Determination of hypovirulence strains of Cryphonectria parasitica with dsRNA analysis by using non-phenol-chloform and their detection by RT-PCR

Chestnut blight, Cryphonectria parasitica, is a serious disease of Castanea spp. and causes bark cankers that progressively kill branches and trunks. Hypovirulent cancers were first identified with the awareness of chestnut trees recovering in Italy in 1964. It has been determined that the hypovirulent isolates of C. parasitica have dsRNA which obtained from chestnut areas of the Black Sea and Marmara regions in our country. The dsRNA isolation and analysis method of Morris and Dodds is usually used to determine the presence of dsDNA in hypovirulent isolates. In this study, to determine the presence of dsRNA in C. parasitica isolates, which may be hypovirulent, method of Balijja and collaborators has been used. Balijja's method has advantages such as low amount of mycelium and chemicals used according to other isolation method, the phenol-chloroform inclusion and the isolation period of dsRNA can be realized in one day, the study was planned in order to facilitate the future research of our country and region. For this purpose, isolates, were collected from healing cankers of chestnut areas of the Aegean, Black Sea and Marmara, in which light-white micelle formation was observed were used. In this study 30 out of 68 isolates analyzed by Balijja's and only identified dsRNA profile in 25 isolates. dsRNA-containing 6 isolates were analyzed by method of Morris and Dodds and confirmed the existence of dsRNA. RT-PCR revealed that 25 out of 15 dsRNAs belonged to the hypovirus Cryphonectria hypovirus 1 (CHV-1).