Citrus mosaic virus (CiMV) and navel orange infectious mottling virus (NIMV) are considered strains of satsuma dwarf virus (SDV). To understand the distribution of different isolates of SDV in China, a field survey was conducted, which allowed for the detection of CiMV and NIMV in citrus plants in Zhejiang and Sichuan provinces, respectively. In the present work, two CiMV isolates, including one named CiMV-GTC1 from Goutoucheng sour orange and one named CiMV-SE1 from Setoka tangor, as well as one NIMV isolate named NIMV-EH1 from Ehime-ken No. 38 tangor were, for the first time, fully sequenced. Among the isolates, CiMV-SE1 and CiMV-GTC1 were closely associated with the partial genomes of known CiMV isolates, while NIMV-EH1 was markedly different from known NIMV isolate. Sequence analysis demonstrated that CiMV, NIMV, and SDV shared similar genome organization and high nucleotide identities at the genome level. In addition, phylogenetic reconstruction of the three viruses also suggested a close relationship. Furthermore, recombination analysis provided evidence for their common cooccurrence in nature. These findings contribute significantly to our understanding of the natural evolution of SDV populations and can provide a basis for preventing the spread of different SDV isolates in the main citrus production regions.
Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch's postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus-host interactions.
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