Molecular Identification Of Six Begomoviruses And Pathogenicity Of Two Begomoviruses In China

Geminiviruses are a group of plant viruses characterized by their unique twinned particles, which encapsidated a circular single-stranded DNA genome. They cause destructive diseases in many crops throughout the world and have emerged as a great threat to crop production. 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 diversity, distribution and spread of begomoviruses in China, molecular identification of begomoviruses collected from Jiangsu, Zhejiang and Yunnan were carried out, and virus pathogenicity were studied.Virus isolates J4, J5 and Zl were collected in Yixing, Jiangsu province and Yuhang, Zhejiang province of China, respectively from field ramie (Bcelmevia nivea L.) samples exhibiting yellow mosaic symptoms. The 500 bp fragments were amplified by the degenerate primer pair PA/PB, and they shared 91.6-99.2% nucleotide sequence identities. Isolates J4 and Z1 were selected for full-length sequencing. The complete DNA-A sequences of J4 and Z1 were determined to be 2736 and 2737 nucleotides (nts) (Accession No. FN396969 and FN396971), respectively, sharing 94.7% nucleotide sequence identity with each other. The DNA-B components were identified for both isolates by PCR. The complete DNA-B sequences of J4 and Z1 comprised 2717 and 2719 nts (Accession No. FN396970 and FN396972), respectively, sharing 88.6% nucleotide sequence identity with each other. Further analysis showed that the complete sequences of DNA-A and DNA-B components of both the isolates had 93.6-94.7% and 90.6-91.2% nucleotide sequence identities, respectively, with isolates of Ramie mosaic virus (RamMV). These molecular data suggest that J4 and Z1 are two different isolates of RamMV. This is the first report of a bipartite begomovirus in Jiangsu and Zhejiang provinces of China.Two begomoviruses were obtained from the Clerodendrum cyrtophyllum Turcz. samples (YX1, YX2, YX3) exhibiting yellow mosaic symptoms in Yixing, Jiangsu province of China. Products of 500 bp were obtained in PCR with the degenerate primer pair PA/PB from all three Clerodendrum cyrtophyllum Turcz. samples. Alignment of the 500 bp sequences revealed that each sample contained two begomoviruses. Representative isolate YX2 was selected for obtaining the full-length DNA components of the two begomovirus isolates (designated as YX2-I and YX2-II). The complete DNA-A sequences of YX2-I and YX2-II were determined to be 2753 and 2775 nts (Accession No. FN396966 and FN396962), respectively, sharing 68.2% nucleotide sequence identity with each other. Sequence comparisons showed that YX2-I DNA-A had the highest sequence identity (79.7%) with A1LCV-[CN:G10:06], an isolate of Allamanda leaf curl virus from Guangdong province. According to the taxonomic criteria of Begomovirus, YX2-I is considered as a distinct begomovirus, for which the name Clerodendrum golden mosaic Jiangsu virus (C1GMJSV) is proposed. YX2-ⅡDNA-A was most closely related to C1GMCNV-[CN:Fz7:08] (93.0%), an isolate of Clerodendrum golden mosaic China virus from Fujian province. Therefore, YX2-Ⅱis considered to be an isolate of C1GMCNV. YX2-ⅡDNA-B molecule was identified by RCA-RFLP with 2728 nts (Accession No. FN396963) in length. The results confirmed YX2-II is a bipartite begomovirus. DNAβmolecule was not detected in any of the samples by PCR. Infectious clones of the two viruses were constructed and used to test their experimental host-range. C1GMJSV alone could induce typical begomoviral symptoms in Nicotiana benthamiana, N. glutinosa, N. tabacum Samsun, Petunia hybrida and Solanum lycopersicum plants. Moreover, C1GMJSV could interact with a heterologous DNAβmolecule associated with Tobacco curly shoot virus (TbCSB) in N. benthamiana plants. C1GMCNV infected N. benthamiana, N. glutinosa and N. tabacum Samsun with severe symptoms and P. hybrida without obvious symptom. C1GMCNV DNA-A alone was also infectious in N. benthamiana, N. glutinosa, N. tabacum Samsun and P. hybrida plants without symptoms. In addition, we illustrated that bipartite begomovirus C1GMCNV DNA-A was capable of functionally interacting with TbCSB to produce symptoms in N. benthamiana and N. glutinosa plants.Two virus samples Y340 and Y341 were obtained from Sida acuta plants showing yellow vein symptoms in Baoshan, Yunnan province of China. The 500 bp fragments amplified by PA/PB showed sample Y340 contained two begomoviruses (designated as Y340-I and Y340-II), whereas sample Y341 contained one begomovirus, which was identical to Y340-I. Both of Y340-I and Y341 were determined to be 2750 nts (Accession No. FN806777 and FN806778) and they had the highest nucleotide sequence identities (90.5-91.0%) with isolates of Kenaf leaf curl virus (KeLCuV), suggesting that Y340-Ⅰand Y341 are two isolates of KeLCuV. Y340-Ⅱwas determined to be 2745 nts (Accession No. FN806779) and it shared the highest nucleotide sequence identities with isolates of Malvastrum yellow vein virus (MaYVV) (87.2-89.7%) and Malvastrum yellow vein Baoshan virus (MaYVBsV) (88.5-89.2%), respectively. Further phylogenetic analyses placed Y340-II in a cluster with isolates of MaYVBsV, thus, it is an isolate of MaYVBsV. This is the first report of KeLCuV and MaYVBsV infecting Sida acuta. RDP analyses on the virus genomic sequences suggested that Y340-I and Y340-II may have arisen by recombination. Both samples were found to be associated with DNAβmolecules by PCR. The complete DNAβsequences from Y340 and Y341 were determined to be 1342 and 1341 nts in length (Accession No. FN806780 and FN806781), respectively, sharing the highest sequence identities (81.7-83.4%) with isolates of Malvastrum yellow vein Yunnan betasatellite (MaYVYnB). Based upon the proposed species demarcation threshold for DNAβ, they are two different isolates of MaYVYnB. Furthermore, DNA1 molecule was identified in sample Y340. Y340 DNA1 was found to be 1370 nts (Accession No. FN806782) and it shared higher sequence identities (61.4-82.2%) with those of previously described DNA1s, whereas lower sequence identities (28.4-30.0%) were found in comparison with nanoviruses.Four viral samples (Y335-338) showing yellow vein symptoms were collected from Ipomoea indica plants in Ruili of Yunnan province and were confirmed to be infected by begomoviruses with PCR detection using universal primer pair BegoAForl/BegoARevl specific for the genus Begomovirus. The four amplified 1.2 kb fragments shared 97.9-99.5% nucleotide sequence identities. The complete sequence of isolate Y338 was determined (Accession No. FN806776). Y338 had the highest sequence identity (97.8%) with SPLCV-[CN:RL31:07], an isolate of Sweet potato leaf curl virus (SPLCV) reported in Yunnan province of China. Therefore, Y335-338 are considered to be isolates of SPLCV. Further phylogenetic analysis show that SPLCV is more close to begomoviruses from the Old World but isolates of this virus seem to form a separate subset.