广西番茄烟粉虱传双生病毒的分布及遗传多样性分析（英文）

李战彪　徐鹏超　秦碧霞　苏琴　谢慧婷　崔丽贤　蔡健和

摘要：【目的】調查研究侵染广西番茄的双生病毒种类、分布及复合侵染情况，为广西番茄育种及抗病品种布局提供理论依据。【方法】自2011年起，对引起广西番茄曲叶病的双生病毒进行调查、鉴定，并采集疑似双生病毒侵染的番茄样品189份，提取样品总DNA，利用双生病毒简并引物对样品进行检测，挑选部分阳性样品的PCR产物，纯化后连接至克隆载体进行测序，并与GenBank已报道的序列进行比对分析。同时选取不同地区的各病毒分离物进行全序列扩增和测定，构建系统发育进化树，研究其进化关系及遗传多样性。【结果】通过PCR检测发现，189份番茄样品中139份为阳性样品，阳性检出率为73.54%。挑选具代表性的番茄样品进行测序比对分析，发现引起广西番茄曲叶病的双生病毒有4种：中国番茄曲叶病毒、中国番木瓜曲叶病毒、中国番茄黄化曲叶病毒和中国胜红蓟黄脉病毒，且样品存在复合侵染现象，存在7种以上不同类型的复合侵染现象；共获得47个各病毒分离物的病毒全长基因组序列。通过系统发育进化树分析发现，各病毒分离物按地域处于不同的分支，表现较丰富的遗传进化关系。【结论】侵染广西番茄的双生病毒有4种，广泛分布于广西主要的番茄种植区，且病毒的侵染多为复合侵染。侵染广西番茄的双生病毒具地域分布性。

关键词：番茄烟粉虱传双生病毒；分布；复合侵染；遗传多样性

0 Introduction

【Research significance】Geminiviruses are the second largest plant viruses family（Geminiviridae） with distinct morphological characteristics of twinned isometric particles consisting of circular single-stranded DNA genomes（Anupam and Malathi， 2003）. These viruses can infect a wide range of important crops and cause severe yield losses. Based on number of genomic components， type of insect vector， host range and phylogenetic relationships， the family Geminiviridae is subdivided into seven genera： Mastrevirus， Curtovirus， Begomovirus， Topocuvirus， Becurtovirus， Turncurtovirus， and Eragrovirus（Varsani et al.， 2014）. Begomovirus， which is transmitted by whiteflies（Bemisia tabaci）， is the largest genus in this family. Begomovirus is recognized as a serious threat to many crops， such as tomato， tobacco and cotton etc.， in many tropical and subtropical regions（Briddon and Markham， 2000； Morales and Anderson， 2001； Li et al.， 2004； Xie et al.， 2006）. Until now， more than 57 species of geminiviruses， mostly Begomovirus， have been described as capable of infecting tomato（Abhary et al.， 2007）. Whitefly transmitted geminiviruses（WTGs） are the most important limiting factor in tomato cultivation and cause severe yield loss in Guangxi. So knowledge of diversity and distribution of tomato-infecting begomoviruses in Guangxi is important for disease management and development of virus-resistant cultivars. 【Research progress】Tomato yellow leaf curl disease （caused by begomoviruses）， was described for the first time in Israel in 1931（Cohen and Antignus， 1994）. Since 1960s， this disease has been reported causing devastating damage to tomato crops in many regions worldwide（Ala-Poikela et al.， 2005； Chowda Reddy et al.， 2005；Tsai et al.， 2011）. In China， begomoviruses infecting tomato was first reported in Guangdong province in 1965， but with low incidence（Ke et al.， 1965）. In recent years， however， the incidence and severity of begomoviruses diseases has increased rapidly in tomato. Many regions of China， such as Guangxi， Guangdong， Yunnan， Jiangsu and Hainan，suffered from this disease（Cai et al.， 1995； Li et al.， 2004； He et al.， 2007； Zhao et al.， 2007； Zhang et al.， 2010； Tang et al.， 2015）. 【Research breakthrough point】Subtropical climate in Guangxi allows year-round tomato cropping， and perennial host plants for both begomoviruses and Bemisia tabaci exist here. Several kinds of begomoviruses were characterized and identified from tomato，tobacco， papaya，weeds， etc.（Ma， 2004； Wang et al.， 2004； Xu and Zhou， 2006； Meng et al.， 2012）. But no survey has been conducted on diversity， distribution and mixed infection of the tomato-infecting begomoviruses in Guangxi. 【Solving problems】This research focused on diversity， distribution and mix-infection of tomato-infecting begomoviruses in Guangxi， in order to provide theoretical support for tomato anti-virus breeding and rational arrangement of tomato anti-virus varieties.

1 Material and Methods

1. 1 Sampling，DNA extraction and virus detection

From 2011 to 2013， 189 tomato samples which showed typical yellow leaf mild yellowing， curling， vein thickening and plant stunting were collected from cities of Baise， Nanning， Qinzhou， Beihai， Fangchenggang， Dongxing and Liuzhou in Guangxi. Total DNA extraction from leaf samples were performed following the method described by Xie et al.（2002）.

The samples were detected by PCR using degene-

rate primer pairs AV494/COPR （location BGMV-PR DNA 1041-1063 nt）（Table 1）（K klü et al.， 2006）. The PCR products were recovered， purified， and cloned using pGEM-T easy vector（Promega， Madison， WI， USA） and sequenced using T7 Promoter Primer or SP6 Promoter Primer.

1. 2 Mixed-infections detecting and full-length DNA-A sequences obtaining

To determine the distribution and mixed-infection of begomoviruses across Guangxi， five pairs of specific primers（Table 1） namely ToL-F/ToL-R[Tomato leaf curl China virus（ToLCCNV）]，AY-F/AY-R[（Ageratum yellow vein China virus（AYVCNV）]，PaL-F/PaL-F[Papaya leaf curl China virus（PaLCuCNV）]，TY-F/TY-R[Tomato yellow leaf curl China virus（TYLCCNV）] and ToLGXV-F/ToLGXV-F（Tomato leaf curl Guangxi virus）， were designed according to conserved sequences of each virus and used to detect mixed infections of 139 positive samples. All of the PCR products were sequenced directly and analyzed by vector NTI.

Begomoviruses genome is circular DNA molecular. In order to obtain full-length DNA-A sequences of begomoviruses， four pairs of overlapping primers， bToL-F/bToL-R（ToLCCNV），bAY-F/bAY-R（AYVCNV），bPaL-F/bPaL-F（PaLCuCNV） and bTY-F/bTY-R（TYLCCNV）， were designed to amply the residual sequences of DNA-A sequences. Contigs were assembled using Vector NTI11.0 software（Thermo Fisher Scientific， USA）.

1. 3 Sequence analysis and phylogenetics tree establishment

Sequences data obtained were compared with those of other geminiviruses sequences available in GeneBank using The National Center of Biotechnology Information basic local alignment search tool or NCBI BLAST （http：//www.ncbi.nlm.nih.gov/BLAST）. Phylogenetic trees were established using full optimal alignment and maximum likelihood method options with 10000 bootstrap replications available in MEGA 6.0. Geminivirus DNA-As used for comparison including abbreviations and GenBank accession numbers were shown parenthetically.

2 Results and Discussion

2. 1 Detection of begomoviruses from tomato

A total of 189 tomato leaf samples observed at different locations in Guangxi which showed symptoms suggesting infection with viruses were collected and detected by PCR using degenerate primer pairs AV494/COPR. PCR amplification yielded a product of expected size of 570 bp（Fig.1）. PCR analysis indicated that 139 tomato plants tested were infected with begomoviruses. Several randomly selected PCR products were cloned， sequenced and compared with the sequences available in GenBank using the tool of BLASTn. The results revealed that four viruses were confirmed to be able to infect tomato in Guangxi. They were ToLCCNV， PaLCuCNV， TYLCCNV and AYVCNV.

DNAβ was also detected from 33 samples using primers Beta 01/02（data not shown）.

In order to make PCR detection assay more cost-effective， convenient and rapid， a simultaneous PCR detection was screened. The PCR reaction system was optimized and the optimum system was confirmed： 2×Taq Master Mix 15.0 μL，ToL-F/ToL-R（10 μmol/L）0.3 μL each， AY-F/AY-R（10 μmol/L）0.6 μL each， PaL-F/PaL-R（10 μmol/L）0.6 μL each， TY-F/TY-R（10 μmol/L）0.6 μL each， template DNA 1.5 μL， ddH2O 9.3 μL to total 30.0 μL. The following protocols were used for PCR： denaturation at 94 ℃ for 3 min and followed by 35 cycles of 94 ℃ for 30 s， 58 ℃ for 30 s and 72 ℃ for 60 s， and further extended by 72 ℃ for 10 min. PCR products were electrophoresed in a 1.2% agarose gel（Fig.2）.

2. 3 Diversity of tomato-infecting begomoviruses

Forty-seven full DNA-A sequences of begomovirus were obtained in this study， and all of the sequences were compared to begomovirus sequences present in GenBank database using BLASTn search tool. Nucleotide sequence homologies were always up to 89% against other begomoviruses， indicating that all the sequences belong to begomovirus. These sequences belonged to four types of viruses. Sequences of type 1 shared the highest nucleotide sequence identity with PaLCuCNV （from 91% to 100%）， while type 2 with AYVCNV（from 95% to 99%）， type 3 with ToLCCNV（from 91% to 99%） and type 4 with TYLCCNV（from 89%to 99%）. All of them were typical monopartite be-

gomoviruses. On the basis of International Committee on Taxonomy of Viruses（ICTV） criteria，>89% DNA-A nucleotide identity was begomovirus species demarca-

tion. Therefore， type 1 was considered as isolate of PaLCuCNV， and type 2 was isolate of AYVCNV， type 3 ToLCCNV and type 4 TYLCCNV， respectively.

Three phylogenetic trees were generated of 47 full sequences together with representative sequences present in GenBank using MEGA 6.0 software to examine the diversity and analyze the phylogenetic relationship with other begomoviruses， and maximum likelihood tree was selected as the best tree. Fig. 4-A showed that all PaLCuCNV were divided into a big clade which contained several sub-clusters with varying bootstrap support. Except for KU892675（isolate BS9） and KU892676（isolate BS10）， almost all of the PaLCuCNV isolated from Baise city in one sub-clade with PaLCuCNV-GX89（JX128102） and PaLCuCNV-HeNZM1（FN256260）； two isolates（KU-

892665 and KU892666） from Liuzhou city together with two isolates（KU892671 and 892670） from Nan-

ning city in one sub-clade with high bootstrap support（86-100）. Three isolates KU892676（isolate BS10）， KU892677（isolate YL1）and KU892664（isolate NN5） were found to relate to PaLCuCNV（DQ641700）from Vietnam and PaLCuCNV（JF682837） from Fujian， China， KU892665 with PaLCuCNV-Ageratum（JX29-

4075） in a sub-clade with high bootstrap support（99）. Another sub-clade included four isolates from Qinzhou[KU892672（isolate QZ1） and KU892674（isolate QZ3）] and Nanning city[KU892667（isolate NN1） and KU892668（isolate NN2）]. KU892673（isolate QZ2） and KU892669（isolate NN4） were basal in this tree along with PaLCuCNV from Vietnam and PaLCuCNV from Guangxi. A maximum likelihood tree depicted variants of ToLCCNVs from this study in a clade containing other ToLCCNV sequences with robust bootstrap support， and TYLCCNVs from this study in another clade with other TYLCCNV sequence（Fig.4-B）. All of the isolates from Nanning city formed one big sub-clade and showed high bootstrap support（100） with ToLCCNV-G63（AJ704603）， other isolates from this study formed another sub-clade and showed high bootstrap support with ToLCCNV-G16（AJ704602）， ToLCCNV-JX-2（JF681158） and ToLCCNV-G32 （AJ-

558118）； TYLCCNV isolate in this study were found to be relate to TYLCCNV from China. According to Fig.4-C， almost all the isolates from Baise city formed a big sub-clade with varying bootstrap support. Isolate NN1 was related to AYVCNV-GX6 with high bootstrap support（100）.

3 Discussion

Begomoviruses have made a worldwide impact on tomato production. The diversity of begomovirus is very important for virus-resistant tomato variety breeding and its layout. In this study， four begomoviruses （TYLCCNV， PaLCuCNV， ToLCCNV and AYVCNV） were found infecting tomato in Guangxi. PaLCuCNV was confirmed as the dominant virus. These results supported the prior reports（Ma， 2004；Liu et al.， 1998；Cai et al.， 2005）. But Tomato leaf curl Guangxi Virus（ToLCGXV）（reported by Xu et al.， 2007） was not detected in this survey（Xu et al.， 2007）. At the same time， the distribution of tomato-infecting in Guangxi was studied， a map which showed the species of tomato-infecting begomovirus was drawn. According to this map， we can see that the viruses mainly distribute in south and west of Guangxi. As is known to all， the first report of tomato-infecting begomovirus was in Nanning city（Liu et al.， 1998）， but now these viruses almost distributed in every city of Guangxi. Only several cities have not found tomato-infecting be-

gomovirus. Weeds are the important intermediate host of geminiviruses. Several cities also found weeds-infecting begomovirus， such as in Hechi city and Gulin city（data not showed）. We suspect that begomovirus is likely to spread to all regions of Guangxi. All these results indicate that some day these viruses will spread to all of the tomato-planting areas in Guangxi and cause serious economic loss.

In recent years， mixed infection of begomovirus were frequently reported on a variety of crops and weeds， most types of mix-infections were caused by a variety of begomoviruses or one virus with a variety of satellite elements（Wang， 2007；Yang et al.， 2008；Ruan et al.， 2011； Yan et al.， 2014）. In this study， four distinct begomoviruses were found co-infection on tomato， and the symptom on mixed infection samples were more serious than the samples infecting one virus. Our results were in accordance with previous reports（Yan et al.， 2014）. The genetic diversity provided insights into the evolution and biology of the plant virus. Mixed infections provided the opportunity for recombination between co-infecting viruses to give rise to new variants or species（Stewart et al.， 2014）. In this research， the diversity of tomato-infecting begomovirus was rich， and complicated mixed infections existed， at least seven types（showed above） of mixed infections were identified. Complicated mixed infections provided facility to virus recombination and variants. So a comparison were conducted by us between sequences we sequenced and sequences in GenBank. All of the sequences showed high identity（>89%） to the sequences available in GenBank. These results indicated that no new recombinations occured. Accor-

ding to the complicated mixed infections in Guangxi， future research should be conducted on the recom-

bination and new species of tomato-infecting bego-

movirus.

Phylogenetic trees were created according to the 47 full sequences， all the sequences determined in this study were located into different clusters， several isolates from same region were located in the same clades， and others from different regions located in another clades. This result showed that the virus isolates may have regional distribution. Such as PaLCuCNV isolate from Baise city， ToLCCNV from Nanning city and AYVCNV from Baise city， these viruses showed regional distribution， and our results support the previous reports（Yue et al.， 2008；Rocha et al.， 2013； Sobrinho et al.， 2014）. Following the phylogenetic trees， we also found that isolates from this study were divided into several sub-clades， and showed relate to different sequences available in GenBank. This result indicated that the diversity of tomato-infecting begomoviruses was very rich.

4 Conclusion

In this study， four distinct begomoviruses were identified infecting tomato in Guangxi， and PaLCuCNV is the dominate virus. All the sequences were analyzed using maximum likelihood tree. Following the trees we could see that the diversity of tomato-infecting begomoviruses was very rich. All the sequences were blasted but no new viruses or new recombination were identified. So further research should be conducted on the evolution of these viruses.

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