First Report of Hop Stunt Viroid Infecting Luffa and Cantaloupe in Taiwan

Luffa (Luffa cylindrica (L.) M. Roem.) and cantaloupe (Cucumis melo L.) are economically important cucurbitaceous crops in Taiwan. In August 2018, luffa samples displaying leaf chlorosis and deformation were collected in Nantou while cantaloupe samples exhibiting the above suspected systemic viral-like symptoms were collected in Chiayi in October 2019, respectively. Reverse transcription (RT)-PCR tests were conducted to investigate potential viral infections in leaf samples from 12 luffa and nine cantaloupe plants. Total nucleic acids were extracted from symptomatic cucurbit tissues using the taco™ DNA/RNA extraction kit (GeneReach Biotechnology, Taiwan), followed by PCR or RT-PCR tests for virus detection. Seven sets of degenerate primers targeting viral genera reported in local cucurbit crops, including genera Begomovirus, Crinivirus, Cucumovirus, Polerovirus, Potyvirus, Orthotospovirus and Tobamovirus, were utilized. However, all tests yielded negative results. Consequently, degenerate primers targeting genus Pospiviroid or hop stunt viroid (HSVd)-specific primers were employed. Among these tests, only HSVd-specific primers HSVd-RTR_F/R (5'-GGAATTCTCGAGTTGCCGCA-3'/5'-CCGCGGCCCTCTCT-3') (van Dorst and Peters 1974) successfully amplified DNA fragments of approximately 130 bp from 19 symptomatic samples (12 from luffa and 9 from cantaloupe), while normal leaf samples of luffa and cantaloupe tested negative. Based on known nucleotide sequences, primers were designed to amplify and obtain the full-length 303-nucleotide sequences of the HSVd genome derived from luffa (accession no. OP331278) and cantaloupe (accession no. OP331279). The two sequences exhibit a 98.7% nucleotide identity with each other while exhibiting 99.3% nucleotide identity with a cucumber isolate from Japan (X00524) (Sano et al. 1984) and 98.0% nucleotide identity with another isolate (X07405) from the Netherlands (Puchta et al. 1988). Comparisons with 13 HSVd citrus isolates from Taiwan revealed nucleotide similarities ranging from 96.1% to 99.0%. To confirm pathogenicity, Nicotiana benthamiana was chosen as the experimental isolation host. Mechanical inoculation of N. benthamiana with diluted sap (20 W/V) from diseased plants consistently produced leaf wrinkling and stunting symptoms in 10-60% of inoculated plants by 12 days post-inoculation (DPI). RT-PCR tests confirmed viroid infection. Additionally, luffa seedlings at the cotyledon stage back-inoculated with the purified HSVd luffa isolate developed leaf deformation symptoms 15-25 DPI, and the infection was confirmed by RT-PCR, fulfilling Koch's postulates. HSVd infections have previously been reported in citrus and grapes in Taiwan (Lin et al. 2015). To our knowledge, this is the first report of HSVd infecting luffa and cantaloupe in Taiwan. The newly identified HSVd cucumber isolates represent a potential threat to cucurbit production and warrant further investigation.