Molecular characterization of a new macluravirus from yam in Yunnan, China

Abstract

Yams (Dioscorea spp.) are important in China as vegetables and as sources of traditional herbal medicine. They suffer from various viral diseases, but there has only been one small study of them in China [6]. We have investigated the viruses associated with diseased yams at two farms 5 km apart in Yongsheng county, Yunnan province. Samples 1–3 from D. zingbierensis had a combination of vein clearing, veinal necrosis and chlorotic symptoms on the expanded leaves. Samples 1 and 2 were from the same farm. Sample 4 from D. parviflora (same site as sample 3) had systemic chlorosis, mottling and mosaic symptoms that also affected the newly emerging leaves. Virus particles and inclusion bodies typical of members of the family Potyviridae were observed in infected leaves. Particles were purified [3], RNA was extracted and the 30-terminal portion of the viral genome was then amplified by RT-PCR by standard methods using universal Potyviridae primers [4]. Products were separated, purified and cloned (into pMD19-T; TaKaRa), and three independent clones were auto-sequenced in both directions using an ABI PRISM 3770 DNA Sequencer. Sequence alignments and phylogenetic analysis were done using MEGA 4.0 [7]. Fragments of 1,742–1,748 nucleotides in length were amplified and sequenced from each sample. Isolates 1, 3 and 4 were almost identical to one another (99.4–99.5% nucleotide identities) while isolate 2 had 78.2% identity to the others. The sequences consisted of an incomplete open reading frame with a stop codon at position 1531–1533 followed by a 30-UTR of 209 nucleotides (215 in sample 2). In comparisons with published sequences, the nearest matches were those for Chinese yam necrotic mosaic virus (ChYNMV, genus Macluravirus), with about 69% nt and 72–73% aa identity. Comparisons with all other macluravirus sequences suggested that the partial ORF of 510 amino acid residues in the new sequences consisted of 222 aa at the C-terminus of the NIb and a coat protein (CP) of 288 aa (32.3 kDa) with a cleavage site of INLQ/M (IDLQ/ M in sample 2). The LQ/M cleavage is typical of members of the genus Macluravirus [1]. The partial NIb gene contained the consensus motifs (T/S)GX3-TX3-N(T/S) and GDD of the RNA-dependent RNA polymerase. The N-terminus of the CP did not contain a DAG (or similar) motif that is known to be associated with aphid transmission in the genus Potyvirus. Molecular criteria for discriminating between species in the family Potyviridae indicate a threshold of 76% (nt) or 82% (aa) identity [2]. Based on these criteria, the new yam sequences belong to a single new species in the genus Macluravirus. These conclusions were confirmed by phylogenetic analysis (Fig. 1). Although isolate 2 is rather different to the others at the molecular level, it was one of the three from D. zingbierensis that had similar symptoms and is not sufficiently different to justify placement in a further new species. Interestingly, there is a similar range of differences between isolates of Cardamom mosaic virus J.-G. Wang X.-J. Zou H.-R. Chen (&) The Phytopathology Laboratory of Yunnan Province, Yunnan Agricultural University, 650201 Kunming, People’s Republic of China e-mail: hrchen44@yahoo.cn