Degenerate PCR primers for potexvirus detection in seagrasses

Abstract

2.AbstractThe genusPotexvirusincludes many species known to infect terrestrial plants and, more recently, a novel species,Turtlegrass virus X(TVX) infecting the seagrassThalassia testudinum. We designed a degenerate primer pair, Potex7F and Potex7-RC, to amplify a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from potexviruses infecting seagrasses. This primer pair was modified from Potex5/Potex2-RC primers published in 2002 that target terrestrial plant potexviruses, by including nucleotide bases found in TVX and related potexvirus sequences identified in seagrass viromes from Tampa Bay, Florida, USA. Using this newly developed primer set, we performed a reverse transcriptase polymerase chain reaction (RT-PCR) survey to screen for potexviruses in 63 opportunistically collected, apparently healthy seagrass samples. The survey examined the host speciesT. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, andZostera marina. PCR products were successfully amplified and sequenced fromT. testudinumsamples collected around Florida, USA, indicating prevailing potexvirus infection in the region.Impact statementPotexviruses are widespread in terrestrial plants; however, the recent discovery of a potexvirus in the seagrassThalassia testudinumextends their host range to marine flowering plants. Here we present degenerate potexvirus PCR primers, Potex7F and Potex7-RC, to enable the identification of potexvirus infections in seagrass habitats. Discovery of potexviruses inT. testudinumhighlights the utility of this new primer set in uncovering the diversity, host range, and geographic range of potexviruses infecting seagrasses.Data summaryAll sequence data are available in NCBI GenBank under the accession numbersOR827689-OR827705,OR854648,OR863396, andOR879052-OR879056. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.