葡萄卷叶病毒的纯化,血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。

PURIFICATION, SEROLOGY AND DETECTION OF GRAPEVINE LEAF ROLL VIRUS

Closterovirus-like particles associated with grapevine leaf roll disease were purified from stem phloem tissue by differential centrifugation and cesium sulphate-sucrose density gradient centrifugation. The particles were between 660-2000 nm long. Most of them were about 1400 nm long and also decorated by the antiserum against GLRV NY-1 isolate. The purified preparation was tested for their serological relatedness in indirect ELISA. The results indicated that these closterovirus-like particles reacted with serotype II, III and IV of GLRV. The antiserum to NY-1 (type III) reacted more strongly than type IV (to VA-4) and type II. (to 1800 nm). In sodium dodecyl sulfate immunodiffusion test, the extract of diseased petiole reacted with serotype III, IV and II. It is possible to be infected by 2 or 3 closterovirus-like particles in China. We have made antiserum to the purified closterovirus-like GLRV and set up a PAS-ELISA to detect GLRV-free grapevine plantelets. We have obtaimed 21 varieties of GLRV-free and GFLV-free grapevine. They shown high quality and quantity in field test.