| Geminiviruses are plant viruses characterized by having small circular single-stranded DNA (ssDNA) genomes, encapsidated in twinned icosahedral particles. In recent years, geminiviruses have caused significant yield loss to many crops and are becoming a serious threat to crops in many parts of the world. In China, several kinds of geminiviruses were identified in tobacco, tomato, squash and papaya in Guangdong, Guangxi, Yunnan and Hainan provinces, and their genomic structure and variation were studied. Geminiviruses have a wide host range and their genomes evolve very fast. In an attempt to provide a better understanding of the diversity, evolution and spread of geminiviruses in China, 32 samples showing typical geminivirus symptoms were collected from papaya, tomato, tobacco, Euphorbia pulcherrima, Sonchus oleraceus Linn and Ageratum conyzoides in Guangxi province. Geminiviruses in these samples were detected and identified with molecular technique.32 samples were collected and confirmed to be infected by geminiviruses with PCR detection using universal primer pair PA/PB for the genus Begomovirus. Comparisons of partial DNA-A sequences showed that these virus isolates could be classified into at least four groups. Nine representative isolates from each group were then chosen for complete DNA analysis.Three viral isolates GX-1, GX-2 and GX-3 were obtained from tomato plants showing leaf curl symptom in Guangxi province of China. The complete sequences of these isolates were determined to be 2752 nucleotides, having a typical genetic organization of a begomovirus. They shared 98.9-99.7% nucleotide sequence identity among themselves, but had only lower than 86.7% nucleotide sequence identity with other reported begomoviruses. The molecular data showed that GX-1, GX-2 and GX-3 were isolates of a distinct begomovirus species, for which the name Tomato leaf curl Guangxi virus (ToLCGXV) is proposed. Further analysis indicates that ToLCGXV is considered to have arisen by recombination among viruses related towas generated by recombination between viruses related to Ageratum yellow vein China virus (AYVCNV), Tomato leaf curl China virus (ToLCCNV) and an unidentified begomovirus. PCR and Southern blot analyses demonstrated that isolates GX-1 and GX-2 were associated with DNAp components, but GX-3 was not.The viral isolates GX-4 and G121-G128 are also collected from tomato disease samples. Complete DNA-A sequences of isolates GX-4 and G121 are 2738 and 2734 nts, respectively. Sequence comparisons showed that DNA-A sequences of GX-4 and G121 had the highest sequence identity with that of Papaya leaf curl China virus (PaLCuCNV, 91.6%) and Tomato yellow leaf curl China virus (TYLCCNV, 96.2%), respectively, indicating that GX-4 is an isolate of PaLCuCNV and G121 is an isolate of TYLCCNV. DNA(3 molecules were amplified from five samples among these nine isolates. Pairwise comparisons showed that DNAP sequences of G121 had the highest sequence identity (90.6%) with that of TYLCCNV. GX-4, G122, G123 and G126 DNAp share high sequence identities and they have 61.5~67.3% sequence identities with that of TYLCCNV.An infectious clone of ToLCGXV isolate GX-1 was constructed. It could efficiently infect Nicotiana benthamiana, N. glutinosa, N. tabacum, N. tabacum cultivars Sumsun and Xanthi, tomato and Petunia hybrida plants, inducing typicalbegomovirus symptoms.The virus isolates G102 and G103 were collected from tobacco showing leaf curl symptoms in Jingxi, Guangxi province. The complete DNA-A sequence of G102 was determined to be 2728 nucleotides. Comparison showed that G102 DNA-A had the highest sequence identity (96.5%) with that of TYLCCNV, suggesting that G102 is an isolate of TYLCCNV. Further studies showed that both G102 and G103 were associated with satellite DNA molecules (DNAP). Sequence analysis revealed that both G102 and G103 DNAp contained 1342 nts and they shared the highest sequence identities (92.9% and 93.4%, respectively) with that of TYLCCNV. This is the first report that TYLCCNV isolates in Guangxi are also associated with satellite DNA molecules.The 12 virus isolates G88~G94 and G105~G109 were collected from papaya showing leaf curl symptoms. The complete DNA-A sequences of G94 weredetermined to be 2739 nts. Comparisons with other begomoviruses showed that G94 DNA-A is most closely related to that of ToLCCNV with 94.7% nucleotide sequences identity, indicating that G94 is an isolate of ToLCCNV. Further studies showed that G91, G94 and G108 were associated with satellite DNA molecules with the lengths of 1340 nts, 1340 nts and 1342 nts, respectively. They all share the highest nucleotide sequence identity (96.9~99.2%) with that of ToLCCNV. The mixed infections of ToLCCNV and PaLCuCNV were existed in the sample G109.The complete DNA-A sequence of isolate G35 from Euphorbia pulcherrima was determined to be 2747 nts, it shares the highest sequence identity with that of ToLCGXV (86.3%), suggesting that G35 is a distinct Begomovirus species, for which the name Euphorbia leaf curl Guangxi virus (ELCGXV ) is proposed.The viral isolate G129 and G130 were collected from Sonchus oleraceus Linn. A 0.5kb fragment was obtained by PCR using primers specific to geminivirus genome. The nucleotide sequence identity of the partial DNA-A sequence between the two isolates was over 98.9%. The complete DNA-A sequence of G129 was determined to be 2747 nucleotides. Comparisons showed that G129 DNA-A had the highest sequence identity (96.5%) with that of AYVCNV, indicated that G129 is an isolate of AYVCNV and Sonchus oleraceus Linnis is a new host of AYVCNV. |
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