Identification Of The Viral Pathogens Of Fujian Narcissus Disease And Complete Genome Sequences Analyses Of Two Viruses

Vius disease is one of the main diseases endangering narcissus production. The result of field survey of narcissus disease in Zhangzhou city of Fujian showed that the disease symptoms of narcissus mainly included chlorotic mottle, chlorotic stripe, yellow stripe and mosaic. By electron microscope observation, many flexuous filamentous particles that were c.550-850nm long were observed. Detection by serological method showed that the samples were infected with Narcissus mosaic virus (NMV), Narcissus yellow stripe virus (NYSV) and Narcissus late season yellows virus (NLS YV). By using molecular biology determination method with the specific primers designed for viruses which have been reported in narcissus and the degenerate primer designed for Potyvirus, combined with biological determination method, there were at least six knowm viruses were detected. The viruses included NMV, NYSV, NLSYV, Narcissus latent virus (NLV), Narcissus degeneration virus (NDV) and Narcissus common latent virus (NCLV). In addition, these were two Potyvirus isolates which were Y13-1and ZZ-2, have also been identified. Y13-1maybe is a new species in the genus Potyvirus and ZZ-2maybe is a strain of NYSV.Using RT-PCR based on degenerate primer of Potyvirus and RACE methods, the complete genome sequences of NLSYVisolate from Zhangzhou (NLSYV-ZZ) and ZZ-2isolate and the3’-end of the genome of Y13-1isolate which is c.4.6kb have been determined.This is the first report of the complete genome sequence of NLSYV (accession number JQ326210). The full-length genome sequence of NLSYV-ZZ comprises9651nucleotides (nt), with123nt in the5’-untranslated region (5’-UTR) and210nt in the3’-untranslated region (3’-UTR). The complete genome sequence of NLSYV-ZZ was compared to those most closely related viruses, NLSYV-ZZ has the highest identity with NYSV at the nucleotide level, with69.9%, and with Scallion mosaic virus (ScaMV) and the isolates of Turnip mosaic virus (TuMV) the identities were62.5%and58.8-59.8%, respectively. In phylogenetic relationship of NLSYV-ZZ and other potyviruses which based on amino acid sequences of the polyproteins, NLSYV-ZZ clustered with NYSV, ScaMV, TuMV, Japanese yam mosaic virus (JYMV). The results indicated that NLSYV-ZZ was most closely with these viruses.The3’-end of the genome of Y13-1isolate comprises4606nt (accession number JQ911735). BLASTn search with the3’-end of the genome of Y13-1, the result showed that Y13-1has the highest identity with NYSV, followed by ScaMV and TuMV. Sequences comparison based on CP gene showed that NLSYV-ZZ has the highest identities with NYSV and NLSYV-ZZ, with73.3%nt (73.6%aa) and72.5%nt (77.1%aa), respectively. These results meet the species demarcation criteria in terms of genome sequence relatedness. So Y13-1isolate is considered as a distanct potyvirus, and Narcissus mottle virus (NMoV) is temporarily proposed.The full-length genome sequence of ZZ-2isloate comprises9654nucleotides (nt), with130nt in the5’-UTR and206nt in the3’-UTR (accession number JQ911732). The complete genome sequence of NLSYV-ZZ has the highest identity with NYSV at the nucleotide level, with71.9%, and identity with NLSYV-ZZ is70.2%. In the CP region, ZZ-2also has the highest identity with NYSV, with77.6%nt (84.4%aa). These values are above the values of the threshold for species demarcation of Potyvirus. So ZZ-2isolate is considerd as a strain of NYSV.