Molecular identification of tobacco vein necrosis pathogen and complete genome sequence analysis based on small RNA deep sequencing

【Objective】 The purpose of the study was to clarify the virus pathogens causing tobacco mosaic, vein necrosis and other symptoms in Nandan County, Hechi City, Guangxi, so as to provide theoretical basis for the comprehensive prevention and control of tobacco virus diseases.【Method】 In May 2022, five tobacco leaf samples showing symptoms such as vein necrosis and mosaic were collected from Nandan, Hechi. The samples were identified and analyzed for genetic evolution by using small RNA deep sequencing, reverse transcription polymerase chain reaction(RT-PCR), friction inoculation, segmented cloning, phylogenetic evolution and recombination analysis.【Result】 Small RNA sequencing obtained five sequences with high nucleotide identity(99.00%-100.00%) with different potato virus Y(PVY) isolates in the GenBank database. Five sequences were assembled to obtain a complete genome sequence with a total length of 9708 nt. The single virus was obtained by identifying a single spot isolate of the host Chenopodium amaranticolor. The purified virus single spot was used to inoculate tobacco variety K326 and Nicotiana benthamiana, in which the new leaves of K326 produced necrotic symptoms, while the new leaves of N. benthamiana showed irregular dark green spot symptoms. Total RNA was extracted from the leaf samples of K326 and N. benthamiana, respectively, and the whole genome sequences of the virus isolates were obtained by RT-PCR detection and segmented cloning. The results showed that the identity of the virus sequences of the two tobacco varieties with the sequences obtained by small RNA deep sequencing was 99.90%. BLAST analysis of the sequences in GenBank showed that the sequences obtained in the study had high nucleotide identity with the PVY isolates that had been published in GenBank, and the highest nucleotide identity was 99.01% with the isolate from Heilongjiang Province, China, PVY HLJ26(MF134425). It indicated that the virus sequence obtained from the study was one isolate of PVY, and named as PVY-GXnd1(OP131591). The results of recombination analysis showed that PVY-GXnd1 was a new PVY isolate derived from the recombination of PVY^O-139(U09509.1), PVY^N-Mont(AY884983.1) and PVY^N-605(X97895.1). The new PVY isolate from recombination mainly had three recombination regions, which were 1-498, 2415-5816 and 8555-9373 nt, respectively, covering P1, P3, 6K1, CI, 6K2, VPg, Nib and CP protein coding regions of PVY. It contained two different recombination structural features of the PVY^NTN and PVY^N-Wi strains, so the PVY-GXnd1 strain was closer to the PVY^NTN-NW strain. Analysis of the phylogenetic evolutionary tree constructed on the basis of the coding region of PVY showed that PVY-GXnd1 was in the same small cluster with PVY HLJ26, an isolate from Heilongjiang Province, China, having close relative relationship. And the isolate was attributed to the PVY^NTN-NW(SYR-II) strain, which was consistent with the recombination analysis. 【Conclusion】 In this study, it is confirmed that the virus PVY-GXnd1 infecting tobacco causing vein necrosis symptom in Nandan County, Hechi City, Guangxi is a recombinant strain of PVY^NTN-NW(SYR-II).