First Report of Zucchini yellow mosaic virus in Ridge Gourd in Pakistan

Zucchini yellow mosaic virus (ZYMV, genus Potyvirus) infects several species of cucurbits, including ridge gourd (Luffa acutangula (L.) Rox), and causes significant yield losses worldwide (Nagendran et al. 2015). During summer 2014, surveys were conducted to document major potyviruses infecting important cucurbits in the Pothwar Region of Pakistan. A total of 25 ridge gourd leaf samples with symptoms of mosaic, leaf malformation, and blistering were collected from three sites in the Rawalpindi-Islamabad area, where incidence of 45 to 60% was recorded based on visual assessment. All symptomatic samples along with two healthy ridge gourd leaf samples were first tested for potyvirus infection with a potyvirus group-specific monoclonal antibody (Bioreba AG, Switzerland) using indirect Plate Trapped Antigen-Enzyme Linked Immunosorbent Assay (PTA-ELISA). Of 25 symptomatic samples, only 13 were positive for potyvirus infection whereas healthy leaf samples were negative. These 13 samples positive for a potyvirus were further screened for the presence of ZYMV, Watermelon mosaic virus, and Papaya ringspot virus by double antibody sandwich (DAS)-ELISA using virus specific polyclonal antisera (Bioreba AG). All samples were positive only for ZYMV. For further confirmation, total RNA from six ELISA-positive samples (two from each site) were isolated using TRIzol Reagent (Life Technologies, Carlsbad, CA). First-strand cDNAs were synthesized using total RNA, Moloney murine leukemia virus reverse transcription (Life Technologies) and Not1(dT)₁₈ (Ali et al. 2004) as first-strand primer. The cDNA of each sample was then used in Polymerase Chain Reaction (PCR) with the ZYMV-specific forward primer (5′-CTACAAGCCCTCCACCAAGA-3′, designed from GenBank Accession No. AB127936) and reverse primer Not1 (dT)₁₈. Fragments of approximately 1100 bp were amplified from each ZYMV-ELISA positive sample. All six amplicons were individually cloned into pGEM-T Easy vector system I (Promega Corporation, Madison, WI) and sequenced in both orientations. A total of 1078 nucleotides were obtained from each clone comprising a full-length coat protein cistron and 3′ untranslated region. Nucleotide sequences of all six clones were identical. The sequence of ZYMV-ridge gourd (AUIKTPK) isolate was submitted to GenBank as Accession No. KR261951. A pairwise comparison of nucleotide sequence (KR261951) with corresponding sequences of ZYMV available in GenBank revealed 99% identity with ZYMV isolates from China (AF513551, AY074809, and EF122498), France (JN861008), and Poland (EU561044), and 96% identity with a ZYMV-bottle-gourd isolate (AB127936) from Pakistan. The occurrence of ZYMV has been reported previously from bottle gourd (Ali et al. 2004) and melon (Malik et al. 2005) from Pakistan. To the best of our knowledge, this is the first confirmed report of ZYMV naturally infecting ridge gourd in Pakistan. The presence of this potentially destructive virus on ridge gourd could represent a serious threat for this important crop, and therefore further surveys are needed from more areas of Pakistan in order to determine its incidence, prevalence, and yield losses in commercial cultivars of ridge gourd.