| Geminiviruses are plant viruses characterized by their unique twinned particles, which encapsidate a circular single-stranded DNA genome They have caused significant yield losses in many economically important crops worldwide. In China, several begomoviruses have been reported infecting crops and weeds in Yunnan, Guangdong, Guangxi, Hainan and Taiwan. In an attempt to provide a better understanding of the species, distribution and spread of begomoviruses in China, molecular identification of begomoviruses collected from Shanghai, Guangdong and Guangxi were carried out, and virus pathogenicity was studied in this dissertation.Two viral samples (SH1 and SH2) showing yellow leaf curl symptoms were collected from tomato plants in Sunqiao Farm of Shanghai and were confirmed to be infected by begomoviruses with PCR detection using universal primer pair PA/PB specific for the genus Begomovirus. The two amplified 500-bp fragments shared 97.4% nucleotide sequence identity. The complete sequence of SH2 isolate was determined (Accession No. AM282874). SH2 had the highest sequence identity (99.8%) with Tomato yellow leaf curl virus (TYLCV) isolate reported in Japan. Therefore, SH1 and SH2 were considered to be isolates of TYLCV. TYLCV has been reported to be widely distributed in the world, but this is the first report of TYLCV in China.Leaf samples of Siegesbeckia glabrescens (isolates GD11, GD12, GD13, GD16, GD21, GD22, GD24, GD25, GD27 and GD30) showing yellow vein, enation and stunting symptoms were collected in Guangdong province, China. The 500-bp fragments amplified by PA/PB shared 95.4-100% nucleotide sequence identity. Isolates GD13, GD24 and GD27 (Accession No. AM183224 and AM230634-5) were selected for determining the complete. The complete nucleotide sequences of GD13, GD24 and GD27 shared 98.3-98.7% identity and had the highest sequence identity (76.8%) with that of Papaya leaf curl China virus (PaLCuCNV). The molecular data showed that GD13. GD24 and GD27 were isolates of a distinct begomovirus species, for which the name Siegesbeckia yellow vein virus (SbYVV) was proposed. All isolates were found to be associated with DNAβmolecules by Southern blot and PCR. The complete DNAβsequences of isolates GD13, GD24 and GD27 were determined (Accession No. AM230643-5). Sequence analysis showed that they had relatively lower sequence identity with other reported DNAβs with the highest (44.0%, 43.9% and 45.6%, respectively) with Eupatorium yellow vein virus (EpYVV) DNAβ. In addition, two DNA 1 species were identified. The sequence identity between these two DNAls was 66.2%, and they had the higest nucleotide sequence identities with DNA1 of Malvastrum yellow vein virus (MYVV, 72.6%) and Tobaco curly shoot virus (TbCSV, 69.1%), respectively. Furthermore, mixed infection of these two DNA1 molecules were identified in GD13,GD21,GD22,GD24 and GD27 samples.Virus isolates GD6-10 were obtained from Malvastrum coromandelianum showing leaf curl symptoms in Guangdong Province of China. The 500-bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 92.8-97.1% nucleotide sequence identity. The complete nucleotide sequences of both GD6 and GD9 were found to be 2767 nucleotides (Accession No. AM236779-80), with all the characteristic features of begomovirus genome organization. The two isolates have less than 85.2% nucleotide sequence identity with other reported begomoviruses. Consequently, GD6 and GD9 are considered to be isolates of a novel begomovirus species, for which the name Malvastrum leaf curl Guangdong virus (MLCuGdV) is proposed. Sequence analyses suggest that MLCuGdV may have arisen by recombination between viruses related to Papaya leaf curl China virus, Tomato leaf curl Philippines virus and other undiscovered virus ancestors. Neither the DNA-B component nor the DNAβmolecule associated with these begomovirus isolates was found. An infectious clone of MLCuGdV isolates GD6 was constructed. MLCuGdV efficiently infected test plants such as Nicotiana benthamiana, N. glutinosa and Petunia hybrida by agro-inoculation inducing typical begomoviral symptoms. MLCuGdV could effectively infect Malvastrum coromandelianum and N. tabacum by whitefly transmission. The interaction between MLCuGdV and Tomato yellow leaf curl China virus (TYLCCNV) DNAβwas studied and MLCuGdV could support the replication of TYLCCNV DNAβ. Virus isolates GD33-38 were obtained from Hibiscus rosa-sinensis showing leaf curl and enation symptoms in Guangdong Province of China. The 500-bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 97.3-99.0% nucleotide sequence identity. The complete sequences of viral DNA and its associated DNAβof GD35 and GD37 were determined with 99.7% identity for viral DNA and 99.8% for DNAβ. Both the viral DNA and DNAβshared the highest sequence with Cotton leaf curl Multan virus (CLCuMV) collected in Hibiscus rosa-sinensis plant in Guangdong with 99% sequence identity for viral DNA and 98% for DNAβ. Therefore, GD33-38 were isolates of CLCuMV. CLCuMV have been widely distributed in Pakistan, and it is a quarantinable in China. An infectious clone of CLCuMV isolate GD37 and DNAβwere constructed. CLCuMV could replicate in test plants such as N. benthamiana, N. glutinosa, S. lycopersicum and P. bybrida without any symptoms. Co-inoculation of CLCuMV and DNAβcould induce typical begomoviral symptoms in these plantsVirus isolates G110-112 were obtained from Siegesbeckia glabrescens plants showing stunting and yellow vein symptoms in Guangxi, China. The 500-bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 95.0-97.3% nucleotide sequence identity. The complete G111 DNA sequence is 2784 nucleotides (Accession No. AM238692) and shares the highest nucleotide sequence identity (85.4%) with SbYVV. The molecular data showed that G110-112 were isolates of a new begomovirus for which the name Siegesbeckia yellow vein Guangxi virus (SbYVGxV) is proposed. DNAβmolecule was indentified in G111 with 1355 nucleotides (Accession No. AM238695) and G111 DNAβmay have arisen by recombination between SbYVV DNAβand other unknown DNA molecule.An infectious clone of Euphorbia leaf curl virus (ELCV) isolate G35 was constructed and its infectivity was tested in host plants. ELCV alone could systemically infect tested plants and induce symptoms, while it could infect more tested plants and induce more severe symptoms when co-inoculated with TYLCCNV DNAβ. Moreover, ELCV and TYLCCNV DNAβcould systemically infect Glycine max, Gossypium hirsutum. Capsicum annuum and Cucumis sativus although symptoms were not observed. |
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