Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Short Communication Host specific preference for low temperature in the multiplication of a tombusvirus, gentian virus A Koki Fujisaki a, *, Yoshiko Abe a , Chika Tateda a , Mari Iwai a , Masanori Kaido b , Kazuyuki Mise b a Iwate Biotechnology Research Center, Kitakami, Iwate, Japan b Laboratory of Plant Pathology, Kyoto University, Kyoto, Japan ABSTRACT Gentian virus A (GeVA), a novel tombusvirus isolated from Japanese gentian, has shown only a limited ability to infect Japanese gentians under experimental conditions. In this study, temperature was found to affect the efficient multiplication of GeVA in Japanese gentians. GeVA efficiently multiplied in inoculated leaves of gentians at 18 °C but not at 23 °C. This low-temperature (18 °C)-preferred GeVA multiplication was specifically observed in Japanese gentians and Arabidopsis thaliana but not in other experimental plants, including Nicotiana benthamiana. In A. thaliana, visible defense responses, including pathogenesis-related protein 1 expression, were not detected at 23 °C. Furthermore, several A. thaliana mutants, including those defective in RNA silencing, with altered plant immunities did not allow GeVA to multiply to detectable levels at 23 °C. Taken together, these data suggest that unique interaction between GeVA and gentians/A. thaliana, which is independent of RNA silencing, may underlie the low-temperature-preferred multiplication of GeVA. Japanese gentians (Gentiana triflora, Gentiana scabra and their hy- brids) are important ornamental flowers in Japan (Nishihara et al., 2017), and have suffered from several viral diseases (Atsumi et al., 2015; Kobayashi et al., 2005, 2013; Nekozuka et al., 2005; Yamamoto, 2012). Gentian virus A (GeVA) is a positive-sense RNA virus that be- longs to the Tombusvirus genus. GeVA has been isolated previously from Japanese gentian (Fujisaki et al., 2018). Nevertheless, inoculating gentians with GeVA did not lead to the development of visible symp- toms, and only limited GeVA infections of Japanese gentians have been observed under experimental conditions (16-h light/8-h dark photo- period at 20-25 °C; Fujisaki et al., 2018). In this study, to determine the virulence of GeVA to Japanese gentians, we examined the effects of environmental conditions on GeVA infection and found that GeVA in- duces necrotic symptoms at a low temperature (18 °C). For the infection assay, gentian plants grown in vitro, which are available all year, were inoculated with GeVA. Previous growth- chamber conditions included an unstable temperature (20–25 °C; Fujisaki et al., 2018). Here, plants were incubated at a stable tem- perature, 18 or 23 °C, in a growth chamber (16-h light/8 h-dark pho- toperiod). At 4 weeks post-inoculation, necrotic symptoms were ob- served on inoculated leaves of gentian cultivars ‘Alta’ and ‘Polano white’ (PW) at 18 °C but not at 23 °C (Fig. 1A), indicating that GeVA is virulent in Japanese gentians at 18 °C. To examine the effects of tem- perature on GeVA multiplication, press-blot assays (Fujisaki et al., 2009) were performed at 10 d post-inoculation (dpi), at which time symptoms were not visible. Using an anti-GeVA antiserum, GeVA infections in inoculated leaves of Japanese gentians (‘Alta’ and ‘PW’) were detected at 18 °C. However, a visible GeVA signal was not de- tected at 23 °C (Fig. 1B), indicating that GeVA multiplication is limited during early infection stages before 10 dpi in inoculated leaves. These data suggest that a lack of GeVA symptoms at 23 °C is due to the re- striction of virus multiplication. This low-temperature-preferred GeVA multiplication was observed in several gentian cultivars/lines, in- cluding G. triflora, G. scabra and their hybrids (Table 1), suggesting that it is a common feature of GeVA infection in Japanese gentians. Cucumber mosaic virus (CMV) is another viral pathogen of Japanese gentian (Piyasak et al., 1994) that is distantly related to tombusviruses, including GeVA. When ‘Alta’ was inoculated with CMV, severe symp- toms were observed at 23 °C, while those observed at 18 °C were re- latively mild (Fig. 1C). Consistent with this result, CMV multiplied more efficiently at 23 °C than at 18 °C (Fig. 1B), indicating that the enhanced susceptibility of Japanese gentians to viral infections at a low tem- perature is virus-species specific. Although many plant viruses efficiently multiply at ∼23 °C in several host plants, some viruses preferentially multiply at low tem- peratures (Dujovny et al., 2009; Ohsato et al., 2003; Yang et al., 2018). For example, red clover necrotic mosaic virus multiplies at 17 °C more efficiently than at 25 °C in several plants, including Chenopodium quinoa and Nicotiana benthamiana (Mizumoto et al., 2002). Consequently, we examined the effects of temperature on GeVA infections in several plant species. Inoculating N. benthamiana with GeVA led to symptoms at both 23 °C and 18 °C (Fig. 2A). Even in Eustoma grandiflorum, which belongs https://doi.org/10.1016/j.virusres.2020.198048 Received 17 March 2020; Received in revised form 2 June 2020; Accepted 5 June 2020 ⁎ Corresponding author at: Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate, 024-0003, Japan. E-mail address: k-fujisaki@ibrc.or.jp (K. Fujisaki). Virus Research 286 (2020) 198048 Available online 06 June 2020 0168-1702/ © 2020 Elsevier B.V. All rights reserved. T