Gene Diagnosis of Viroids: Comparisons of Return-PAGE and Hybridization Using DIG-labeled DNA and RNA Probes for Practical Diagnosis of Hop Stunt, Citrus Exocortis and Apple Scar Skin Viroids in Their Natural Host Plants.

Abstract

Return-PAGE was reliable for diagnosis of HSVd in hop, CEVd in citron and ASSVd in apple, but was not reliable for HSVd in grapevine and citron, and ASSVd in pear, because of their low concentration. A disadvantage of return-PAGE was possible appearance of double or triple bands in case of mix-infecting samples such as hop or citron. Although DIG-labeled DNA probe was 2.5 to 25 times more sensitive than return-PAGE, the practical reliability of this method was not so superior to return-PAGE. DIG-labeled RNA probe was the most sensitive of the three methods examined in this experiment, which was 25-125 times and 100-625 times more sensitive than DIG-DNA probe and return-PAGE, respectively. Furthermore, DIG-RNA probe was sensitive enough to detect HSVd in grapevine, a symptomless host of the pathogen, although negative sample was emphasized to be re-examined by the other sensitive method such as cucumber bioassay or RT-PCR. DIG-RNA probe, however, is not sensitive enough to detect HSVd in citrus and ASSVd in pear. Based on the results obtained in this experiment, we proposed appropriate amounts of plant tissues for the successful diagnosis of the three viroids.