First Report of Natural Infection of Watermelon mosaic virus on Lagerstroemia indica in China

Lagerstroemia indica (crepe myrtle) is an important garden and border tree. In May 2017, a new virus-like disease was observed on L. indica in Hangzhou, Zhejiang province, China. Symptoms observed included patches of vein clearing and chlorosis on L. indica leaves. A survey in the region indicated that more than 98% (n = 1,485) of L. indica plants showed similar symptoms. To determine the causal agent, a small RNA (sRNA) library was constructed using symptomatic L. indica leaf tissue and sequenced using the Illumina Hiseq next-generation sequencing platform (Lianchuan Bio, Hangzhou, Zhejiang, China). After adapter trimming and removing low-quality sequences, the sRNAs were assembled using Velvet 1.1 and Oases 0.2.09 with k-mers of 15, 17, and 19 (Schulz et al. 2012). Assembled contigs were searched against NCBI nucleotide and amino acid databases using the BLASTn and BLASTx programs (Johnson et al. 2008). Forty-seven contigs showed high homologies to the genomic sequence of Watermelon mosaic virus (WMV). No transcript that was homologous to the genomic sequence of other plant-infecting viruses was identified. The entire genome of this WMV isolate was then amplified by the reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends with primers designed from the sequence of the assembled contigs. The amplified fragments were ligated into the pEASY-Blunt vector and confirmed by DNA sequencing. Excluding the poly-A tail, the genome of this L. indica-infecting WMV (WMV-Li) is 10,032 nt in length (GenBank accession no. MG194418), and it shares the highest similarity (95%) with a French WMV isolate (GenBank accession no. EU660581). Further analysis showed that the 47 sRNA-derived contigs covered the entire genome, except the 5′-terminal 58 nt. Nicotiana benthamiana seedlings were mechanically inoculated with sap extracted from symptomatic L. indica leaves. Mild mosaic and vein clearing symptoms were observed at 7 days postinoculation. The systemic infection of WMV-Li on N. benthamiana plants was confirmed by RT-PCR using primers WMVcp-F and WMVcp-R, which amplify the entire coat protein coding region. RT-PCR was also performed to confirm the presence of WMV-Li on eight symptomatic and two healthy L. indica plants. Results showed that an amplicon of about 900 bp was amplified from all eight symptomatic plants but not the two healthy plants. The RNA sequences of six out of the eight amplicons were identical to that of WMV-Li, whereas the other two amplicons each contained a single nucleotide substitution (GenBank accession nos. MG194419 and MG194420). To the best of our knowledge, this is the first report of WMV infecting a woody host. Notably, WMV-Li was also detected infecting Luffa cylindrica with typical symptoms of vein clearing in a subsequent survey. Thus, special attention should be paid to the damage that WMV-Li may cause to both crepe myrtle and cucurbitaceous crops.