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First report of Dahlia latent viroid and Potato spindle tuber viroid mixed-infection in commercial ornamental dahlia in Japan
1 Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori 036-8561, Japan
2 National Agricultural and Food Research Organization, Tsukuba, Ibaraki, 305-8519, Japan
3 Faculty of Science and Technology, Akita Prefectural University, Yurihonjo, Akita 015-0055, Japan
Received: 07 Jan 2015; Published: 30 Mar 2015
Dahlia (Asteraceae) has been cultivated since 1841 in Japan. Two viroids belonging to the family Pospiviroidae, Potato spindle tuber viroid (PSTVd) and Dahlia latent viroid (DLVd), were identified separately from dahlia plants in Japan and in the Netherlands, respectively (Tsushima et al., 2011; Verhoeven et al., 2013). Because DLVd was characterised as a novel species, the findings drew attention towards the study of DLVd infection in dahlia and its potential risk in domestic dahlia cultivation. A total of four dahlia samples infected with PSTVd-dahlia (Tsushima et al., 2011) were collected, and total RNA was extracted from approximately 200 mg of leaf tissue using TRIzol® reagent (Life Technologies, Carlsbad, USA) according to manufacturer's instructions. RNA was separated in 2-dimensional polyacrylamide gel (2D-PAGE) (Schumacher et al., 1983) and two specific bands appeared, one corresponding to PSTVd infection and the other being unknown (Fig. 1). The unknown circular RNA samples were gel-excised and first tested with PSTVd-specific primers using a two-step reverse-transcription-polymerase chain reaction (RT-PCR) (Tsushima et al., 2011), with, however, negative results. Therefore, a DLVd-specific primer set, DLVd-P1 (5'-GGGGCTCCTCAGAGAGTCTC-3') and DLVd-P2 (5'-GGGGCAACTCCGAGAGTGCTG-3') (Verhoeven et al., 2013), was then used in two-step RT-PCR for the unknown viroid-like circular RNAs. The amplified DNA bands of the expected size (~340 bp) from these positive samples were purified, ligated to pGEM®-T easy vector (Promega, Madison, USA) and sequenced. Identical 342 bp sequences were obtained from all four dahlia isolates (GenBank Accession No. LC036322), corresponding with DLVd reported from the Netherlands (JX263426). Furthermore, a total of 78 dahlia leaf samples were collected from fields around Japan, irrespective of disease symptoms, and verified by two-step RT-PCR using the same DLVd-specific and pospiviroid-specific primers (Tsushima et al., 2011). Of these samples, DLVd infection was detected in 38, suggesting that DLVd is widespread in Japan. New PSTVd infection was not found. To evaluate the pathogenicity of DLVd and PSTVd, these viroids were co-inoculated into wild hop (Humulus lupulus var. cordifolius) and dahlia seedlings. DLVd alone was inoculated into members of the Asteraceae family, Chrysanthemum pacificum, Tithonia rotundifolia, Helianthus annuus and Glebionis coronaria. Inoculated dahlia seedlings were positive for both DLVd and PSTVd, but wild hop and the remaining members of Asteraceae were negative. Dahlia seedlings that were positive for DLVd and PSTVd showed mild leaf curling in the early stage of growth (Fig. 2). To the best of our knowledge, this is the first report of DLVd infecting commercial dahlia in Japan and also of the mixed-infection of DLVd and PSTVd in commercial dahlia plants. A possible negative synergistic interaction of DLVd and PSTVd during dahlia cultivation must be considered.
This work was supported in part by Japan Society for the Promotion of Science KAKENHI grant no. 24380026. TT received Research Fellowship of the Japan Society for the Promotion of Science.
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- Tsushima T, Murakami S, Ito H, He YH, Raj APC, Sano T, 2011. Molecular characterization of potato spindle tuber viroid in dahlia. Journal of General Plant Pathology 77, 253-256. [http://dx.doi.org/10.1007/s10327-011-0316-z]
- Verhoeven JThJ, Meekes ETM, Roenhorst JW, Flores R, Serra P, 2013. Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification. Journal of General Virology 94, 711-719. [http://dx.doi.org/10.1099/vir.0.048751-0]
To cite this report: Tsushima T, Matsushita Y, Fuji S, Sano T, 2015. First report of Dahlia latent viroid and Potato spindle tuber viroid mixed-infection in commercial ornamental dahlia in Japan. New Disease Reports 31, 11. [http://dx.doi.org/10.5197/j.2044-0588.2015.031.011]
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