The First Report of Cucumber Mosaic Virus Infecting Water Chestnut in China

Water chestnut (Eleocharis dulcis), which is cultivated worldwide today, first originated in India and China. It is a popular seasonal aquatic vegetable valuable to people for its sweet crisp taste and rich nutrition. In October 2012, field-grown water chestnut seedlings (E. dulcis) showing mosaic, chlorotic, dwarfing, and malformed symptoms were observed in Fanggaoping Town, Tuanfeng County, Hubei Province, China. Sap from leaf-like stems of two symptomatic seedlings (BQ6 and BQ7) were mechanically inoculated onto Nicotiana glutinosa plants using 0.01 M phosphate buffer (pH 7.4) to investigate whether viral etiology was responsible for the disease. Typical symptoms of chlorosis and systemic mosaic similar to that inflicted by Cucumber mosaic virus (CMV) were observed on inoculated N. glutinosa leaves 13 days post inoculation, whereas mock inoculated seedlings remained symptomless. Three naturally field-grown symptomatic water chestnut and the inoculated N. glutinosa seedlings, together with a healthy water chestnut plant as negative control, were sampled. Double-antibody sandwich (DAS)-ELISA with antisera against CMV using commercial kits (Agdia, Elkhart, IN) was carried out to detect and confirm the presence of CMV. The symptomic water chestnut and inoculated N. glutinosa seedlings tested positive for CMV. Total RNAs were extracted using the SDS column isolation method from leaves of the inoculated N. glutinosa and stems of 13 field-grown symptomatic water chestnuts. The extracted RNAs were subjected to reverse transcription. The first-round PCR was carried out using the obtained cDNAs as template with the CMV specific primer set CMV-3F (5'-GCGATGYCGTGTTGAGAAG-3') and CMV-3R (5'-TTTAGCCGTAAGCTGGATGGA-3') targeting a 983-bp fragment covering 657 nt of the whole CP and partial flanking sequence within RNA3 referred as 'Fny' strain in GenBank (Accession No. D10538). The resulting amplicons were diluted 1:20 and further amplified with the nested-primer set CMV-P1 (5'-ATGGACAAATCTGAATCAACC-3') and CMV-P2 (5'-TAAGCTGGATGGACAACCCGT-3') targeting a fragment of 777 bp corresponding to the complete CP followed by part of 3'-UTRs of RNA3 (1). The amplicons of the expected size of ~777-bp were consistently amplified from 13 naturally infected water chestnuts and inoculated N. glutinosa. The PCR product derived from BQ6 isolate was cloned and three clones sequenced in both directions. The sequence (GenBank Accession No. KF268463) was analyzed by MEGA5 software (3). Sequence comparison of the complete CP gene of BQ6 isolate showed 98% nt and 99% amino acid (aa) identity with CMV isolate RP6 from South Korea (GenBank Accession No. KC527735) in subgroup I and had low similarities of 76% nt and 80% aa to that of CMV isolate infecting Trifolium from Hungary (GenBank Accession No. L15336) belonging to subgroup II of CMV. Phylogenetic analysis showed BQ6 isolate was more closely related to the isolates belonging to IB subgroup of CMV (GenBank Accession Nos. EF153739, DQ302715, and KC576805) (2). To our knowledge, this is the first report of CMV infecting water chestnut (E. dulcis) in China. CMV infection may pose a significant threat to water chestnut production. This result provide information to the producer that the CMV-free seedlings should be chosen for cultivation of water chestnut. References: (1) P. Palukaifis et al. Adv. Virus Res. 41:281, 1992. (2) S. K. Raj et al. Plant Dis. 92:171, 2008. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.