First Report of Squash leaf curl China virus Associated with Mosaic and Mild Leaf Curl Disease of Pumpkin in Bangladesh

Pumpkin (Cucurbita pepo) in the Cucurbitaceae family is a popular vegetable and has many benefits, including a high content of vitamin A. Pumpkin is a natural host to both RNA and DNA viruses, including squash leaf curl China virus (SLCCNV). This bipartite begomovirus exists as two strains, China and India, and causes yellow mosaic and leaf distortion symptoms on pumpkin (Singh et al. 2008). SLCCNV is becoming a major threat in many cucurbit crops in South East Asia (Wu et al. 2020). In Bangladesh, SLCCNV was identified from whiteflies collected in a brinjal field (Khatun et al. 2020). During a survey for geminivirus in August 2019, we observed mild leaf curl and mosaic symptoms on pumpkin in a commercial field in Chittagong (22° N, 91° E) in southeastern Bangladesh and collected one representative leaf sample. Then, 10 additional (eight symptomatic and two asymptomatic) samples were collected in different areas including Golachipa, Barguna, Patuakhali, and Dumki in 2021. Total DNA was extracted using a Viral Gene-spin DNA/RNA Extraction kit (iNtRON Biotechnology, Seongnam, South Korea) and used as a template for PCR with begomovirus-specific primers (Lal et al. 2020). The resulting 1 kb DNA fragment was sequenced (Macrogen, Seoul, South Korea) in eight out of nine symptomatic samples and showed high identity (>94%) with SLCCNV DNA A (DQ026296) isolated from pumpkin in Lucknow, India (Singh et al. 2008). Next, Southern blot was performed with the PCR product as the probe. Together with an ssDNA band, the result also showed supercoiled and open circular forms of dsDNA, thus confirming the presence of SLCCNV infection in the collected sample. Rolling circle amplification (RCA) was performed using a TempliPhi 100 Amplification kit (Cytiva, Marlborough, MA, U.S.A.) with the extracted DNA of five positive samples as the template. The RCA products were digested with BamHI (TaKaRa Bio, Japan) resulting in a 2.8 kb fragment in three out of five samples that were cloned into the pGEM-Teasy vector (Promega, Madison, WI, U.S.A.) and sequenced. A BLASTn search of the GenBank database showed the putative full-length sequence (accession number: MT081229) to have the highest nucleotide sequence identity (95.2%) to the DNA A of an SLCCNV isolate (EU573715) from Varanasi, India (Singh et al. 2009). With the same approach, the putative full-length of DNA B was obtained after RCA products were digested with HindIII (TaKaRa). The DNA B sequence (MT081230) had 90.2% nucleotide identity with the DNA B component of an Indian strain of SLCCNV (JN624306). Moreover, we demonstrated that there was no satellite DNA associated with SLCCNV in the collected plants based on failure to amplify expected fragments in a PCR test with alphasatellite and betasatellite universal primers (Briddon et al. 2002; Bull et al. 2003). Also, no mixed infection with other reported viruses including papaya ringspot virus, watermelon mosaic virus, cucumber mosaic virus, and zucchini yellow mosaic virus was detected by the reverse transcription PCR method with specific primers described in previous reports (Cho et al. 2019; Lee et al. 2015). Thus, this is the first report that an isolate of the Indian strain of squash leaf curl China virus infects pumpkin in Bangladesh. The presence of SLCCNV in pumpkin poses a new potential threat to cucurbit production and should be monitored in Bangladesh.