AMELIORATION OF A REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) FOR THE DETECTION OF ASSVD, PBCVD AND PLMVD VIROIDS, AND THEIR PRESENCE IN CULTIVATED AND WILD POME AND STONE FRUITS IN GREECE

Research in Greece on pome and stone fruit viroids is limited compared to the extent and the severity of the problem. On the other hand, the existence of sensitive, reliable, fast and mass diagnostic methods is a prerequisite for the development of a certification system which can contribute to the restriction of viroid spread and also to the production of healthy and of high quality propagation material. Present research focuses on Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd) and Peach latent mosaic viroid (PLMVd). Reverse transcription polymerase chain reaction (RT-PCR) was selected and optimized for the detection of the three viroids. Primer pairs were designed for each viroid from conserved regions of the viroid genomes and with the lowest possible secondary structure. Concerning the sample preparation, the protocols described by Rowhani et al. (1995), Coffin and Coutts (1997) and Rott and Jelkmann (2001) were applied with minor modifications. The last method exhibited the best detection results. In the RT step, in order to reduce the detrimental effects of the viroid high secondary structure in primer annealing, we used several detergents. The use of Tertamethylene Sulfoxide, both in RT and in PCR, was found necessary for the detection of ASSVd isolates. Problems related to primer dimers were faced successfully by applying Hot-Start PCR. During the years 2003 and 2005, a significant number of samples were collected from rosaceous trees (pome and stone fruit trees) and shrubs from orchards and wild nature of Peloponnesus (Arkadia, Argolis, Korinthia and Elia), Etoloacarnania, Magnesia and Kilkis. The results showed, for the first time in Greece, the presence of PLMVd in apricot and peach, PBCVd in quince and Crataegus sp., ASSVd in apple and wild apple (M. baccata) and the geographically extended natural spread of ASSVd and PBCVd in central and northern Greece, beyond the so far known area of Peloponnesus of southern Greece.