First Report of Telosma Mosaic Virus Infecting Emperor’s Candlesticks (Senna alata) in China

Abstract

Emperor’s candlesticks (Senna alata L.) is native to Mexico and is widely distributed in the tropics. It is an important medicinal tree, as well as an ornamental flowering plant in the family Caesalpinioideae. In July 2017, virus-like symptoms, including mosaic, mottle, and crumpling on leaves, were observed in emperor’s candlesticks in Xishuangbanna, Yunnan. Small RNAs (sRNAs) from the infected emperor’s candlesticks leaves were extracted and sequenced using an Illumina HiSeq 2000 system (Majorbio, China). All clean reads with lengths from 18 to 30 nucleotide sRNAs were assembled into contigs using Velvet software with k-mer value of 17. After searching against the GenBank database, 18 of 24 contigs were found that shared 84 to 100% nucleotide identities (approximately 38% coverage) with the full-length genome sequences of Telosma mosaic virus (TeMV) (GenBank accession no. DQ851493). The other six contigs showed more than 84% nucleotide identities with TeMV or more than 76% nucleotide identities with other potyviruses genome sequences owing to different program selection choices. For the amino acid sequence alignment, five of these six contigs shared 92% and above amino acid identities with the genome sequences of TeMV using BLASTx. To verify the result, reverse transcription polymerase chain reaction (PCR) was performed using a specific forward primer (TeMV-F, 5′-AGGAACTTGCAGCTGAAGGGAAAG-3′) designed based on the sequence of assembled contigs of virus-derived sRNAs, and the reverse primer M4 described before (Chen et al. 2001). Total RNAs were extracted from infected leaves using the RNeasy Plant Mini Kit (Qiagen, German), and reverse transcription was performed using M-MLV reverse transcription (RNase H-) (TaKaRa, Japan) and primer M4T, as described (Zhang et al. 2016). The 3′-proximal sequence (containing part of the NIb gene, the complete CP gene, and 3′-UTR) was amplified by PCR using Ex Taq polymerase (TaKaRa). Fragments of the expected size (1,300 bp) were observed after electrophoresis in 1.0% (w/v) agarose gels in infected but not in healthy plants. This PCR fragment was purified, cloned, and three independent clones were sequenced using Sanger methodology (GenBank accession no. MH137276). BLASTn analysis of the amplicon sequence revealed nucleotide identity of 89% (Hanoi-DQ851493) and 92% (GX1-KJ789129) with previously characterized TeMV isolates. Nucleotide and amino acid sequence analysis of the putative CP gene revealed 86.0 to 91.4% nucleotide and 85.3 to 94.9% amino acid identity, respectively, to TeMV isolates Hanoi, GX1, Pangda15 (AM409188), and Pangda12 (AM409187). The filamentous virus particles (700 to 800 × 13 nm) and pinwheel inclusions typical of members of the genus Potyvirus were observed in infected emperor’s candlesticks leaves by transmission electron microscopy. Four infected samples tested positive in antigen-coated plate ELISA with potyvirus group-specific antibodies (Agdia, U.S.A.),whereas three healthy plants tested were negative. These results further confirmed the presence of TeMV in infected emperor’s candlesticks leaves from Yunnan; hence, the isolate was named TeMV-YN. TeMV was reported to infect telosma (Telosma cordata), passion fruit (Passiflora edulis), and patchouli (Pogostemon cablin) in Asia (Chiemsombat et al. 2014; Ha et al. 2008; Noveriza et al. 2016). To our knowledge, this is the first report of TeMV infecting emperor’s candlesticks.