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First Report of Taro vein chlorosis virus on Taro (Colocasia esculenta) in the U.S. Territory of American Samoa

Atibalentja, N., Fiafia, S. T., Gosai, R. C., Melzer, M. J.
Plant disease 2018 v.102 no.4 pp. 828
Colocasia esculenta, Dasheen mosaic virus, RNA, RNA-directed RNA polymerase, Taro bacilliform virus, Taro vein chlorosis virus, amino acids, chlorosis, complementary DNA, denaturation, farms, genes, germplasm, nucleic acid hybridization, oligodeoxyribonucleotides, petioles, plant diseases and disorders, plant viruses, protocols, reverse transcriptase polymerase chain reaction, sequence diversity, staple crops, taro, viral load, viruses, American Samoa, Germany, Hawaii
Virus diseases of taro (Colocasia esculenta) are a serious cause for concern wherever taro is grown, not only for the extent of yield losses they inflict, but also for the restrictions imposed to the international exchange of taro commodities and germplasm in order to limit the spread of new viruses in countries where they have not yet been reported. The stakes are even higher for small South Pacific island communities like American Samoa where taro is the most important staple food crop and a highly valued gift in many cultural functions. Dasheen mosaic virus (DsMV, family: Potyviridae) and Taro bacilliform virus (TaBV, family: Caulimoviridae) are the only two viruses previously known to infect taro in American Samoa (Revill et al. 2005a). In August of 2016, taro plants (cv. Samoa red 2) exhibiting leaf-vein chlorosis and streaks along the petioles, similar to the symptoms caused by Taro vein chlorosis virus (TaVCV, family Rhabdoviridae, genus Nucleorhabdovirus) (Revill et al. 2005b), were observed on the extension plot (14.319176°S, 170.740749°W) of the American Samoa Community College’s Division of Agriculture, Community, and Natural Resources. Only a few (<5%) plants exhibited symptoms, and no symptoms were observed at two nearby taro farms. Total RNA was extracted from the symptomatic leaves using the RNeasy Plant Mini kit (Qiagen, Valencia, CA), and the extracts were used as template, with random hexamers, for the synthesis of the first-strand cDNA, following the SuperScript III First-Strand Synthesis System for RT-PCR protocol (Life Technologies, Carlsbad, CA). Two primer pairs, DCGF3 (5′-GTCTGGTAAAGAGGGGTTGA-3′)/DCGR3 (5′-GTCGAGGGTCATGATAGAGT-3′) and DCGF5 (AGGGGYTGAGRCAAAAGGGGT-3′)/DCGR5 (5′-CGCTCYTTCATACATACATGCSGCCTT-3′), designed to specifically amplify 399- and 442-bp fragments of the TaVCV RNA-dependent RNA polymerase gene, respectively, were used for PCR amplification of the synthesized cDNA. PCR conditions comprised an initial cycle of denaturation at 94°C for 3 min; followed by 45 cycles each of 94°C for 1 min, 53°C (DCGF3/DCGR3) or 60°C (DCGF5/DCGR5) for 1 min, and 72°C for 2 min; ending with a final cycle of extension at 72°C for 10 min. PCR products of the expected size were cloned into pCR 2.1-TOPO (Life Technologies) and sequenced. Consensus sequences of the DCGF3/DCGR3 (GenBank accession KY614143) and DGCF5/DGCR5 (KY614144, KY614145) were subjected to similarity searches using the NCBI BLAST program. All sequences aligned with the Hawaiian TMo1-1 (KF921086) and Fijian (AY674964) isolates of TaVCV, and shared 90 to 93% amino acid identity with these isolates. To confirm the presence of TaVCV, a DCGF3/DCGR3 amplification product was DIG-labeled (Roche, Mannheim, Germany) and used to probe the cDNA of three symptomatic taro samples from American Samoa, as well as three TaVCV-positive taro samples from Hawaii, using a dot blot hybridization assay. Only cDNA of the sample from which the probe was derived tested positive using this assay, suggesting either low viral titer in the other samples and/or the high sequence diversity between TaVCV isolates observed here and reported previously (Revill et al. 2005b) reduced the efficacy of this procedure. This is the first report of TaVCV infecting taro in American Samoa and only the second report in the U.S. (Long et al. 2014). It is not clear how widespread this virus is in the territory, but with the RT-PCR assays described in this report, it should now be possible to investigate the incidence of TaVCV in American Samoa.