LITTLE CHERRY CLOSTEROVIRUSES-1 AND -2, THEIR GENETIC VARIABILITY AND DETECTION BY REAL-TIME-PCR

Two distinctly different members of the family Closteroviridae have been identified from little cherry disease-affected cherry trees and were designated Little cherry virus-1 (LChV-1) and -2 (LChV-2). Variability studies were conducted for LChV-1 and in addition to published data for LChV-2. For this purpose, amplification primers were designed to both viruses in potentially conserved regions of the coat protein, truncated coat protein, HSP70 gene and the RNA dependent RNA polymerase (RdRp). These primers were used in PCR assays on a wide range of isolates obtained from Europe and North America. Nucleotide sequence analysis of cloned PCR fragments showed considerable genetic variation for both viruses, with higher variability for LChV-1. Based on alignments of conserved regions within the RdRp, PCR primers were designed for the purpose of developing both a real-time-PCR and a standard RT-PCR multiplex assay. These assays were able to identify all available isolates in single and mixed infections. Field data from woody indexing trials on indicator plants and preliminary data from application of the RT-PCR test in plant protection services of German provinces, suggested that LChV-1 infections either remained latent or were delayed in symptoms when compared to LChV-2 with typical symptoms as described in the literature.