First report of chickpea chlorotic dwarf virus in watermelon (Citrullus lanatus) in Tunisia

In Tunisia, watermelon (Citrullus lanatus) is one of the most common and popular summer fruit crops. The crop is widely cultivated in the central and southern parts of the country with a yearly production of about 400,000 tons. Hard fruit syndrome of watermelon is a well-known syndrome, reported in Tunisia since 1994, when up to 70% incidence was reported in some areas; in recent years, the incidence ranged between 10 and 40%, leading growers to switch to alternative crops. The etiology of the hard fruit syndrome of watermelon is uncertain up to now, but two begomoviruses (Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus) have been recently found associated with this syndrome (Mnari-Hattab et al. 2014), suggesting a viral etiology for symptoms initially attributed to physiological and/or nutritional disorders. Between 2005 and 2015, watermelon fields were surveyed in different areas of Tunisia (Kairouan, Zaghouan, Gabes, Gafsa, and Beja). Samples were collected from unmarketable fruits exhibiting chlorotic mottling on rind and white hard portions inside the flesh. Total DNA from 11 samples were subjected to rolling circle amplification using ?29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, U.K.). A single DNA fragment of about 2,500 bp was obtained following SpeI digestion from three samples. The SpeI-cut DNA derived from a sample collected in Zaghouan Province in 2015 was cloned in pBluescript KS(+) and fully sequenced in both directions. The sequence was 2,571 nt long and exhibited the genomic organization typical of mastreviruses. Alignment analysis against 63 mastrevirus genome sequences using STD program (Muhire et al. 2013) showed high nucleotide sequence identity values with Chickpea chlorotic dwarf virus (CpCDV) genome isolates (about 86%), with the maximum identity score of 98% with an isolate from chickpea collected in Syria (FR687959); identity percentage toward other mastreviruses was less than 70%. The new CpCDV sequence was submitted to GenBank under accession number KX580024. A primer pair CpCDV-CP-F (5?-GCAGAATCAAGGGCGAAGAG-3?) and CpCDV-CP-R (5?-CGGACCGGGACCATAGTAAG-3?) was used to check for CpCDV infection in field watermelon samples; 10 of 11 samples were positive in PCR amplifying a 501-bp diagnostic fragment in the coat protein gene. PCR analyses were also conducted for 39 additional watermelon samples collected in Kairouan (21 samples), Gafsa (4 samples), and Zaghouan (14 samples), the main growing areas in Tunisia, during 2005, 2014, and 2015. Thirty-two of them tested positive for CpCDV. A 1.8-mer construct was engineered in the binary plasmid pBin19, used to transform Agrobacterium tumefaciens LBA4404, and then used to challenge Nicotiana benthamiana plants in infectivity assays. Symptoms of downward leaf curling were observed at 7 days post inoculation in upper uninoculated leaves that later became crumpled, with the plant exhibiting marked chlorosis and dwarfing. CpCDV infection was confirmed by tissue print hybridization using a digoxigenin-labeled probe. CpCDV infecting several species has been reported from many tropical and subtropical countries and was recently found in Tunisia on chickpea (Kumari et al. 2015). To our knowledge, this is the first report of CpCDV infecting watermelon. The finding of CpCDV in samples with the hard fruit syndrome adds an unexpected piece to the complex puzzle of its etiology issue.