Molecular Characterization Of Geminiviruses And Their Associated DNAβ Molecules Infecting Crops And Weeds In Yunnan

Members of the genus Begomovirus in the family Geminiviridae have circular single-stranded DNA genome encapsidated in twinned icosahedral particles. Begomoviruses have caused significant yield loss to many crops and are becoming a serious threat to crops in south China since 1990s. The current study was carried outto identify begomoviruses in several crops and weeds in Yunnan province.Forty begomovirus samples were collected from Nicotiana tabacum,Lycopersicon esculentum, Malvastrum coromandelianum, Solarium aculeatissimum, Ageratum conyzoides and Siegesbeckia orientalis plants. PCR detection using universal primer pair PA/PB for begomoviruses indicated that these samples were infected by begomoviruses. Based on the nucleotide sequences of the 500 bp fragments amplified by PA and PB, the 40 isolates consist of four main types: Y231, Y238, Y240, Y241, Y244, Y248, Y261, Y262, Y263, Y264, Y265, Y266, Y268, Y295, Y296, Y297, Y298, Y318, Y319, Y320, Y321, Y322, Y323 and Y324 are isolates of Tomato yellow leaf curl China virus (TYLCCNV);Y288, Y289, Y291 and Y315 are isolates of Tobacco curly shoot virus (TbCSV);Y290, Y309, Y310, Y311, Y314, Y316 and Y317 are isolates of Tobacco leaf curl Yunnan virus (TbLCYNV);Y304, Y305, Y306, Y307 and Y308 are isolates of Malvastrum yellow vein Yunnan virus (MYVYNV).The virus isolate Y322 was collected from Solanum aculeatissimum showing leaf curl and enation symptoms in Yanshan, Yunnan Province. The complete nucleotide sequence of the DNA-A like molecule of Y322 is determined to be 2730 nucleotides. Y322 DNA-A is most closely related to isolates of TYLCCNV with 88.3%~99.2% nucleotide sequence identities, while less than 79.6% nucleotide sequence identities are found in comparisons with other begomoviruses. Therefore, Y322 is taxonomically considered as an isolate of TYLCCNV. Further studies show that Y322 is associated with a DNAp molecule. The DNAP of Y322 contains 1331 nucleotides having the highest nucleotide sequence identity (93.1%) with DNAβ associated with TYLCCNV, while less than 55.4% nucleotide sequence identities arefound in comparisons with other DNAP molecules. This is the first report of TYLCCNV infecting Solarium aculeatissimum.The virus isolates Y231 and Y244 were obtained from Nicotiana tabacum and Y295 from Lycopersicon esculentum. Complete DNA-A sequences of Y231 has 99.4% nucleotide sequence identity with that of Y244, Y231 and Y244 have high nucleotide sequence identities (99.6% and 99.2%) with TYLCCNV-[Y64]. Y295 DNA-A has the highest sequence identity (95.2%) with TYLCCNV-[Y25] and shares only 86.6% and 86.4% nucleotide sequence identities with Y231 and Y244. It reveals that Y231, Y244 and Y295 are isolates of TYLCCNV but variations exsit among different isolates because of different hosts.27 DNAP molecules were isolated from these 40 isolates. Comparisons of the nucleotide sequences indicate that these DNAP consist of three main types which are associated with three Chinese begomovirus species (TbCSV, TYLCCNV and MYVYNV). High DNAP nucleotide sequence identities were found among 21 isolates of TYLCCNV (70%~99%), 3 isolates (Y289, Y291 and Y317) of TbCSV (95%~99%) and 3 isolates (Y304, Y307, Y308) of MYVYNV (96%). We demonstrated that PCI ORF has the same degree of variability as DNAP molecules and the degree of variability among DNAP molecules are greater than that of DNA-A.Previously constructed infectious clones of TYLCCNV-[Y10] and DNAP molecules of TYLCCNVp-[Y5], TYLCCNVp-[Y10], TYLCCNVp-[Yll], TYLCCNVp-[Y25] and TYLCCNVp-[Y64] were used to infect N. benthamiana and N. glutinosa. In N. benthamiana, TYLCCNV-[Y10] co-inoculated with five DNAP molecues induced downward leaf curl symptoms while TYLCCNV-[Y10] co-inoculated with TYLCCNV-[Y10] induced enation and yellow vein phenotype. TYLCCNV-[Y10] co-inoculated with DNAp molecules of TYLCCNV-[Y10] and TYLCCNVp-[Y64] induced severer symptom than other DNAP molecues. In N. glutinosa, only TYLCCNV-[Y10] co-inoculated with DNAP molecues of TYLCCNV-[Y10] and TYLCCNV-[Y64] induce downward leaf curl symptoms and TYLCCNV-[Y10] co-inoculated with other DNAP molecues didn't produce obvioussymptoms.