Preliminary Analysis Of The Evolution And Origin Of Citrus Tristeza Virus

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 detectionTotal 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 citrus2.1 RFLP analysisSingle 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 analysisThe 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 isolatesThe 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 analysisBased 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 analysisThe 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 analysisTaking 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 ConclusionIn 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.