First Report of Tomato zonate spot virus in Iris tectorum in China

Abstract

Iris tectorum Maxim, a very popular Chinese traditional medicinal perennial herb belonging to the Iridaceae family, is widely grown as a year-round ornamental in China. During May to August 2014, as part of a survey for tospoviruses (family Bunyaviridae) in flue-cured tobacco, symptoms suspected to be caused by tospoviruses were observed on I. tectorum around farmers' fields in Kunming, Yunnan province. Symptoms were chlorotic spots on younger leaves and necrosis on older leaves. Since Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are two common tospoviruses in flue-cured tobacco fields in Yunnan, ELISA with monoclonal TSWV antibody (provided by J. X. Wu, Zhejiang University, China) and polyclonal TZSV antiserum (provided by J. H. Dong, Yunnan Academy of Agriculture Science, China) was performed to identify the presence of virus. Positive extinction values (ODλ405nm 0.835 ± 0.121 and 1.024 ± 0.193, as compared with the negative 0.153 ± 0.076 and the positive control 0.510 ± 0.109 at a confidence interval of P ≤ 0.05) were obtained from two symptomatic samples with TZSV antibody but not with TSWV. The absence of TSWV was confirmed with a commercially available immune-strip (Agdia, Elkhart, IN), following the manufacturer's instructions. To further verify the causal agent of these symptoms, total RNA was isolated from two symptomatic and one asymptomatic samples and reverse transcribed using degenerate primer J13 (1). These cDNAs were then used as a template in a universal PCR assay using specific primers TZSVNF (5′-ATGTCTAACGTCCGGAGTTTAACAC-3′) and TZSVNR (5′-TTAAAAAGACAGATCATTGCTG-3′), which amplify the complete nucleocapsid (N) protein. The PCR was carried out for denaturation at 94°C for 3 min, and subsequently 30 cycles were carried out, with each cycle consisting of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. An 0.8-Kb DNA fragment was amplified from symptomatic samples and cloned into a pGEM-T Easy (Promega, Madison, WI) vector. Three clones of each sample were selected and sequenced. BLAST analysis of the obtained sequences (Accession Nos. KM452916 and KM452917) revealed that the N sequences of these isolates have 96 to 99% nucleotide identity and 99 to 100% amino acid identity with the deposit TZSV sequence in NCBI from Yunnan (JN116580 to JN116583 and EF552433) (2). These combined results provide further confirmation of TZSV infection. It is known that perennial herb or ornamental plants may act as reservoirs for tospoviruses that can infect cultivated crops because tospoviruses have a very broad host range. Therefore, elaborate inspections for tospoviruses and appropriate management strategies to limit virus spread are necessary for production of crops. To our knowledge, this is the first report of TZSV in I. tectorum Maxim. References: (1) I. Cortez et al. Arch Virol. 146:265, 2001. (2) J. Dong et al. Arch Virol. 153:855, 2008.