POSTERS: Pathogen detection, quantification and diagnosis
Near complete genome sequence of Tulip virus X obtained by high-throughput sequencing from naturally-infected lemon balm (Melissa officinalis)
John Hammond - USDA ARS Floral and Nursery Plant Research Laboratory. Ramon Jordan- USDA-ARS, USNA, Floral & Nursery Plants Research, Connor Gulbronson- USDA-ARS, USNA, Floral and Nursery Plants Research Unit, Michael Reinsel- USDA ARS, Floral and Nursery Plants Research Unit
Three plants of Melissa officinalis with mosaic symptoms were obtained from a local nursery. Transmission electron microscopy (TEM) of extracts revealed flexuous particles typical of potexviruses; TEM of thin sections showed loose cytoplasmic arrays of similar particles. M. officinalis is known to be susceptible to Alternanthera mosaic virus (AltMV), and to tulip virus X (TVX). ELISA with AltMV-specific antibodies yielded negative results, while ELISA with antibodies to Plantago asiatica mosaic virus (PlAMV; closely related to TVX) was positive. Inoculation to bioassay plants susceptible to AltMV and/or PlAMV yielded no symptoms or positive ELISA results. A cDNA library for high-throughput sequencing was generated from total RNA from pooled leaves of the three lemon balm plants, and sequenced by paired-end protocols (Illumina MiSeq); raw reads (3,337,739; of 80-251 nt) were trimmed to remove adapter linkers, and de novo assembled into contigs using Geneious Pro R9. Contigs were analyzed by BLASTX against viral sequences from GenBank; those with high identity to potexviruses were further edited and assembled into a 6034 nt nearly full-length sequence, lacking 3-17 nt at the 5’, and 2 nt at the 3’, by comparison to the reference (TVX-J) and other TVX isolates. The predicted RdRp, TGB1, TGB2, TGB3, and CP share 89%, 91%, 86%, 75%, and 94% amino acid identity to those of TVX-J. RT-PCR and sequencing of cloned PCR products to validate the genome sequence is in progress.