Improved detection of petunia vein clearing caulimovirus

Petunia vein clearing virus (PVCV), a possible member of the caulimovirus g roup, was detected in several cultivars of vegetatively propagated petunias (Petunia xhybrida Hort. Volm.-Andr.) grown in commercial nurseries. Leaf d ip preparations and ultrathin sections of leaf tissue were analyzed by tran smission electron microscopy (TEM). Spherical virus particles, 45-50 nm in diameter, were observed in samples taken from symptomatic petunia plants. T he virus was purified and a polyclonal antiserum was prepared. In immune-sp ecific electron microscopy (ISEM), the PVCV antiserum-treated samples react ed with a distinct decoration on the virus suspect particles. A polymerase chain reaction (PCR)-based assay was used to detect PVCV in total nucleic a cid extracts derived from infected petunia plants. Two primer pairs were de signed to flank a 736-base-pair sequence located in the RNA-dependent RNA p olymerase gene of the PVCV genome. A DNA fragment of predicted size was vis ualized in agarose gels. The authenticity of the amplified DNA fragment was confirmed by restriction analysis and by hybridization with the virus-spec ific PVCV DNA probe. The virus could be detected efficiently in high diluti ons of sap extracted from infected petunia plants.