Purification of Barley Yellow Streak Mosaic Virus and Detection by DAS-ELISA and ISEM Using Polyclonal Antibodies

Abstract

Skaf, J. S., and Carroll, T. W. 1995. Purification of barley yellow streak mosaic virus and detection by DAS-ELISA and ISEM using polyclonal antibodies. Plant Dis. 79:1003-1007. A purification procedure was developed for barley yellow streak mosaic virus (BaYSMV), the causal agent of a disease in barley and wheat. The procedure utilizes differential and density gradient centrifugation in Percoll gradients and yields highly concentrated virus preparations. These preparations were infectious in Nicotiana benthamiana. Electron microscopy of non-glutaraldehyde-fixed virus particles negatively stained or detergent-treated revealed the presence of a finely granular viral nucleocapsid and an envelope presumed to be lipoidal in nature. Internal cross striations characteristic of rhabdovirus particles were not observed. Concentrated virus preparations were used to produce rabbit polyclonal antibodies that were useful in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) for the detection of the virus in barley, wheat, N. benthamiana, as well as in its vector, the brown wheat mite Petrobia latens. The virus was detected by ELISA and ISEM in the Pocatello Valley, Idaho, in 1992 and 1993, verifying our 1991 detection of the virus in the same valley. Leaf extracts from BaYSMV-infected barley or N. benthamiana plants injected via hypodermic needle and syringe were infectious, causing characteristic disease symptoms in barley. By contrast, viral nucleic acid preparations used to rub-inoculate N. benthamiana plants were non-infectious.