| Allium chinense(G.Don),the genus Allium,family Liliaceae,is an economically important vegetable that has been considered to be of Asian origin.It can be usually used as edible bulb or medicinal herb.However,virus diseases of A.chinense are sever,causing the serious losses in bulbs production and poor quality.Some plants of cultivated A.chinense showing mosaic,chlorotic streak,twist,and crinkle on leaves were collected from fields at Jiangxia,a suburban district of Wuhan, Hubei Province,China,and transplanted into pots in a greenhouse.Crude sap from naturally infected plants were mechanically inoculated to six indicator plants.Results showed that the indicator chenopodium quinoa was the best host for virus propagation indoor.Three types of negatively stained virus particles from 4 infected sample leaves, including rod shape,lightly straight and curved filamentous shape,were observed under an electron microscope.Pinwheel or cylindrical inclusions,typical of a potyvirus infection,were also examined in the electron microscope by using ultrathin sections of diseased leaves.A potyvirus was detected in diseased A.chinense using degenerate primer sprimer and M4.The 3'-termined part(1625nts) of its genome was cloned and sequenced. Sequence analysis showed it contained the partial NIb gene(636 bp) and the complete coat protein(CP gene;771 bp),excluding its poly(A) tails.The 3'-termined part of obtained virus genome was closely similar to Onion yellow dwarf virus(OYDV)(highest identity 99%).The CP gene had 80%-99%identity at nucleotide level and 87%to 99% identity at the amino acid level with corresponding regions of known OYDV isolates from other hosts.Analysis of CP phylogenetic tree indicated the isolate,named tentatively OYDV-WH,showed high identity(no less than 97%) at nts level with Yunnan isolate (OYDV-YN2,GenBank accession number AJ409313),Jiangsu isolates(OYDV-JS1, OYDV-JS2,GenBank accession number AJ293278 & AJ409310,respectively) and Japan isolate(OYDV-Gzm,AB000840),and became closely the same group.It was first report of OYDV infecting A.chinense in China.Nine clones of carlavirus were obtained using carlavirus specific degenerate primer(pCar-1(|)) and p(?)imer M4,the 3'-terminal part(1808-1812 nts,respectively) of its genome RNA were amplified and sequenced,excluding its poly(A) tails.Sequences analysis showed that the partial genome contained ORF3-6 and 3'-UTR of the RNA genome of carlavirus genus.Sequence comparison indicated that the 3'-termined part of obtained virus clones were closely similar to Garlic latch t virus(GLV),showing 75%-82%identity to other known GLV.7 clones out of obtained clones were great variable in the 3'-termined same region of genome,showing 77%-93%identity at nucleotide level between themselves.The CP gene had 75%-87%identity at nucleotide level and 88%to 97%identity at the amino acid level with corresponding regions of known GLV isolates from other hosts,and had 76%-89%identity at nucleotide level and 88%to 100%identity at the amino acid level between the 7 clones from this paper.The isolates P2 and carl-5 had high identity(97%) at the amino acid level with the Japan isolate GLV-Rjpn(GenBank accession number AB004565) from the same host A. chinense.Analysis of CP phylogenetic tree suggested that the clone P4 was a distant group,and other clones were grouped respectively with other reported GLV isolates. More ORFs sequence comparison indicated that the ORF3,ORF4,ORF6 of its genome had 71%-90%,59%-85%and 82%-95%identity at the amino acid level between the clones and the reported GLV strains,respectively.Based on ORF3(TGB2),ORF4(TGB3) and ORF6(nucleic acid binding protein,NABP) amino acid sequences respectively,the ORF5(coat protein) gene was most conserved and the ORF4(TGB3 protein) gene was most great variable.The variable region of CP was mainly N-end of its amino acid sequences.According to the strains and species demarcation criteria for the genus carlavirus formulated by ICTV including CP amino acid identity(＜65%for distinct species and 75%-90%for distinct strains),the clones from A.chinense,in this paper were different GLV strains.This was first report of GLV occurring in A.chinense in China.Based on the obtained sequences above,a specific virus downstream primer pCarl-2dw(-) with another upstream degenerate primer pCarl-2(+) were designed for PCR. the fragment including partial ORF1 and ORF2(708nts) of GLV genome was amplified and sequenced.There was a non-translated region(30nts) between ORF1 and ORF2,and a overlapping region(23nts) between ORF2 and ORF3.Comparing with other known isolates showed that the ORF2 gene was also variable,and had 72%-78%identity at nucleotide level and 76%-83%identity at the amino acid level.Two Allexivirus were detected from the infected leaves in A.chinense using the degenerate primer pGV-3t(+) and M4.The 3'-end of its clone pGTdw-8 RNA genome had 939nt long,including partial CP(441nts) and complete NABP gene(381nts) and 3'-UTR,excluding its poly(A) tails.The same region of another deputy clone pGTdw-15 RNA genome had 949nt long,also including partial CP(441nts) and complete NABP gene(381nts) and 3'-UTR,excluding its poly(A) tails.Sequence comparisons showed that the two clone from A.chinense in this paper had 94%and 91%respectively nucleotides identity with the published China isolate of GarV-E(AJ292230) in garlic and the Korean isolate of GarV-X(U89243) in garlic.Analysis of NABP and 3'-UTR gene phylogenetic tree suggested that the isolates in this paper had close relationship with the reported garlic China isolate(AJ292230) and Korean isolate(U89243),and closely grouped respectively.Moreover,their 3'-UTR gene had 94%identity to GarV-E and 98%identity to GarV-X.According to the species demarcation criteria for the genus Allexivirus formulated by ICTV including CP amino acid identity(＜90%) and 3'-UTR nucleotide identity(＜90%),the two clones pGTdw-8 and pGTdw-15 in this paper were different strains of GarV-E and GarV-X respectively.This was first report of Allexivirus occurring in A.chinense in China. |
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