Differentiation among garlic viruses in mixed infections based on RT-PCR procedures and direct tissue blotting immunoassays

Abstract

A total of six viruses belonging to at least three distioct genera, Potyvirus, Carlavirus, and a novel unclassified genus, were identified from infected garlic plants in Japan based on partial cDNA coloning and sequencing of genomes. We developed procedures that combined reverse-transcription polymerase chain reaction (RT-PCR) with restriction analysis for indentification of each of the six viruses. The respective viral coat protein genes were expressed as fusion proteins in Escherichia coli, and the products were used as immunogens for producing antibodies that reacted against viral particles. The antisera obtained specifically recognized each of three types of rod-shaped virus particles according to immunoelectron microscopy and enzyme-linked immunosorbent assay. Furthermore, a convenient serological method based on direct tissue blotting immunoassy (DTBIA) was developed. Survey of virus incidence using both DTBIA and RT-PCR presented direct evidence for mixed infections of garlic plants with several viruses. In addition, DTBIA is suitable for large-scale and routine diagnosis of garlic viruses.