柑橘衰退病毒的进化与起源初步分析
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摘要
柑橘衰退病(Tristeza)是由柑橘衰退病毒(Citrus trtisteza virus，CTV)引起的一种具有经济重要性的病害，广泛分布于世界各柑橘产区，对全世界的柑橘产业造成了严重的威胁。CTV属于长线形病毒属(Closterovirus)，病毒粒子呈11×2，000nm的长线形，基冈组约19，300个核苷酸组成的正义单链RNA(+ssRNA)，是目前已知植物病毒基因组中最大的病毒，具有12个开放读码框(ORF)，可以编码19种以上的蛋白质产物，住5’端和3’端各有107nt和275nt的非翻译区(URT)。CTV存在复杂的株系分化现象，不同的CTV分离株能引起包括茎陷点、酸橙作砧木的甜橙和葡萄柚植株快速死亡在内的多种症状。随着品种结构调整，茎陷点型柑橘衰退病为害日益加重。由于CTV在田间通过蚜虫传播，对其最有效的防治方法是应用弱毒株系交叉保护技术(MSCP)，而对其株系研究是应用该技术的基础。
本研究从我国野生柑橘资源丰富的地区收集CTV分离株，经直接组织点免疫(DTBIA)和反转录-聚合酶链式反应(RT-PCR)检测，阳性样品嫁接保存于网室锦橙实生苗上。应用RT-PCR扩增CTV分离株的主要衣壳蛋白基因(p25基因)，结合限制性内切酶HinfⅠ对PCR产物进行限制性片段长度多态性(RFLP)分析，同时对CTV分离株的p25基因进行单链构象多态性(SSCP)分析，初步明确我国野生柑橘资源感染CTV及其分离株组群构成情况。同时，通过对野生柑橘上的CTV分离株及国内栽培甜橙及柚上的强弱毒CTV代表分离株的部分基因片段(p23、p20、p13、p18、p25、p27、POL、HEL、k17)进行扩增测序，并同国外典型CTV分离株相应基因片段进行序列比对，绘制基因系统发育进化树状分类图进行遗传进化分析，初步明确野生柑橘上的CTV分离株同国内外栽培品种上CTV分离株间的序列差异程度，为弱毒株保护技术的应用和衰退病毒的进化及起源分析提供相关依据。
主要研究结果：
1野生柑橘品种的收集和CTV检测
分别从我国云南、四川、重庆、广西、湖南、江西等野生柑橘主要分布地区收集到79个野生柑橘样品，经中国农业科学院柑橘研究所国家果树种质(重庆)柑橘圃鉴定后进行DTBIA和RT-PCR检测，筛选出共有11个野生柑橘样品携带CTV，带毒率为13.9％，说明野生柑橘存在CTV的感染。
2野生CTV分离株的组群构成分析
2．1 RFLP分析
对野生柑橘上的11个CTV分离株p25基因经RFLP分析，有72.7％的样品出现单—p25／HinfⅠRFLP谱型，其中以第3组群为主，第1组群次之；有27.3％的样品表现出p25／HinfⅠRFLP第1、3组群和第2、3组群混合谱型。
有一个样品含有p25／HinfⅠRFLP第4组群，可能是具有潜在保护作用的弱毒株；另外一个样品为第6组群。
2．2 SSCP分析
通过对野生柑橘上的11个CTV p25基因的扩增产物进行SSCP分析，结果表明RFLP分析为混合组群的3个CTV分离株具有4条特征性谱带外，其余分离株均只有两条特征性谱带，说明在野生状态下生存的柑橘植株主要受CTV株系的单一组群为害，并且SSCP分析与RFLP分析的结果相一致。
3进化起源分析
将野生柑橘上的11个CTV分离株和国内栽培甜橙和柚上的强弱毒代表分离株位于基因组3’端的p23、p20、p13、p18、p25、p27和位于中部的RdRp和p33基因的交叠部分POL片段以及5’的HEL、k17基因片段进行扩增和测序。
3．1同源性分析
将上述9个基因片段的序列进行同源性比对分析，结果表明11个野生CTV基因组的3’端的序列同源性较高，保守性较强，5’端的序列变异较大。在分析的9个基因片段中位于3’端的p25基因的保守性最高，野生柑橘上11个CTV分离株间的核苷酸和氨基酸序列的同源性分别为92.1％～99.5％和94.8％～100％，同国内栽培品种上的4个代表分离株以及国外栽培品种上的21个代表株之间的核苷酸和氨基酸序列的同源性分别为91.3％～99.5％和95.2％～100％，91.3％～99.6％和94.3％～100％；11个野生CTV分离株位于基因组5’端的HEL基因片段的核苷酸和氨基酸序列同源性最低为77.0％和86.3％，k17基因片段的核苷酸和氨基酸序列同源性最低只有65.5％和63.2％。
3．2遗传进化分析
9个基因片段序列的遗传进化分析表明，碱基含量GC＜AT，以T结尾的密码子使用频率较高。四种碱基在密码子第一、二、三位的含量不同，野生柑橘样品和国内外栽培晶种上的CTV分离株p25、p27、p23基因片段核苷酸序列中碱基A的含量最高，C的含量最低；p20、p13、p18、POL、HEL、k17基因片段核苷酸序列中碱基T的含量最高，C的含量最低。低GC含量说明序列的突变率较低。
其中p25基因片段核苷酸序列中变异位点占总位点的25.2％，氮基酸序列中变异位点占总位点的17.5％；k17基因片段的变异最大，核苷酸序列中变异位点占总位点的50.7％，氨基酸序列中变异位点占总位点的55.1％；从野生柑橘样品和国内外栽培品种上的CTV分离株密码子第一、二、三位的碱基替换的情况看，总的来说，具有编码序列的共同特点：密码子第三位的碱基替换数最多，其次为第一位，最少是第二位，转换数多于颠换数，比率在2以上；p25、p27、p23、p20、p13、p18、POL、k17基因片段序列间的同义突变(d_S)和非同义突变(d_N)比较表明：序列间总的比较d_S大于d_N，而且差异显著(P＜0.05)，只有在含有国外栽培品种上的CTV分离株的分析组群的HEL基因片段比对序列中非同义突变与同义突变相比，无明显的差异(P＞0.05)。
CTV分离株间p25基因遗传距离最小，5’端的遗传距离逐渐增大。同源性较高的序列间的遗传距离较小，同源性较低的分离株间遗传距离较大。
从分析的位于CTV整个基因组不同位置的9个基因片段的同源性及遗传变异的情况来说，在基因组的3’端同源性较高，遗传变异较小，从3’端到5’端同源性逐渐降低，遗传变异逐渐增大。d_N均显著低于d_S(d_N／d_S＜1)，低d_N值表明分析的CTV9个基因片段在进化过程中承受着净化选择，说明它们对CTV来说是重要的。
3．3系统发育树构建
以BYV或LIYV的相应基因核苷酸序列为外群对分析的CTV分离株9个基因片段核苷酸序列构建MP树、NJ树，每个基因片段核苷酸序列构建的MP、NJ系统发育树除个别支系间的聚合关系有所不同外，两种系统树的拓扑结构基本是相似的。在系统发育树的拓扑结构中，聚类在同一支上的分离株间的核苷酸和氮基酸序列同源性均高于处于不同分支上的分离株。当把9个基因片段合并构建系统发育树时，所得的进化树和单个基因构建的系统发育树中CTV分离株间的聚类情况基本是一致的。在单个基因和基因片段合并构建的系统发育树中，收集到的野生柑橘样品上的11个CTV分离株并未在进化树中聚类在同一簇，而是分散在不同的组群中，和国内外栽培品种上的CTV分离株聚类在一起，具有较复杂的亲缘关系。然而具有相同症状的分离株聚类在同一簇上，说明致病性是构建系统发育树的关键因素，与CTV起源和进化往往紧密相关。
4总结
综上所述，柑橘在野生状态下存在CTV的侵染，通过RFLP、SSCP分析发现感染的CTV分离株主要以p25／HinfⅠRFLP单一组群构成，构成的主要组群同栽培品种上的主要组群相似。
从我国分离株和国外分离株间的核苷酸和氨基酸序列同源性、分离株序列间的遗传变异分析以及构建的系统发育树表明，分析的CTV9个基因片段在进化过程中承受着净化选择，并且我国柚的弱毒分离株CT9可能是不同于所有其它分离株的我国独有株系。我国野生柑橘上CTV的主要构成组群同栽培品种上的相似，与国外栽培柑橘上的分离株有较高的序列相似性，在系统发育树上同国内外栽培品种上的CTV分离株亲缘关系较近，分析的11个野生CTV分离株分散在由不同国家分离株聚类的组群中，且往往有相似生物学特性的CTV分离株聚类相同组群，表明病原致病性与CTV的起源和进化有紧密关系，结合我国是柑橘的主要起源中心及柑橘的传播过程，CTV在国内外不同环境条件选择压力下的生存特点，推测我国可能是CTV的主要起源地。
Citrus tristeza virus (CTV) is distributed worldwide, and is the causal agent of one of the most economically important diseases of citrus. CTV, a member of the genus Closterovirus within the family Closteroviridae, is phloem-limited and is dispersed to new areas by grafting of buds infected and then locally spread by aphids in a semi-persistent manner. CTV virions are flexuous filamentous of around 11×2,000nm, composed of a single-stranded, positive-sense genomic RNA(gRNA) molecule of approximately 19,300nt, the largest known for plant RNA viruses. The gRNA is organized in 12 open reading frames (ORFs), potentially encoding at least 19 protein products, and untranslated regions (UTRs) of 107 and 275 nucleotides at the 5' and 3' termini. Complicated diversity of CTV strains or isolates has occurred, which cause such a range of symptoms as decline and death of sweet orange and grapefruit on sour orange rootstock, stem pitting, seedling yellows, etc. Along with the development of some superior in China, stempitting tristeza has been becoming a severe threat to some susceptible cultivars. So far, according to the experience obtained in developed countries, mild strain cross protection (MSCP) has been proven to be the most effective way in preventing sensitive citrus varieties from CTV damage where both severe CTV strains and brown citrus aphids co-exist. The application of MSCP bases on priority to investigation, isolation and identification of the severe and mild CTV strains from infected citrus.
Direct tissue blot immuno-assay (DTBIA) and reverse transcription polymerase chain reaction (RT-PCR) were approached to pre-select CTV isolates from the samples of wild citrus plants collected in Yunnan, Guangxi, Sichuan, Hunan and Jiangxi provinces of China in this study. CTV-infected samples were then preserved in the Jingcheng sweet orange by graft-inoculation. The target sequences of major coat protection gene (p25 gene) were amplified from the CTV-infected samples using RT-PCR, and the p25 genes of these isolates were analyzed by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) for investigating the p25/Hinf I RFLP groups and molecular characteristics of CTV isolates found in Chinese wild type citrus. Further assessment of the genetic differences and genetic relationships, the sequences of 9 genomic regions (p23, p20, p13, p18, p25, p27, POL(the overlapping region of RdRp and p33 gene), HEL(region of the Helicase gene), kl7 (region of the ORF1a)) of the 11 CTV isolates from wild type citrus, 4 Chinese CTV isolates from cultivated sweet oranges and pummelos were amplified and sequenced using RT-PCR and sequencer. Their sequences were subject to sequence analysis and compared with those of foreign isolates from GenBank each other by BioEidt and MEGA softwares. By sequence analysis the differences were therefore estimated among the CTV isolates sourced from wild type citrus and cultivated citrus, to provide theoretical data for controlling tristeza by MSCP and analyzing the evolution and origin of CTV isolates.
The main experimental results are as follows,
1 Collecting the wild citrus samples and CTV detection
Total 79 wild citrus samples were collected in Yunnan, Guangxi, Sichuan, Hunan and Jiangxi provinces of China and identified by the National Citrus Germplasm Repository located at CRI, CAAS, of which 11 (13.9% of all the samples) were detected CTV-positive by DTBIA and RT-PCR, indicating that the wild type citrus could be infected by CTV.
2 RFLP and SSCP analysis of the CTV isolates from wild type citrus
2.1 RFLP analysis
Single p25/Hinf I RFLP groups were detected in 72.7% of 11 CTV-positive samples, the dominants were p25/Hinf I RFLP group 3, followed by the group 1. Mixtures of p25/Hinf I RFLP groups 1 and 3, groups 2 and 3 were found in 27.3% of positive samples.
One positive sample was found to be with p25/Hinf I RFLP group 4, with potential usage as mild protective isolate. Another positive sample with p25/Hinf I RFLP group 6.
2.2 SSCP analysis
The polymorphism of p25 genes of the 11 CTV isolates in wild type citrus was also analyzed by SSCP, the results showed that the mixed infections had four characteristic bands, those with single p25/Hinf I RFLP groups had two characteristic bands, the consistent results both by RFLP and SSCP analyses, further confirmed that most of CTV-infected wild type citrus carried single CTV isolates in this study.
3 Analysis of the evolution and origin of CTV isolates
The sequences of p23, p20, p13, p18, p25, p27, POL, HEL, k17 genomic regions of 11 CTV isolates from wild citrus plants and 4 Chinese isolates from cultivated sweet oranges and pummelos were sequenced. Their sequences were analyzed and compared with those corresponding foreign isolates from GenBank by BioEidt and MEGA softwares.
3.1 Sequence identity analysis
Based on the sequence alignments of the 9 genomic regions of all the analyzed CTV isolates, the results showed that the sequence identity in the 3' half of the genome was higher than that in the 5' half of the genome, the genomic RNA at the 3' half was relatively conserved, whereas the sequences of the HEL, k17 regions in the 5' proximal of the genome showed much more dissimilarity, with higher diversity. P25 gene sequences were generally well conserved among the 9 genomic regions. The nucleotide sequences and their amino acid sequences of p25 genes of the 11 wild CTV isolates were found to have identity ranging from 92.1% to 99.5% and 94.8% to 100%, respectively. The nucleotide sequence identity ranged from 91.3% to 99.5% as compared with 4 Chinese CTV isolates of cultivated citrus, while ranged from 91.3% to 99.6% in contrast to 21 foreign CTV isolates of cultivated citrus, and of their amino acid sequences ranges from 95.2% to 100% and 94.3% to 100%, respectively.
Sequence comparisons also suggested that the nucleotide sequences and amino acid sequences of HEL regions of 11 wild CTV isolates had the lowest identity of 77.0% and 86.3%, respectively. Further analysis found that the lowest nucleotide sequences and amino acid sequences identity were 65.5% and 63.2% in the k17 regions of the 11 wild CTV isolates, which was the lowest among the analyzed 9 genomic regions.
3.2 Genetic evolution analysis
The sequences of the 9 genomic regions of the Chinese isolates were analyzed and compared with those corresponding foreign isolates from GenBank. The results showed that GC% of the 9 genomic regions was less than AT%; base A content was the highest among the four bases in the p25, p27,p23 genomic regions, while base T content was the highest in the p20,p13,p18, POL, HEL, k17 genomic regions, base C content was the lowest in the above 9 genomic regions, lower content of bases GC means less variability of sequences.
The number of variable sites of nucleotide sequences and amino acid sequences were 25.2% and 17.5% in the p25 genes, respectively; those of k17 regions were 50.7% and 55.1%, respectively, the most variable region among the 9 regions analyzed. More transitions were found in the 9 genomic regions than transversions with the transitions/transversions ratio over 2.0 among species. The most frequency of substitution occurred at the third codon, followed by that at the first and second codons. The significant difference was found among the rates of non-synonymous mutations(d_N) and of synonymous mutations(d_S) in the p25,p27,p23,p20,p13,p18, POL, k17 genomic regions (P<0.05), although the former was lower than the latter, whereas no significant difference was shown in the HEL regions of the CTV isolates with identity to the foreign isolates (P>0.05).
The genetic distance of the 9 genomic regions were compared with each other, and found that the shortest one is of p25 gene, there was an gradual increment in genetic distance towards the 5' proximal of the CTV genome, the genetic distance of the isolates with high homology was low, contrarily, of those with low homology was high. In general, d_N values were much lower than d_S values(d_N/d_S<1), lower d_N values suggest that the 9 genomic regions have been under purifying selection in the evolution process, which are of importance to CTV.
3.3 Phylogenetic analysis
Taking Beet yellow virus (BYV) and/or Lettuce infectious yellows virus (LIYV) as outgroups, the phylogenetic trees of nucleotide sequences of the 9 genomic regions were constructed by using maximum parsimony method (MP) and neighbor-joining method (NJ). Both methods produced consistent trees with equivalent topology, which showed that most CTV isolates shared the same grouping in the phylogenetic trees obtained from the 9 genomic regions analyzed in the study, regardless of their geographical origins. The results showed that the 11 CTV isolates in Chinese wild type citrus were located different phylogenetic clusters, and shared higher homology and closer relationships with foreign CTV isolates, the CTV isolates classified into one cluster shared higher homology than those into different clusters in phylogenetic trees. In comparison to the phylogenetic trees of the sequences of 9 genomic regions pieced together, MP and NJ trees had similar topology with those of each genomic regions, which suggest these exist complicated genetic relationships among the CTV isolates. Phylogenetic relationship consistency in different genomic regions and statistical analysis indicated that the homology among the 11 wild CTV isolates from the same area was much lower than that between those and the foreign CTV isolates from different geographical origins. Whereas, those CTV isolates with similar biological characteristics usually dropped into the same clusters, it is suggested that pathogenicity is critical to the evolution and origin of CTV.
4 Conclusion
In summary, there existed CTV infections of wild type citrus. RFLP and SSCP analyses on the p25 gene demonstrated that most of CTV-infected wild type citrus carried single CTV isolates, and the dominant p25/Hinf I RFLP groups in wild type citrus were similar to those in cultivated citrus.
Sequence identity & genetic evolution assays and phylogenetic analyses of the CTV isolates sourced from Chinese wild type citrus, and cultivated citrus, in comparison to those overseas showed that the 9 genomic regions of CTV isolates analyzed have been under purifying selection in the evolution process, and mild isolate CT9 sourced from Chinese pummalo may be a specific one different from all isolates analyzed so far. Phylogenetic analyses showed that the 11 CTV isolates in Chinese wild type citrus were located at different clusters, and shared higher homology and closer relationships with foreign CTV isolates from different geographical origins, isolates with similar biological characteristics usually dropped into the same clusters, it is suggested that there exists complicated genetic relationships among the CTV isolates, and that pathogenicity is critical to the evolution and origin of CTV, and China is the most important origin of CTV based on the above analyses and that China is one of most important origin centers of citrus.
本研究从我国野生柑橘资源丰富的地区收集CTV分离株，经直接组织点免疫(DTBIA)和反转录-聚合酶链式反应(RT-PCR)检测，阳性样品嫁接保存于网室锦橙实生苗上。应用RT-PCR扩增CTV分离株的主要衣壳蛋白基因(p25基因)，结合限制性内切酶HinfⅠ对PCR产物进行限制性片段长度多态性(RFLP)分析，同时对CTV分离株的p25基因进行单链构象多态性(SSCP)分析，初步明确我国野生柑橘资源感染CTV及其分离株组群构成情况。同时，通过对野生柑橘上的CTV分离株及国内栽培甜橙及柚上的强弱毒CTV代表分离株的部分基因片段(p23、p20、p13、p18、p25、p27、POL、HEL、k17)进行扩增测序，并同国外典型CTV分离株相应基因片段进行序列比对，绘制基因系统发育进化树状分类图进行遗传进化分析，初步明确野生柑橘上的CTV分离株同国内外栽培品种上CTV分离株间的序列差异程度，为弱毒株保护技术的应用和衰退病毒的进化及起源分析提供相关依据。
主要研究结果：
1野生柑橘品种的收集和CTV检测
分别从我国云南、四川、重庆、广西、湖南、江西等野生柑橘主要分布地区收集到79个野生柑橘样品，经中国农业科学院柑橘研究所国家果树种质(重庆)柑橘圃鉴定后进行DTBIA和RT-PCR检测，筛选出共有11个野生柑橘样品携带CTV，带毒率为13.9％，说明野生柑橘存在CTV的感染。
2野生CTV分离株的组群构成分析
2．1 RFLP分析
对野生柑橘上的11个CTV分离株p25基因经RFLP分析，有72.7％的样品出现单—p25／HinfⅠRFLP谱型，其中以第3组群为主，第1组群次之；有27.3％的样品表现出p25／HinfⅠRFLP第1、3组群和第2、3组群混合谱型。
有一个样品含有p25／HinfⅠRFLP第4组群，可能是具有潜在保护作用的弱毒株；另外一个样品为第6组群。
2．2 SSCP分析
通过对野生柑橘上的11个CTV p25基因的扩增产物进行SSCP分析，结果表明RFLP分析为混合组群的3个CTV分离株具有4条特征性谱带外，其余分离株均只有两条特征性谱带，说明在野生状态下生存的柑橘植株主要受CTV株系的单一组群为害，并且SSCP分析与RFLP分析的结果相一致。
3进化起源分析
将野生柑橘上的11个CTV分离株和国内栽培甜橙和柚上的强弱毒代表分离株位于基因组3’端的p23、p20、p13、p18、p25、p27和位于中部的RdRp和p33基因的交叠部分POL片段以及5’的HEL、k17基因片段进行扩增和测序。
3．1同源性分析
将上述9个基因片段的序列进行同源性比对分析，结果表明11个野生CTV基因组的3’端的序列同源性较高，保守性较强，5’端的序列变异较大。在分析的9个基因片段中位于3’端的p25基因的保守性最高，野生柑橘上11个CTV分离株间的核苷酸和氨基酸序列的同源性分别为92.1％～99.5％和94.8％～100％，同国内栽培品种上的4个代表分离株以及国外栽培品种上的21个代表株之间的核苷酸和氨基酸序列的同源性分别为91.3％～99.5％和95.2％～100％，91.3％～99.6％和94.3％～100％；11个野生CTV分离株位于基因组5’端的HEL基因片段的核苷酸和氨基酸序列同源性最低为77.0％和86.3％，k17基因片段的核苷酸和氨基酸序列同源性最低只有65.5％和63.2％。
3．2遗传进化分析
9个基因片段序列的遗传进化分析表明，碱基含量GC＜AT，以T结尾的密码子使用频率较高。四种碱基在密码子第一、二、三位的含量不同，野生柑橘样品和国内外栽培晶种上的CTV分离株p25、p27、p23基因片段核苷酸序列中碱基A的含量最高，C的含量最低；p20、p13、p18、POL、HEL、k17基因片段核苷酸序列中碱基T的含量最高，C的含量最低。低GC含量说明序列的突变率较低。
其中p25基因片段核苷酸序列中变异位点占总位点的25.2％，氮基酸序列中变异位点占总位点的17.5％；k17基因片段的变异最大，核苷酸序列中变异位点占总位点的50.7％，氨基酸序列中变异位点占总位点的55.1％；从野生柑橘样品和国内外栽培品种上的CTV分离株密码子第一、二、三位的碱基替换的情况看，总的来说，具有编码序列的共同特点：密码子第三位的碱基替换数最多，其次为第一位，最少是第二位，转换数多于颠换数，比率在2以上；p25、p27、p23、p20、p13、p18、POL、k17基因片段序列间的同义突变(d_S)和非同义突变(d_N)比较表明：序列间总的比较d_S大于d_N，而且差异显著(P＜0.05)，只有在含有国外栽培品种上的CTV分离株的分析组群的HEL基因片段比对序列中非同义突变与同义突变相比，无明显的差异(P＞0.05)。
CTV分离株间p25基因遗传距离最小，5’端的遗传距离逐渐增大。同源性较高的序列间的遗传距离较小，同源性较低的分离株间遗传距离较大。
从分析的位于CTV整个基因组不同位置的9个基因片段的同源性及遗传变异的情况来说，在基因组的3’端同源性较高，遗传变异较小，从3’端到5’端同源性逐渐降低，遗传变异逐渐增大。d_N均显著低于d_S(d_N／d_S＜1)，低d_N值表明分析的CTV9个基因片段在进化过程中承受着净化选择，说明它们对CTV来说是重要的。
3．3系统发育树构建
以BYV或LIYV的相应基因核苷酸序列为外群对分析的CTV分离株9个基因片段核苷酸序列构建MP树、NJ树，每个基因片段核苷酸序列构建的MP、NJ系统发育树除个别支系间的聚合关系有所不同外，两种系统树的拓扑结构基本是相似的。在系统发育树的拓扑结构中，聚类在同一支上的分离株间的核苷酸和氮基酸序列同源性均高于处于不同分支上的分离株。当把9个基因片段合并构建系统发育树时，所得的进化树和单个基因构建的系统发育树中CTV分离株间的聚类情况基本是一致的。在单个基因和基因片段合并构建的系统发育树中，收集到的野生柑橘样品上的11个CTV分离株并未在进化树中聚类在同一簇，而是分散在不同的组群中，和国内外栽培品种上的CTV分离株聚类在一起，具有较复杂的亲缘关系。然而具有相同症状的分离株聚类在同一簇上，说明致病性是构建系统发育树的关键因素，与CTV起源和进化往往紧密相关。
4总结
综上所述，柑橘在野生状态下存在CTV的侵染，通过RFLP、SSCP分析发现感染的CTV分离株主要以p25／HinfⅠRFLP单一组群构成，构成的主要组群同栽培品种上的主要组群相似。
从我国分离株和国外分离株间的核苷酸和氨基酸序列同源性、分离株序列间的遗传变异分析以及构建的系统发育树表明，分析的CTV9个基因片段在进化过程中承受着净化选择，并且我国柚的弱毒分离株CT9可能是不同于所有其它分离株的我国独有株系。我国野生柑橘上CTV的主要构成组群同栽培品种上的相似，与国外栽培柑橘上的分离株有较高的序列相似性，在系统发育树上同国内外栽培品种上的CTV分离株亲缘关系较近，分析的11个野生CTV分离株分散在由不同国家分离株聚类的组群中，且往往有相似生物学特性的CTV分离株聚类相同组群，表明病原致病性与CTV的起源和进化有紧密关系，结合我国是柑橘的主要起源中心及柑橘的传播过程，CTV在国内外不同环境条件选择压力下的生存特点，推测我国可能是CTV的主要起源地。
Citrus tristeza virus (CTV) is distributed worldwide, and is the causal agent of one of the most economically important diseases of citrus. CTV, a member of the genus Closterovirus within the family Closteroviridae, is phloem-limited and is dispersed to new areas by grafting of buds infected and then locally spread by aphids in a semi-persistent manner. CTV virions are flexuous filamentous of around 11×2,000nm, composed of a single-stranded, positive-sense genomic RNA(gRNA) molecule of approximately 19,300nt, the largest known for plant RNA viruses. The gRNA is organized in 12 open reading frames (ORFs), potentially encoding at least 19 protein products, and untranslated regions (UTRs) of 107 and 275 nucleotides at the 5' and 3' termini. Complicated diversity of CTV strains or isolates has occurred, which cause such a range of symptoms as decline and death of sweet orange and grapefruit on sour orange rootstock, stem pitting, seedling yellows, etc. Along with the development of some superior in China, stempitting tristeza has been becoming a severe threat to some susceptible cultivars. So far, according to the experience obtained in developed countries, mild strain cross protection (MSCP) has been proven to be the most effective way in preventing sensitive citrus varieties from CTV damage where both severe CTV strains and brown citrus aphids co-exist. The application of MSCP bases on priority to investigation, isolation and identification of the severe and mild CTV strains from infected citrus.
Direct tissue blot immuno-assay (DTBIA) and reverse transcription polymerase chain reaction (RT-PCR) were approached to pre-select CTV isolates from the samples of wild citrus plants collected in Yunnan, Guangxi, Sichuan, Hunan and Jiangxi provinces of China in this study. CTV-infected samples were then preserved in the Jingcheng sweet orange by graft-inoculation. The target sequences of major coat protection gene (p25 gene) were amplified from the CTV-infected samples using RT-PCR, and the p25 genes of these isolates were analyzed by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) for investigating the p25/Hinf I RFLP groups and molecular characteristics of CTV isolates found in Chinese wild type citrus. Further assessment of the genetic differences and genetic relationships, the sequences of 9 genomic regions (p23, p20, p13, p18, p25, p27, POL(the overlapping region of RdRp and p33 gene), HEL(region of the Helicase gene), kl7 (region of the ORF1a)) of the 11 CTV isolates from wild type citrus, 4 Chinese CTV isolates from cultivated sweet oranges and pummelos were amplified and sequenced using RT-PCR and sequencer. Their sequences were subject to sequence analysis and compared with those of foreign isolates from GenBank each other by BioEidt and MEGA softwares. By sequence analysis the differences were therefore estimated among the CTV isolates sourced from wild type citrus and cultivated citrus, to provide theoretical data for controlling tristeza by MSCP and analyzing the evolution and origin of CTV isolates.
The main experimental results are as follows,
1 Collecting the wild citrus samples and CTV detection
Total 79 wild citrus samples were collected in Yunnan, Guangxi, Sichuan, Hunan and Jiangxi provinces of China and identified by the National Citrus Germplasm Repository located at CRI, CAAS, of which 11 (13.9% of all the samples) were detected CTV-positive by DTBIA and RT-PCR, indicating that the wild type citrus could be infected by CTV.
2 RFLP and SSCP analysis of the CTV isolates from wild type citrus
2.1 RFLP analysis
Single p25/Hinf I RFLP groups were detected in 72.7% of 11 CTV-positive samples, the dominants were p25/Hinf I RFLP group 3, followed by the group 1. Mixtures of p25/Hinf I RFLP groups 1 and 3, groups 2 and 3 were found in 27.3% of positive samples.
One positive sample was found to be with p25/Hinf I RFLP group 4, with potential usage as mild protective isolate. Another positive sample with p25/Hinf I RFLP group 6.
2.2 SSCP analysis
The polymorphism of p25 genes of the 11 CTV isolates in wild type citrus was also analyzed by SSCP, the results showed that the mixed infections had four characteristic bands, those with single p25/Hinf I RFLP groups had two characteristic bands, the consistent results both by RFLP and SSCP analyses, further confirmed that most of CTV-infected wild type citrus carried single CTV isolates in this study.
3 Analysis of the evolution and origin of CTV isolates
The sequences of p23, p20, p13, p18, p25, p27, POL, HEL, k17 genomic regions of 11 CTV isolates from wild citrus plants and 4 Chinese isolates from cultivated sweet oranges and pummelos were sequenced. Their sequences were analyzed and compared with those corresponding foreign isolates from GenBank by BioEidt and MEGA softwares.
3.1 Sequence identity analysis
Based on the sequence alignments of the 9 genomic regions of all the analyzed CTV isolates, the results showed that the sequence identity in the 3' half of the genome was higher than that in the 5' half of the genome, the genomic RNA at the 3' half was relatively conserved, whereas the sequences of the HEL, k17 regions in the 5' proximal of the genome showed much more dissimilarity, with higher diversity. P25 gene sequences were generally well conserved among the 9 genomic regions. The nucleotide sequences and their amino acid sequences of p25 genes of the 11 wild CTV isolates were found to have identity ranging from 92.1% to 99.5% and 94.8% to 100%, respectively. The nucleotide sequence identity ranged from 91.3% to 99.5% as compared with 4 Chinese CTV isolates of cultivated citrus, while ranged from 91.3% to 99.6% in contrast to 21 foreign CTV isolates of cultivated citrus, and of their amino acid sequences ranges from 95.2% to 100% and 94.3% to 100%, respectively.
Sequence comparisons also suggested that the nucleotide sequences and amino acid sequences of HEL regions of 11 wild CTV isolates had the lowest identity of 77.0% and 86.3%, respectively. Further analysis found that the lowest nucleotide sequences and amino acid sequences identity were 65.5% and 63.2% in the k17 regions of the 11 wild CTV isolates, which was the lowest among the analyzed 9 genomic regions.
3.2 Genetic evolution analysis
The sequences of the 9 genomic regions of the Chinese isolates were analyzed and compared with those corresponding foreign isolates from GenBank. The results showed that GC% of the 9 genomic regions was less than AT%; base A content was the highest among the four bases in the p25, p27,p23 genomic regions, while base T content was the highest in the p20,p13,p18, POL, HEL, k17 genomic regions, base C content was the lowest in the above 9 genomic regions, lower content of bases GC means less variability of sequences.
The number of variable sites of nucleotide sequences and amino acid sequences were 25.2% and 17.5% in the p25 genes, respectively; those of k17 regions were 50.7% and 55.1%, respectively, the most variable region among the 9 regions analyzed. More transitions were found in the 9 genomic regions than transversions with the transitions/transversions ratio over 2.0 among species. The most frequency of substitution occurred at the third codon, followed by that at the first and second codons. The significant difference was found among the rates of non-synonymous mutations(d_N) and of synonymous mutations(d_S) in the p25,p27,p23,p20,p13,p18, POL, k17 genomic regions (P<0.05), although the former was lower than the latter, whereas no significant difference was shown in the HEL regions of the CTV isolates with identity to the foreign isolates (P>0.05).
The genetic distance of the 9 genomic regions were compared with each other, and found that the shortest one is of p25 gene, there was an gradual increment in genetic distance towards the 5' proximal of the CTV genome, the genetic distance of the isolates with high homology was low, contrarily, of those with low homology was high. In general, d_N values were much lower than d_S values(d_N/d_S<1), lower d_N values suggest that the 9 genomic regions have been under purifying selection in the evolution process, which are of importance to CTV.
3.3 Phylogenetic analysis
Taking Beet yellow virus (BYV) and/or Lettuce infectious yellows virus (LIYV) as outgroups, the phylogenetic trees of nucleotide sequences of the 9 genomic regions were constructed by using maximum parsimony method (MP) and neighbor-joining method (NJ). Both methods produced consistent trees with equivalent topology, which showed that most CTV isolates shared the same grouping in the phylogenetic trees obtained from the 9 genomic regions analyzed in the study, regardless of their geographical origins. The results showed that the 11 CTV isolates in Chinese wild type citrus were located different phylogenetic clusters, and shared higher homology and closer relationships with foreign CTV isolates, the CTV isolates classified into one cluster shared higher homology than those into different clusters in phylogenetic trees. In comparison to the phylogenetic trees of the sequences of 9 genomic regions pieced together, MP and NJ trees had similar topology with those of each genomic regions, which suggest these exist complicated genetic relationships among the CTV isolates. Phylogenetic relationship consistency in different genomic regions and statistical analysis indicated that the homology among the 11 wild CTV isolates from the same area was much lower than that between those and the foreign CTV isolates from different geographical origins. Whereas, those CTV isolates with similar biological characteristics usually dropped into the same clusters, it is suggested that pathogenicity is critical to the evolution and origin of CTV.
4 Conclusion
In summary, there existed CTV infections of wild type citrus. RFLP and SSCP analyses on the p25 gene demonstrated that most of CTV-infected wild type citrus carried single CTV isolates, and the dominant p25/Hinf I RFLP groups in wild type citrus were similar to those in cultivated citrus.
Sequence identity & genetic evolution assays and phylogenetic analyses of the CTV isolates sourced from Chinese wild type citrus, and cultivated citrus, in comparison to those overseas showed that the 9 genomic regions of CTV isolates analyzed have been under purifying selection in the evolution process, and mild isolate CT9 sourced from Chinese pummalo may be a specific one different from all isolates analyzed so far. Phylogenetic analyses showed that the 11 CTV isolates in Chinese wild type citrus were located at different clusters, and shared higher homology and closer relationships with foreign CTV isolates from different geographical origins, isolates with similar biological characteristics usually dropped into the same clusters, it is suggested that there exists complicated genetic relationships among the CTV isolates, and that pathogenicity is critical to the evolution and origin of CTV, and China is the most important origin of CTV based on the above analyses and that China is one of most important origin centers of citrus.
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