Gentian (Gentiana sp.) is an important perennial flower in Japan. In terms of cut flower cultivation area size it occupies 2nd to 3rd place. Cucumber mosaic virus (CMV), broad bean wilt virus (BBWV), tobacco rattle virus and clover yellow vein virus have been reported to infect gentians. We have analyzed gentian plants collected from the largest production area in Japan, Ashiro-cho in the Iwate prefecture, and found that most plants with severe virus symptoms were infected either by CMV alone or by both CMV and BBWV. The association of CMV with severe symptomatolgy was also confirmed in greenhouse experiments in which CMV and BBWV isolates obtained from symptomatic gentian plants were inoculated to gentian seedlings. CMV induced severe symptoms, while BBWV did not cause any. Dual inoculation of CMV and BBWV slightly aggravated symptoms in comparison with those caused by CMV alone. These results suggested that CMV is the most destructive virus in gentian. In greenhouse experiments three satellite RNA containing attenuated CMV strains (CMV vaccines), CMV-PV1, CMV-NDM1 and CMV-KO3 were tested to evaluate their possible adverse or protective effects on gentian plants. The vaccines were pre-inoculated to lower leaves of gentian seedlings, and two weeks later upper leaves were challenge-inoculated with virulent CMV and/or BBWV. The results showed that CMV vaccines either caused no symptoms or very mild symptoms on gentian. The majority of vaccinated seedlings were protected against challenge-inoculation by CMV or by CMV and BBWV. The protective effect of CMV vaccines was also examined in two farmers' fields in Ashiro-cho, Iwate. CMV-KO3, CMV-NDM1 and another satellite containing CMV strain, CMV-KO1 were inoculated to gentian seedlings before their transplantation to the field. Such plants were compared for a period of 5 to 6 years with nonvaccinated plants cultivated near the vaccinated ones. The number of plants with severe virus symptoms increased over time in both vaccinated and non-vaccinated plants. However, the percentage of plants with severe symptoms was always lower in vaccinated plants than in non-vaccinated ones, and this difference became more apparent year after year. Typically, the number of cut flowers harvested from vaccinated plants in a farmer's field was 2 to 3 times larger than from nonvaccinated plants in the 6(th) growing year. These results suggest that vaccination of gentian plants protects them from virulent CMV and also extends their commercial cultivation period by at least 2 more years.
