First Report of Broad bean wilt virus 2 in Echinacea purpurea in China

Field-grown Echinacea purpurea plants showing necrosis, leaf roll, yellow mosaic, and mosaic symptoms in leaves were collected in June 2010 in Huairou, Beijing, China. ELISAs of extracts of four samples showed that one sample with mosaic symptoms had a positive reaction with Broad bean wilt virus 2 (BBWV-2) monoclonal antibody provided by Professor X. P. Zhou (1). The monoclonal antibody recognized the 44.7 kD coat protein subunit of BBWV-2. We used Chenopodium quinoa as an assay species to isolate the virus by sap transmissions and to maintain the virus strain. Sap from infected C. quinoa, when inoculated onto indicator plant species, induced the following symptoms: C. quinoa: local lesions in inoculated leaves, systemic chlorotic mottle in upper leaves, deformation, and apical necrosis; C. amaranticolor: chlorotic local lesions, systemic mosaic and leaf distortion; Nicotiana benthamiana: systemic mosaic; Gomphrena globosa: local purple spots in inoculated leaves and systemic infection in upper leaves; Tetragonia expansa: local lesions, but no symptoms of systemic infection; Physalis floridana: systemic mosaic. No symptoms were observed on Capsicum annuum, Datura stramonium, N. glutinosa, or N. tabacum cv. White Burley. To confirm BBWV-2 infection, total RNAs extracted from infected C. quinoa leaves were reverse transcripted to cDNA using oligo-dT primer (T17V). The primer pair Fab5'R1F (5'-AAATATTAAAACAAACAGCTTTCGTT-3') and Fab5'R1R (5'-TTCAAAGCTCGTGCCATNTYATTKGC-3') for specific detection of the Fabavirus genus (2) was used for PCR analysis. The amplified fragment is between the 5'-terminal non-translatable region (NTR) and the beginning of the coding region of RNA1. Amplicons of approximately the expected size (~391 bp) were produced from the virus-infected C. quinoa and a BBWV-2 positive control (ATCC PV131, PV0537). Amplicons of approximately the expected size (~350 bp) were produced from the BBWV-1 positive control (ATCC PV132). However, no such amplicons were observed from healthy C. quinoa plants and water control. The 391-bp amplicons of RNA1 obtained from the infected C. quinoa were cloned and sequenced. Comparison with sequences of other BBWV-2 isolates showed that the isolate we obtained (No. JX070674) had approximately 99% nt identity (98% amino acid identity) with Chinese BBWV-2 isolate BC (No. FJ485686.1) (3). As an ornamental and medicinal plant, E. purpurea is widely cultivated in northern China. Up until now, Tomato ring spot virus, Tobacco rattle virus, Cucumber mosaic virus, and Tomato spotted wilt virus have been detected or isolated from E. purpurea in the world (4). To our knowledge, this is the first report of BBWV-2 infecting E. purpurea in China. BBWV-2-infected E. purpurea may have less secondary metabolites, which could influence the quality and therapeutic efficacy of this herbal medicine. References: (1) L. Qing et al. Acta Microbiologica Sinica 40:166, 2000. (2) R. M. Ferrer et al. J. Virol. Methods 144:156, 2007. (3) C. Sui et al. Plant Dis. 93:844, 2009. (4) B. Dikova. Bulgarian J. Agric. Sci. 17:306, 2011.