDVaRNA）.The roles of each agent played on tobacco for disease symptom development and disease transmission are still unclear.Based on the differences of aphid transmission characteristics among the complex agents,three biological methods were tested to isolate TVDV from tobacco bushy top disease virus complex.In method A,aphids were firstly fed on the diseased tobacco plants for dozens of days to gain the complex pathogens,and then the aphids were starved for 24 h.The single starved aphid was transferred to a single tobacco plant for agent transmission,and the aphids were killed by pesticide 24 h later.In method B,virus-free aphids were firstly starved for 2-4 h,then fed on the diseased tobacco plant for 48 h.Thesingle aphid was transferred to a single plant for agent transmission for 24 h.In method C,virus-free aphids were fed on the diseased plant for 48 h,and then single aphid was transferred to a single healthy tobacco plant for disease complex pathogen transmission for 24 h.The aphid was moved to another healthy tobacco plant every 24 h till the aphid was dead.The results showed that TVDV could be separated by methods A and B from tobacco bushy top disease virus complex,and method A was more simple and effective than method B.No bushy top disease symptoms could be observed from the tobacco plants infected with TVDV except shrinking in the area near the main vein of the leaf.The leaves of the infected plants tended to be longer and thinner,and the upper leaves showed vein-distorting at the later growth stage.The single TVDV isolated by using these methods could provide useful experimental material for studying the interaction among TVDV with other agents of TBTD,with host plants,or transmission vectors.The methods also might provide a guide for aphid-transmitted virus isolation from complex agent.