Proc. 6 th Conf. EFPP 2002, Prague. Plant Protect. Sci., 38 (Special Issue 2), 2002: 249–251 249 Plant Protection Science – 2002 INTRODUCTION Peach latent mosaic viroid (PLMVd) is one of the most important viroids in stone fruit trees. For the first time it was isolated in 1988 (FLORES & LLACER 1988) even when the disease it causes has been known since thirties of the last century (DESVIGNES 1986). The main host of PLMVd is peach with symptoms like leaf mosaic, flower breaking, deformation and cracking of fruits. Presence of PLMVd in other stone fruit species (apricots, cherries, plums) was also re- ported ( FAGGIOLI et al . 1997; HADIDI et al . 1997) but experimentally only peaches could be infected (DESVIGNES 1986). PLMVd is probably widespread all over the world as it could be detected in 25% of trees from Europe, USA, China and Japan (DESVIGNES 1986). Currently PLMVd is detected either by bio- logical tests by grafting onto GF 305 rootstock or by revers PAGE. Both these methods are rather time consuming (for biological test several months) or the sensitivity is low (electrophoresis). For certification of seed material there is need to have fast, sensitive and reliable method of PLMVd determination. That is why PCR is being developed in several laboratories (SHAMLOUL & HADIDI 1999). MATERIAL AND METHODS Four isolates of PLMVd were obtained from Dr Di Serio (CNR Bari) and multiplied by budding onto GF 305 rootstocks (3 buds per rootstock). One part of plants was transferred to glasshouse at the begin- ning of December to initiate the growth. Leaves were taken during vegetation both from glasshouse grown and later also from outside grown plants and RNA was extracted. Four methods of RNA extrac- tion were used: method of ROBERTSON et al . (1991), method of ROBINSON (1992) and extraction by kits from Qiagen and Adgen. Healthy leaves and also plant material infested by Potato spindle tuber viroid and Hop latent viroid were used as controls. On the basis of published sequences of PLMVd three prim- ers were designed using Williamstone Enterprises program, one reverse (PLMVdR) and two forward (PLMVdF1 and F2). One of them (F1) gives with reverse primer product 208 bp long and second (F2) gives product 114 bp long. Reverse transcription was done in 25 µl reactions containing 5 µl reaction buffer, 0.5 µl (5 U) AMV reverse transcriptase (Promega), 0.5 µl (20 U) Rnasin (Promega), 10 pmol of reverse primer, 0.1mM of each dNTP, 1 µl RNA and DEPC Detection of Peach Latent Mosaic Viroid by PCR P. RYŠÁNEK * , M. ZOUHAR and M. HASSAN Department of Plant Protection, Czech University of Agriculture, 165 21 Prague-Suchdol, Czech Republic * E-mail: rysanek@af.czu.cz Abstract Peach latent mosaic viroid (PLMVd) is widespread in peach all over the world. It has never been reported from the Czech Republic. That is why we adapted specific and sensitive method for its detection, PCR, to be able to prove its possible occurrence and for certification purposes. Primers PLMVdR, PLMVdF1 and PLMVdF2 were designed on the basis of published RNA sequences. Products of amplification are 208 and 114 bp long for PLMVdF1 and PLMVdF2, respectively. Four PLMVd isolates from Dr Di Serio (CNR Bari) were used as standards. Potato spindle tuber viroid and Hop latent viroid infected plant material and also healthy material were used to check detection specifity. Both RNA extraction from plant material and PCR were optimalized so that this method of PLMVd detection can also be used for certification purposes. Keywords: PLMVd; PCR; peach; certifcation