Improved Detection of Petunia Vein Clearing Caulimovirus

Abstract

Petunia vein clearing virus (PVCV), a possible member of the caulimovirus group, was detected in several cultivars of vegetatively propagated petunias (Petunia ×hybrida Hort. Volm.-Andr.) grown in commercial nurseries. Leaf dip preparations and ultrathin sections of leaf tissue were analyzed by transmission electron microscopy (TEM). Spherical virus particles, 45-50 nm in diameter, were observed in samples taken from symptomatic petunia plants. The virus was purified and a polyclonal antiserum was prepared. In immuno-specific electron microscopy (ISEM), the PVCV antiserum-treated samples reacted with a distinct decoration on the virus suspect particles. A polymerase chain reaction (PCR)-based assay was used to detect PVCV in total nucleic acid extracts derived from infected petunia plants. Two primer pairs were designed to flank a 736-base-pair sequence located in the RNA-dependent RNA polymerase gene of the PVCV genome. A DNA fragment of predicted size was visualized in agarose gels. The authenticity of the amplified DNA fragment was confirmed by restriction analysis and by hybridization with the virus-specific PVCV DNA probe. The virus could be detected efficiently in high dilutions of sap extracted from infected petunia plants.