First Report of Wild tomato mosaic virus Infecting Tobacco (Nicotiana tabacum) in China

Abstract

Wild tomato mosaic virus (WTMV), a potyvirus, has been reported in Laichau, Vietnam, infecting Solanum torvum (wild tomato) in 2008 (3), and Kanchanaburi, Thailand, infecting Capsicum spp. in 2013 (KF250353). In mid-May 2013, Nicotiana tabacum showing yellowing, mosaic, and/or ringspot symptoms were found in natural tobacco fields of Nanxiong, Guangdong Province, China. Total RNA was extracted from symptomatic leaves and reverse transcribed with M4T (5′-GTTTTCCCAGTCACGAC (T)15-3′) as the 3′ anchoring primer (1). The cDNA was used as template in a PCR assay using primers M4: 5′-GTTTTCCCAGTCACGAC-3′ and Sprimer: 5′-GGXAAYAAYAGYGGXCAZCC-3′, which amplifies a region comprising part of the NIb protein gene, the entire coat protein (CP) gene and the 3′ nontranslated region (UTR) of a potyvirus (1). A ~1,700-bp product was amplified from the cDNA derived from three of the five diseased plants. The product (KF639967) showed 87% and 84% nucleotide sequence identities with those of WTMV isolates KAN and Laichau, respectively. The CP deduced from the sequence of the product shared 87% and 86% nucleotide and 94% and 93% amino acid sequence identities with those of WTMV isolates KAN and Laichau, respectively. The 3′-UTR of the putative virus shared 93% and 92% nucleotide sequence identities to those of WTMV isolates KAN and Laichau, respectively. Thus, according to the molecular criteria for potyvirus species demarcation (2), the virus we identified should be an isolate of WTMV (isolate GD1). One of the diseased samples was homogenized in 0.1 mol/liter phosphate buffer (pH 7.0) and used to inoculate the potyvirus to healthy, two to four leaf-stage Capsicum annuum L., N. tabacum, and N. benthamiana. The inoculated, as well as mock-treated plants, which were inoculated only with phosphate buffer, were grown in soil under 12 h day/12 h night at 25°C. All inoculated N. tabacum and N. benthamiana plants developed yellowing and mosaic symptoms by 14 days post inoculation (dpi). For N. benthamiana, the symptom became very severe by 21 dpi and some diseased plants died prematurely. About 10% of inoculated C. annuum L. developed very mild veinal chlorosis 18 dpi. Cloning and sequencing experiments showed that all the symptomatic plants tested were WTMV positive, but Cucumber mosaic virus, Tobacco mosaic virus, and Tobacco etch virus negative. To our knowledge, this is the first report of WTMV in China. Also, it is the first report that WTMV infects Nicotiana spp. Although further experiments are needed to definitively attribute the disease observed in the field to WTMV, our results indicate that WTMV, which forms a monophyletic clade with a number of other potyviruses infecting Solanaceae species in phylogenetic analysis, is widely distributed, or is spreading in Southeast Asia. It may pose a threat to Solanaceae species cultivation in this region. References: (1) Chen et al. Arch. Virol. 146:757, 2001. (2) Adams et al. Arch. Virol. 150:459, 2005. (3) Ha et al. Arch. Virol. 153:25, 2008.