MOLECULAR ANALYSES OF BETAFLEXIVIRIDAE VIRUSES ASSOCIATED WITH RUSTY MOTTLE AND TWISTED LEAF DISEASES OF SWEET CHERRY (PRUNUS AVIUM)
Villamor, Dan Edward Veloso
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Virus(es) associated with the rusty mottle group, cherry twisted leaf (CTL) and apricot ring pox (ARP) diseases of sweet cherry were characterized. A primer pair, Fovea2-AdPr, designed to amplify a broad range of viruses in the genus Foveavirus, amplified the 3'-end region of genomic RNAs of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) as well as the corresponding regions of virus-like RNAs detected in trees affected with CTL, ARP, cherry necrotic rusty mottle (CNRM), cherry rusty mottle (CRM) and cherry green ring mottle (CGRM) diseases. Phylogenetic analyses of sequences encoding a putative coat protein (CP) along with published sequences of CGRMV and CNRMV from other geographic regions revealed segregation into four major clades, each corresponding to one of the diseases and correlated with symptoms induced in Prunus avium `Bing', `Sam', and Prunus serrulata `Kwanzan'. These clades of virus sequences were designated as clade I: Cherry twisted leaf associated virus (CTLaV) with two subpopulations designated as CTLaV-Ia and CTLaV-Ib, clade II: CNRMV, clade III: Cherry rusty mottle associated virus (CRMaV), and clade IV: CGRMV. Any inconsistencies between the virus sequence profile and symptoms on biological woody indicators were resolved by reverse transcriptase-polymerase chain reaction (RT-PCR) assays using newly designed primers specific for each virus sequence. The clustering is further supported by the phylogenetic analyses of the coding regions and putative protein sequences from different viral open reading frames (ORFs) of representative members of the above viruses. Deep sequencing analyses of representative source trees for CRM, CTL and ARP diseases did not reveal novel viruses that may be associated with these diseases other than those revealed by RT-PCR, thereby further supporting the status of CRMaV and CTLaV as the causes of CRM and CTL-ARP diseases, respectively. Based on careful examination of the pairwise nucleotide and amino acid sequence identity values on whole CP and replicase region between and within members of each virus, genome organization and symptoms of diseases associated with individual viruses, it is proposed that a new genus (Robigovirus) within the family Betaflexiviridae is created to accommodate these viruses.