First Report of Broad bean wilt virus 2 and Turnip mosaic virus in Tropaeolum majus in China

Tropaeolum majus, an herbaceous plant in the family Tropaeolaceae, is widely cultivated in China as an ornamental and medicinal plant. T. majus plants showing chlorotic spots and mosaic symptoms were collected in July 2016 from a park at Chaoyang district, Beijing, China. Leaves from six symptomatic plants were tested for Cucumber mosaic virus (CMV), Tomato aspermy virus (TAV), Broad bean wilt virus 1 (BBWV-1), BBWV-2, Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), Lettuce mosaic virus (LMV), Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Zucchini yellow mosaic virus (ZYMV) by ELISA (Agdia, Elkhart, IN, U.S.A.). All six samples tested negative for CMV, TAV, TSWV, INSV, IYSV, LMV, PVY, and ZYMV. All six samples were positive for BBWV-1, BBWV-2, and TuMV. Total RNA was extracted from symptomatic leaves of T. majus using an RNA extraction kit (Bioteke, Beijing, China). Primer pairs BBWV1-4758F/BBWV1-5245R to detect BBWV-1, BBWV2-5692F/BBWV2-5907R to detect BBWV-2 (†), and degenerate potyvirus primers to detect TuMV (†) were used for RT-PCR analysis. Amplicons of the expected size were obtained for BBWV-2 (238 bp) and for TuMV (1,800 bp), but not for BBWV-1. The purified PCR fragments of BBWV-2 were directly sequenced. The purified PCR TuMV fragments were cloned into pMD18-T vector (TaKaRa, Dalian, China) and sequenced. BLASTN analyses of the T. majus BBWV-2 isolate sequence (GenBank accession no. KX698095) showed 96 to 99% nucleotide identity with BBWV-2 isolates in GenBank (FJ485684.1, U65984.2, KC625499.1). The TuMV isolate sequence (KX698096) showed 98 to 99% nucleotide identity with TuMV isolates in GenBank (KC119189.1, HQ446216.1, AJ297629.2). The two viruses were separated using local lesions on Chenopodium amaranticolor. BBWV-2 and TuMV were maintained on Nicotiana benthamiana and Brassica pekinensis, respectively, for further mechanical inoculation experiments. The BBWV-2 isolate induced the following symptoms: C. quinoa and C. amaranticolor: chlorotic lesions in inoculated leaves, systemic chlorotic mottle in upper leaves, and apical necrosis; N. benthamiana: chlorotic ringspots in inoculated leaves, systemic mottle in upper leaves, and leaf distortion; Physalis floridana, Petunia hybrid, and T. majus: mild mosaic in upper leaves; Vicia faba: leaflet necrosis, wilt, and often death; Vigna unguiculata: reddish-brown local lesions in inoculated leaves; Phaseolus vulgaris: chlorotic ringspots in inoculated leaves. The TuMV isolate induced the following symptoms: C. quinoa: chlorotic lesions in inoculated leaves, followed by systemic veinal flecks and spots; C. amaranticolor: chlorotic local lesions in inoculated leaves; N. benthamiana: systemic mottle; P. floridana and P. hybrida: systemic mosaic; B. pekinensis and T. majus: chlorotic spots in inoculated leaves and mosaic in upper leaves. All indicator host plants producing symptoms were tested by ELISA and confirmed to be positive. TuMV was previously reported in T. majus in Brazil (†). To our knowledge, this is the first report of the BBWV-2 and TuMV infections of T. majus in China and the first report of T. majus as a natural host of BBWV-2. © 2017, American Phytopathological Society. All rights reserved.