Molecular and serological characterization of a distinct potyvirus causing latent infection in calla lilies

Abstract

A virus (isolate: Ca-M19) capable of inducing local lesions on Chenopodium quinoa Willd. was isolated from calla lilies (Zantedeschia spp.). Subculture of Ca-M19 was easily maintained in C. quinoa, but a back inoculation from single lesion of C. quinoa to calla lilies has so far not been successful. Typical potyvirus-like flexuous particles were consistently detected in Ca-M19 infected plants, and a 1.3-kb DNA fragment was amplifiedfrom these plants by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus degenerate primers. The PCR product was cloned and its sequence analyzed (AF469171). The amplicon was revealed to correspond to the 3’ terminal region of a potyviral genome. After comparing this sequence with known potyvirus sequences in the GenBank, we considered the virus a new species of Potyvirus based on the uniqueness in its coat protein gene (CP) and the 3’ non-coding region (NCR). Comparative studies showed that Soybean mosaic virus (SMV) and Watermelon mosaic virus 2 (WMV 2) were the two most similar potyviruses with Ca-M19, but they shared only 80% of nucleotide identities in CP and NCR with Ca-M19. Attempts to purify a sufficient quantity of Ca-M19 from C. quinoa for preparation of antibodies were unsuccessful. Alternatively, Ca-M19 CP was expressed by the vector pET28b and puri-fied from E. coli culture, and polyclonal antibodies were prepared in rabbits. The antibody was applied in ELISA, Western blotting, SDS-immunodiffusion and immuno-specific electron microscopy for the detection of Ca-M19 in calla lilies. It did not react with at least five calla lily infecting potyviruses, including Dasheen mosaic virus, Bean yellow mosaic virus, Konjak mosaic virus, Turnip mosaic virus, and Zantedeschia mild mosaic virus. Indirect ELISA and SDS-immunodiffusion tests showed that Ca-M19 was serologically related, but distinct from Bean common mosaic virus (BCMV), Black cowpea mosaic virus (BlCMV), Melon vein banding mosaic virus (MVbMV), Passionfruit mottle virus (PaMV), Passionfruit crinkle virus (PCV), Pas-sionfruit woodness virus (PWV), Soybean mosaic virus (SMV), Watermelon mosaic virus 2 (WMV 2), and Zucchini yellow mosaic virus (ZYMV). Besides serological techniques, a primer pair (M19u/M19d) and a DNA probe were designed which could also specifically detect and differentiate Ca-M19 from other viruses. By the use of specific antibodies in ELISA, Ca-M19 was frequently detected in calla lily plants collected from several major calla lily production townships in Taiwan. Among 86 field samples positively reacting to the antibody, 77 of them exhibited evident systemic mosaic symptoms, but these symptomatic plants were confirmed to be infected simultaneously by other viruses. Nine plants were found to be infected by Ca-M19 alone. These plants were confirmed to have remained symptomless throughout a 6-month observation period. Therefore, we propose naming this isolate Calla lily latent virus (CLLV) for its inability to develop any visible symptoms on calla lily