| Two kinds of geminivirus disease complexes, Tobacco curly shoot virus (TbCSV) and Tomato yellow leaf curl China virus (TYLCCNV), which were associated with their satellite DNAβ (TYLCCNB and TbCSB), have caused serious damages on tobacco, tomato. Studies have shown that the pathogenesis of TbCSV/TbCSB and TYLCCNV/TYLCCNB are different. TYLCCNB is needed for induction of symptoms, and all field-collected samples of TYLCCNV are associated with DNAβ. While TbCSB is not necessary for pathogenticity but intensifies symptoms, and some TbCSV isolates were found to be associated with DNAβ in the field. To identify the occurrence, distribution, hosts and variation of these two kinds of begomovirus/DNAβ complexes,306samples with severe symptoms were collected from11crops and14weed species in Yunnan, Sichuan and other six regions, which then were addressed for detections of these two kinds of begomovirus/DNAβ complexes with specific primers of TbCSV/DNAP and TYLCCNV/DNAβ. The genome organization, mutation and genetic structure of viruses and betasatellites were analyzed.PCR detection using universal primer pair PA/PB for begomoviruses showed that235of306samples were infected by begomoviruses, of which67samples were infected with TYLCCNV and20with TbCSV, mixed-infection ratio of these two viruses was1.3%. Five plant species collected from Zhaotong, Chuxiong, Baoshan, Wenshan, Honghe in Yunnan and Panzhihua in Sichuan were found to be infected by TYLCCNV, while most Ageratum conyzoides samples from Dehong in Yunnan, as well as Nicotiana tabacum and Mirabilis jalapa from Yunnan and Capsicum annuum from Sichuan were found to be infected with TbCSV, but neither TbCSV or TYLCCNV was found in crops and weeds from other six provinces such as Guangdong, Guangxi and Fujian. The distribution and host ranges of TYLCCNV are wider than those of TbCSV. Three kinds of betasatellites (MYVYNB, MYVB and TYLCCNB) were found to be associated with all TYLCCNV isolates using abutting primers β01/β02specific for DNAβ. While some TbCSV isolates were found to be associated with betasatellites (TbLCYNB and TbCSB).The genetic diversity of the two kinds of virus isolates (including6TbCSV isolates,23TYLCCNV isolates in this study and others from GenBank) from different hosts and locations were analyzed. Results showed that the population diversity of both TbCSV and TYLCCNV were rich. TYLCCNV had a higher level of genetic diversity than that of TbCSV, and the evolution rates of different regions in the genome were divergent. To further investigate the putative recombination, the complete sequences of47TYLCCNV isolates were analyzed using the recombination detection program RDP3. Results indicated that recombination events were common in TYLCCNV but there was none in TbCSV isolates. Many recombination sites are distributed in IR or AC1region in the genome. Neutral tests and mismatch distribution of isolates (in this study and GenBank), involving analysis of population size from different hosts or geographic origin, indicated that population size of TYLCCNV had formed into a stable situation with an expansion trend. Phylogenetic analysis suggested that genetic variation and diversity were varied in TbCSV and TYLCCNV isolates collected from different hosts or different regions. The intraspecific genetic diversity in the same virus phylogenetic analysis suggested geographical selection force played a more significant role in virus evolution than host.In order to understand the genetic structure and population variation of virus, in this study, TbCSV isolate Y35A and TYLCCNV isolate YlOA associated with their cognate satellite DNAp (Y35β or Y10β) were agroinoculated to N. benthamiana and N. glutinosa. Results showed that the populations of TbCSV and TYLCCNV in plants were genetically heterogeneous. In a laboratory-maintained population, it was found that about8.33%of Y35A clones mutated and with6.1×10-5of mutation frequency in N. glutinosa, while with1.2×10-4of mutation frequency and14.3%of clones mutated in N. benthamiana. For Y10A, about25%clones mutated and the mutation frequency was2.2×10-4in N. glutinosa, while the mutation frequency was4.3×10-4and41.5%clones mutated in N. benthamiana. The variation in TbCSV or TYLCCNV infected field Nicotiana tabacum, Solanum lycopersicum, Malvastrum coromandelianum were analyzed and the result would be helpful for further confirmation the quasispecies features of virus population. The mutation frequency of TbCSV is2.8×10-4and the mutation frequency of TYLCCNV ranged from6.5×10-5to4.5×10-4, compared to those reported for plant RNA viruses and detected in laboratory-maintained populations.The mutation sites were distributed throughout the coding regions and non-coding region of TbCSV and TYLCCNV, and the IR region exhibited a most variable mutation frequency across all virus populations compared with mutation frequencies in ACI and AC4coding region. The types of mutations in TbCSV and TYLCCNV populations were different in N. benthamiana and N. glutinosa. Most mutations were base substitution while deletion and insertion occurred much less frequent in all populations. The population variation levels of these two kinds of viruses in N. benthamiana were higher than those in N. glutinosa., suggesting that the selection pressures were different for these two kinds of virus and host may play important role in evolution of virus.It was reported that DNAP were co-evoluted with their cognate viral DNA-A. In order to understand the genetic structure and population variation of satellite DNAβ, Y35A+Y35β, Y10A+Y10β and Y35A+Y10β were infiltrated into N. benthamiana and N. glutinosa to investigate genetic variation of Y35β and Y10β. Results showed that both the populations of Y35β and Y10β were heterogeneous and had a higher level of mutation than their helper virus. When associated with cognate helper virus, the mutation frequency of Y35β was4.5×10-4and43.6%clones mutated, while nearly all clones of Y10β population (93.3%) mutated and the mutation frequency of Y10β was1.5×10-3, indicating that the variation level of Y10β populations was higher than that of Y35β. When associated with heterogenous helper virus, all clones of Y10β population (100%) mutated and the mutation frequency of Y10β was1.9×10-3. The population diversity level of which was a little bit higher than that of with cognate helper virus. In the terms of DNAβ, variation levels varied in different hosts. Besides substitution, deletion and insertion mutations were detected in DNAβ populations, which were distributed throughout the genome of DNAβ. Most mutation sites were found in the A-rich region, especially the section from889to901of Y10β genome, in which deletion or insertion of A or G occurred frequently. The base transitions from G to T, from A to T or from T to A occurred frequently in the mutation types of Y35β populations while deletion and insertion mutations occurred much less frequent in Y35β populations. |
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