Construction Of Infectious HSVd Clone And Molecular Characterization Of Two Newly Identified Citrus Viroids

Viroids are plant subviral pathogens,of which genomes are constituted by a single-stranded and covalently closed small RNA molecule with a high degree of self-complementation resulting in a compact folding. Viroids, in contrast with viruses, do not encode for any proteins, and entirely rely on the host factors to complete genome transcription and duplication. Despite this genomic simplici-ty, consisting of246-401nucleotides (nt) only, they are able to induce devastating symptoms in susceptible plants.Information from the9th ICTV (International Committee on Taxonomy of Viruses) indicated that viroids are classified into2different families (.Pospiviroidae and Avsunviroidae) based on the secondary structures and functional properties of RNA genome. With a central conserved re-gion(CCR) and no hammer-headed ribozymes, the rodlike or quasi-rodlike Pospiviroidae family contributes28species within5genera, while Avsunviroidae family, which is able to self-cleave dur-ing duplication through hammer headed ribozymes and lacking of CCR,is composed with only4species.So far, all7citrus viroids identified belong to family Pospiviroidae. Citrus exocortis vi-roid(CEVd), Hop stunt viroid(HSVd), Citrus bark cracking viroid(CBCVd) are classified into the genera Pospiviroid, Hostuviroid, Cocadviroid respectively, while Citrus bent leaf viroid(CBLVd, formerly namedCitrus viroid I, CVd-I), including strains of citrus viroid I low sequence similarity, CVd-I-LSS, Citrus dwarfing viroid(CDVd), Citrus viroid â…¤(CVd-V) and Citrus viroid â…¥(CVd-VI) are in the same genus, Apscaviroid, based on phylogenetic trees and nucleotide sequence homologies within CCR.1) Citrus cachexia caused by special strains â…¡b,â…¡c of HSVd induced serious symptoms on cit-rus plants, including stunning and gummingin sicon and bark cracking on rootstock. The mix infe-tion of cachexia and non-cachexia (Ha) were predomindant in citrus orchards in China. This study identified strains â…¡a and â…¡b through clone technique and doubled the inserted cDNA in tandem connection to construct infectious clones. Biological indexing showed that those â…¡a and â…¡b infec-tious clones were able to cause significant dwarfing in young cucumber plants (Cucumis sativus L. cv. Suyo), but no symptoms on Parson special mandarin (Citrus reticulata) wasobserved. Sequences of HSVd descendant variants derived from strains on cucumber and citrus were in correspondence with those of the original strains, suggesting that infectious clones in this study successfully com-pleted their infection cycle at molecular level.2) CVd-VI was a new viroid found onlyin citrus and persimmon from Japan previously. Here we reported the molecular characterization of CVd-VI isolates collected from different citrus varie-ties and different geographical regions in China. A total of90cDNA clones were sequenced includ-ing10sequences from each isolate. Sequence alignment showed that one or two predominant se-quences were found.Allpredominant sequences and variants from NCBI were classified into two main clades by phylogenetic analysis.3)49cDNA of CVd-I-LSS from Pakistan and China were analyzed. Sequence alignments showed that one to two predominant sequences were found. All predominant sequences and variants from NCBI were classifiedinto three main clades by phylogenetic analysis. Based on these phyloge-netic data and records of citrus cultivars derived from Pakistan, it is suggested that Pakistan may be one of the original source of the CVd-I-LSS.