Comparisons of gene diagnostic methods for the practical diagnosis of chrysanthemum stunt viroid in chrysanthemum plants

Abstract

The sensitivity of gene diagnostic methods for detecting chrysanthemum stunt viroid (CSVd) in 2 M LiCl‐soluble nucleic acids extracted from chrysanthemum plants was examined. Dot blot hybridization using DIG‐labeled cRNA and cDNA probes was compared. The cRNA probe was at least 25 times more sensitive than the cDNA probe and had a superior signal‐to‐noise ratio. Although reverse transcription and polymerase chain reaction (RT‐PCR) is much more sensitive in theory, in practice it was only 5 times more sensitive than hybridization using the cRNA probe. In addition, the nucleic acid extracts used inhibited the cDNA amplifications, probably due to natural inhibitors such as polysaccharides and polyphenolic compounds in the plants. HC1 treatment following ethanol precipitation of the nucleic acid extracts was effective in rapidly eliminating the inhibitory factors. The combination of RT‐PCR and hybridization on microplate well expanded the detectable range and was at least 25 times more sensitive than agarose gel analysis of the RT‐PCR products. Based on these results, the use of practical diagnosis for protecting chrysanthemum from CSVd and for certifying CSVd‐free chrysanthemum is discussed.