Simultaneous Detection of Two Viroids Infecting Grapevines in Taiwan by Multiplex RT-PCR

Abstract

Results of this work demonstrated the potential of multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for detecting Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid-1 (GYSVd-1) simultaneously in naturally infected grapevines. A total of one hundred grapevine leaf samples with yellowing or mosaic symptoms collected from different vineyards in Changhua County (GPS coordinates N204976, E2653708) Taiwan, R.O.C from May to June 2015. Only GYSVd-1 and the grapevine isolate of HSVd were detected. Specific primer pairs for detecting HSVd and GYSVd-1 were selected from previous reports. New primers were designed as forward (5’CTGGGGAATTCTCGAGTTGC-3’), and reverse (5’AGGGGCTCRAGAGAGGMTC-3’) for HSVd and as forward (5’ACGAA GGGGTGCACTCCGAGTG-3’), and reverse (5’ CGACGACGAGGCTCACTCCC-3’) for GYSVd-1 according to the sequences retrieved from the National Center of Biotechnology Information (NCBI). The amplicons obtained through RT-PCR were examined by 1% agarose gel electrophoresis. Subsequently, the sequences revealed that HSVd-DY and GYSVd-1-DY contained 301 and 367 nucleotides, respectively. The sequence identity of HSVd-DY with other HSVd viroids from GenBank ranged from 37.6% to 99.7% while that of GYSVd-1-DY with other viroids from the GenBank ranged from 82.9% to 99.7%. A phylogenetic tree derived from the HSVd sequence indicated that HSVd-DY closely matched an Iranian isolate (KF916041), while GYSVd-1-DY coincided with a Chinese isolate (JF746188). Multiplex RT-PCR amplification was successfully performed to identify HSVd and GYSVd-1 from grapevine samples.