First Report of Zucchini yellow mosaic virus in Round Gourd (Praecitrullus fistulosus) in Pakistan

Praecitrullus fistulosus (Stocks) Pangalo, commonly known as ‘tinda’ or Indian round gourd, is a cucurbit vegetable mainly grown in India and Pakistan (Sharma et al. 2014). P. fistulosus, like other species of cucurbits, is also infected by Zucchini yellow mosaic potyvirus (ZYMV) causing high yield losses across the world (Nagendran et al. 2015; Sharma et al. 2014). In summer 2014, as a part of cucurbit and potyvirus survey in Pothowar, Pakistan, a total of 40 leaf samples of P. fistulosus with symptoms of leaf blistering, shoe-string, and severe mosaic were collected from five fields in the Islamabad Capital territory with an average incidence of 60% based on visual observations. All of the round gourd disease samples as well as two samples from healthy plants were subjected to indirect PTA-ELISA using the “Potyvirus group test” kit (Bioreba AG, Switzerland). Only 20 samples showed positive reaction for potyvirus infection. These 20 positive samples were further serologically tested for the infection of three major cucurbit potyivurses named ZYMV, Papaya ringspot potyvirus (PRSV), and Watermelon mosaic potyvirus (WMV) by DAS-ELISA using virus specific polyclonal antisera (Bioreba AG, Switzerland). Only ZYMV was serologically detected in all 20 samples. The presence of ZYMV in naturally infected leaf samples was further confirmed by RT-PCR. Total RNA from five ELISA-positive samples (one from each field) were isolated using TRIzol reagent (Life Technologies, Calrsbad, CA) and subjected to RT-PCR using a primer set F-8193 (forward primer) and Not1 (dT)₁₈ (downstream primer) (Ali et al. 2004). All five PCR amplicons were purified using QIAquick PCR purification kit (Qiagen) and subsequently cloned into pGEM-T Easy vector system I (Promega Corporation, Madison, WI). All these clones were sequenced in both orientations and a total of 1,416 nucleotides were obtained from each clone consisting of 360 nucleotides from 3′ end of Nib region, full coat protein gene, and 216 bases of 3′ nontranslated region. All five clones showed 100% nucleotide identity. The sequence of ZYMV AIRGPK isolate from P. fistulosus was deposited in GenBank with accession number KR261952. BLASTn of ZYMV isolate AIRGPK (KR261952) revealed 99% sequence identity with ZYMV isolate AUIKTPK (KR261951) from Pakistan, ZYMV isolate Liaocheng (EF122498) from China, 98% with ZYMV isolate Zuy (EU561044) from Poland, 97% with ZYMV isolates (AB188115, AB188116) from Japan, and 97% identity with ZYMV isolates (D00692, L31350) from the United States. This destructive virus has also been reported from ridge gourd, melon, and bottle gourd in earlier studies from Pakistan (Ali et al. 2004; Ashfaq et al. 2015; Malik et al. 2005), but to our knowledge, this is the first documented molecular evidence for the occurrence of ZYMV in P. fistulosus in Pakistan. The large scale occurrence of this notorious virus on P. fistulosus would suggest further investigations from other cucurbits growing areas of Pakistan.