Determination of Hypovirulence Strains of Cryphonectria parasitica by Using Non-Phenol-Chloroform dsRNA Analysis Method and Detection of Cryphonectria hypovirus 1 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, and followed by hypovirulent strains containing the dsRNA virus used as a biological control agent. 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 of Turkey. The dsRNA isolation and analysis method of Morris ve Dodds (1979) is usually used to determine the presence of dsRNA in hypovirulent isolates. In this study, to determine the presence of dsRNA in C. parasitica isolates, which may be hypovirulent, the method of Balijja and collaborators has been used (Balijja et al., 2008). This study was planned in order to investigate whether this method is applicable for conducting research in the future due to its advantages such as shorter time requirements, less amount of mycelium and chemicals used, and not involving phenol-chloroform compared to other isolation methods. For this purpose, the isolates collected from healing cankers of chestnut areas in the Aegean, Black Sea and Marmara regions of Turkey, where light-white mycelium formation was observed, were used. In this study, 30 isolates out of 68 were analyzed by Balijja et al.’s method and only 25 isolates were identified to contain dsRNA profile. dsRNA-containing six isolates were also analyzed by the method of Morris and Dodds, and the existence of dsRNA was confirmed. It was found that 15 of 25 dsRNAs belonged to the hypovirus Cryphonectria hypovirus 1 (CHV-1) by RT-PCR