First report of mosaic disease associated with zucchini tigre mosaic virus in wax gourd (Benincasa hispida) in Taiwan

Wax gourd (Benincasa hispida) is a popular cucurbitaceous summer crop used for culinary purposes, confectionery, and refreshing drinks in Asia. During a field survey for cucurbit viral diseases at Beidou, Changhua in May 2017, B. hispida plants with symptoms of mosaic, chlorosis and leaf curl were found. Because more than 90% of plants showed leaf curl symptoms, samples were first examined by PCR with degenerate primers for begomoviruses (Forward: 5'-TGTGAAGGNCCDTGTAARGT-3' and Reverse: 5'-GCRTDGGTACADGCCATATA-3'). Sequence analysis of amplicons revealed that all of tested plants were infected by squash leaf curl Philippine virus (SqLCPhV) (Liao et al., 2007). However, electron microscopic examination revealed potyvirus-like particles and pinwheel-like inclusion bodies on some of begomovirus-infected leaves showing mild curling and mosaic symptoms. RT-PCR with potyvirusdegenerate primers (PNIbF1: 5'-GGBAAYAATAGTGGNCAACC-3' and PCPR1: 5'-GGGGAGGTGCCGTTCTCDATRCACC-3') were used to detect the potyvirus. Among 8 samples examined, all were infected by SqLCPhV (100%) and 5 samples were mixed infection of SqLCPhV and a potyvirus (62.5%). The filamentous virus was isolated from field samples by sap inoculating plants of B. hispida to separate from nonmechanically-transmissible SqLCPhV. Inoculated B. hispida plants showed severe mosaic and deformation. Total RNA was extracted from inoculated B. hispida and used for RT-PCR with a degenerate potyvirus primer PNIbF1 (5'- GGBAAYAATAGTGGNCAACC) pairing with oligo-dT. A 1.8-kb DNA fragment containing partial NIb gene, full-length coat protein (CP) gene, and 3'-UTR was amplified, cloned, and sequenced. The CP gene (293 aa) shared 82.1% nucleotide and 81% amino acid sequence identity to that of zucchini tigre mosaic virus (ZTMV) isolate E11045 (KC345608). Two degenerate primers (fZTMV-F: 5'- AGCRTGTGGYAHCC-3' and gourd-R: 5'-TCCCACCAYTTYTCRAAHGT-3') target the flanking region between the middle of P3 and 5' of CIP genes of ZTMV strains were designed and used to detect ZTMV. An expected 0.7 kb fragment could be amplified from 2 of 5 field-collected samples and all 6 mechanically inoculated B. hispida plants. Upon sap inoculation, ZTMV was also able to infect pumpkin (Cucurbita pepo var. pepo), zucchini (C. pepo var. cylindrica), squash (C. moschata), bottle gourd (Lagenaria siceraria) and Cucumis metuliferus, but not bitter gourd (Momordica charantia), cucumber (Cucumis sativus), watermelon (Citrullus lanatus), luffa (Luffa