Molecular Identification Of Four Geminiviruses

Geminiviruses are a group of plant viruses characterized by their unique twinned particles, which encapsidate a circular single-stranded DNA genome. They cause destructive diseases in many economically important crops throughout the world. In China, several economically important crops, such as tobacco, tomato, squash and papaya, have suffered infections by geminiviruses in Yunnan, Guangxi provinces since 1990s. In an attempt to provide a better understanding of occurrence, distribution and damage of these begomoviruses, plant samples expressing virus infection symptoms were collected from Hainan and Guangxi provinces and the viruses were detected and identified with molecular technique.Thirty-three virus samples collected from Hainan and Guangxi provinces were confirmed to be infected by geminiviruses by PCR detection with universal primer pair PA/PB for geminiviruses. Sequences comparisons showed that these viruses could be classified into eight groups. Group II to group V are closely related with previously reported begomoviruses, while Group I, groupâ…¦ and groupâ…§ are distinct from reported begomoviruses. Group VI belongs to Squash leaf curl China virus (SLCCNV), however the existence of SLCCNV DNA-B component has not been reported in China. So genomic structure of virus isolates in group I, group â…¥, group â…¦ and group â…§ were studied in this dissertation.Group I contains only one sample (Hn51) obtained from Alternanthera philoxeroide showing yellow vein symptom in Hainan province. The complete nucleotide sequence of Hn51 DNA-A was determined to be 2745 nucleotides, it is most closely related to that of Ageratum yellow vein virus (65.2% sequence identity). In accordance with the current species demarcation criteria for geminiviruses, Hn51 is considered as a distinct begomovirus species, for which the name Alternanthera yellow vein virus (AlYVV) is proposed. This is the first report of a begomovirus infecting an aquatic plant in China. PCR and Southern blot analysis demonstrated that AlYVV is not associated with DNA-B or DNAp.GroupVDI samples were isolated from Sida cordifolia showing mild upward leaf-curling symptoms in Hainan province. The complete nucleotide sequence of Hn57 DNA-A was determined. Sequence comparison with other reported begomoviruses revealed that Hn57 DNA-A has the highest sequence identity (71.0%) with that of Tobacco leaf curl Yunnan virus. Consequently, Hn57 was considered to be a new begomovirus species, for which the name Sida leaf curl virus (SiLCV) is proposed. In addition to DNA-A molecule, two additional circular single-stranded satellite DNA molecules corresponding to DNAP and DNA1 were found to be associated with SiLCV isolates. Sequence analysis shows that DNAps of Hn57 and Hn60 share 93.8% nucleotide sequence identity, and have the highest sequence identity (51.5 and 52.1%, respectively) with DNAP associated with ageratum leaf curl disease (AYLCDP02). Hn57 DNA1 shares 83.8% nucleotide sequence identity with that of Hn60, and they share 58.2-79.3% nucleotide sequence identities with other previously reported DNA1, and only 21.9-34.0% identities with nano viruses.GroupW consists of six virus isolates obtained from Malvastrum coromandelianum showing yellow mosaic symptoms in Hainan province. Isolates Hn36 and Hn37 were selected for full length sequencing. Complete sequence comparisons show that Hn36 and Hn37 share 99.1% nucleotide sequence identity, and they had the highest sequence identity (81.3% and 81.5%, respectively) with Malvastrum yellow vein virus (MYVV). The two isolates are therefore considered as isolates of a distinct begomovirus for which the name Malvastrum yellow mosaic virus (MalYMV) is proposed, hi addition, four kinds of DNA forms were found to be associated with MalYMV: DNAP, DNA1, defective DNAp and recombinant DNA molecule (RecDNA-AP). The results showed that Malvastrum yellow mosaic disease was caused by a complex, comprising of MalYMV and its associated small DNA molecules.GroupVI including two samples (Hn61 and G25) was collected from squash exhibiting leaf curl symptoms. The genomes of Hn61 and G25 consisted of two components (DNA-A and DNA-B). The nucleotide sequence identity of DNA-A between Hn61 and G25 is 95.0%, while the identity of DNA-B between them is 84.8%. Furtheranalysis shows that the complete sequences of DNA-A and DNA-B components of the both isolates have 89.4-98.4% and 77.4-91.3% nucleotide sequence identity, respectively, with other SLCCNV isolates, indicating that variations of DNA-B are much more than that of DNA-A. This is the first report of bipartite geminivirus in China.