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Title: Molecular and biological characterization of Mexican papita viroid

item Li, Rugang
item Ling, Kai-Shu

Submitted to: Tomato Disease Workshop
Publication Type: Proceedings
Publication Acceptance Date: 9/18/2012
Publication Date: 10/16/2012
Citation: Li, R., Ling, K. 2012. Molecular and biological characterization of Mexican papita viroid. Tomato Disease Workshop. p28.

Interpretive Summary: N/A

Technical Abstract: Mexican papita viroid (MPVd), a member of the genus Pospiviroid, family Pospiviroidae, was first isolated from wild papita (Solanum cardiophyllum Lindl) plants in 1996. Beginning in 2009, several disease outbreaks caused by this viroid have been reported in greenhouse tomatoes growing in North America (including Canada and Mexico). Despite of the availability of complete genomic sequences for several MPVd isolates, numerous questions as to their molecular and biological properties remain unanswered. The lack of a local lesion host makes it difficult to isolate MPVd from field samples infected with multiple viruses and viroids; thus, we decided to create infectious cDNA clones from selected MPVd isolates and then use these infectious clones to characterize the molecular and biological properties of each isolate. In the current study, we constructed infectious cDNAs from two MPVd isolates by insertion of full-length PCR products into the BamHI site of plasmid pGEM-3zf(+). Sanger-sequencing showed that 3 of 4 MPVd-MX and 1 of 6 MPVd-Mex8 cDNAs were in proper orientation for expression of potentially infectious RNA transcripts. Using a previously prepared infectious Potato spindle tuber viroid (PSTVd) cDNA clone as a positive control, infectivity of the four engineered MPVd constructs were tested by mechanical inoculation of ‘Rutgers’ or ‘Moneymaker’ tomato plants with either purified plasmid DNA or in vitro RNA transcripts. Typical symptoms of MPVd infection (chlorosis and stunting) were observed on the inoculated tomato plants 2-8 weeks post inoculation. The presence of MPVd in the symptomatic plants was confirmed through MPVd-specific RT-PCR and additional genomic sequencing. Using these infectious clones, a host range study is currently underway, and preliminary results will be discussed.