Archivio Journal of Plant Pathology

Abstract:

Little cherry virus 2 (LChV-2) is considered an important pathogen of cherries and, as such, testing of plant material is required according to the EPPO certification scheme PM 4/29 for cherries. In Croatia sour cherry Marasca is a well-known native variety with total production of 1500-1800 tons of fresh fruit per year, used mainly for production of renowned liqueurs and juices. In June 2014, 19 trees were selected and total RNA was extracted from leaves with the RNeasy plant mini kit (Qiagen, Germany). RT-PCR for LChV-2 was performed using two sets of primers: LCHV2LO2/LCHV2UP2 (Rott and Jelkmann, 2001), amplifying a 438 bp fragment of the methyltransferase (MT) gene, and LC26L/LC26R (Eastwell and Bernardy, 2001), amplifying a 409 bp fragment of the RdRp gene. Six trees were positive, four with the first primer set and two with the second. However, none of the trees tested positive by both sets, suggesting the presence of significant sequence variability among LChV-2 isolates (Theilmann et al., 2004) and the presence of at least two virus variants in Croatia. One PCR product for each primer set was sequenced from both directions and sequences were compiled using MEGA6 software. BLAST searches indicated that the part of the MT gene of Croatian isolate M-79 (GenBank accession No. KT369315) shares 88% identity with the USA6b isolate of LChV-2 (AF531505), while isolate M-75 (KT369316) was closest to Canadian strain LC5 (AF416335), sharing 96% identity with part of the RdRp gene. During the 2015 harvest period, some of the LChV-2-infected trees displayed the characteristic symptoms of uneven ripening. To the best of our knowledge this is the first report of LChV-2 in Croatia.