Genetic Structure And Variability Of A Virus In Geminivirade

A plenty of studies demonstrated that the population of plant, especial animal RNA virus is genetically diverse, termed as quasispcies. Generally, the characteristic of high potential for diverse population for an RNA virus is regarded as the lack of proofreading of RNA-dependent RAN polymerase (RdRp), which encoded by virus self. The genomes of viruses in family Geminiviridae are single stranded DNA, and their replication strategies are distinctly different from that of RNA viruses. The geminiviruses use the replication machinery of the host and hence have been presumed to have a high fidelity of replication. However, this view has been challenged by several publications published recently, which using the natural infected materials for population structure analysis. Considering the evidence to describe population structure of geminiviruses is extremely limited, and the unavoidable mixture infection or repeated infection for the field plants may cover up some reality, we used unique sequence infectious clone of Tomato yellow leaf curl China Virus (TYLCCNV) isolate Y10 to start the infection, and the genetic structure, diversity and the (?)ariation strategy of its population were evaluated at 60 dpi and 120 dpi in plants of Nicotiana benthamiana and tomato (Lycopersicon escultentum) by amplifying a fragment of the virus genome and randomly sequencing 16-24 clones in the virus progeny population.Our results indicate that the populations of TYLCCNV in the 3 analyzed N. benthamiana are heterogeneous, consisting of the initial sequence (consensus sequence) and a set of minor sequences that are not identical to the consensus sequence but closely related to the consensus sequence. The population diversity values, indicated by the percentage of mutation clone and the mutation frequency, are very close in the 3 analyzed N. bethamiana, averaged 40% and 4.54 X 10^-4, respectively, which are comparable with those reported in the RNA virus populations. The quasispecies structure for TYLCCNV population is also observed in infected tomato, in which the diversity level of population is slightly lower than that in the N.benthamiana.To confirm the quasispecies feature of TYLCCNV population further, the population variability in a TYLCCNV-infected field tomato sample was investigated. The results also showed the presence of quasispecies for field virus population. The percentage of mutated clone and mutation frequency were 28.6%and 2.63 X10"4, which is comparable with that detected in the controlled experimental condition. Moreover, we detected the occurrence of mixture infection by two genetically distinct subpopulation of TYLCCNV in this plant.To learn if the population structure and diversity level change as the infection progression, the population variation of TYLCCNV in an analyzed N. benthamiana plant was assayed at 120 dpi. The results demonstrated that the diversity level increased by some degree, but not statistically significant, suggesting that the diversity rapidly reach to a certain value for a specific virus in a specific host plant. The consensus sequence was not changed over the infection, but the spectrum of variants in the population was distinctly different from that found at 60 dpi. Majority variants detected at 60 dpi did not appear at 120 dpi, except of a few variants, indicating the population structure of TYLCCNV is equilibrium dynamically. The variants carried the mutations at nt 127 and nt 2718 (IR), and nt 2312 (overlapping region of AC1 and AC4), and were found in all analyzed plants, suggesting these variants possess the selection advantage over the other variants during the infection.Soft rot with symptoms of initial brown spots on the surface and soft rot inside of melon (Cucumis melo L.) fruits was often reported in Yiwu and other regions of Zhejiang, China. The infected melon fruits corrupted consequently. In some areas this disease caused significant yield loss to melon industry. Four isolates (Ml to M4) with two types of morphology of bacterial colonies were isolated and purified from diseased melon fruits. The tests of hypersensitive reaction in tobacco (Nicotiana tabacum L.) and pathogenicity in the melon fruit for these isolates demonstrated that Ml and M3 were pathogenic, whereas M2 and M4 were not. Both Ml and M3 were identified to be an isolate of Erwinia carotovora subsp. carotovora based on their properties of morphology, flagella, gram reaction, host range and the sequencealignment of 16S rRNA gene. In addition, inoculation test showed that this pathogen could induce soft rot to tomato, cucumber, watermelon and pumpkin fruits.