Molecular Characterization Of Citrus Tristeza Virus Isolate S45 And The Effect Of Aphid Transmission On CTV Molecular Variants Composition

Citrus tristeza virus persists worldwidely in citrus production areas. The virus is highly divergent and differentiated into three isolates, including quick decline (QD), stem pitting (SP) and seedling yellow (SY) isolates. QD isolate causes decline of citrus trees on orange rootstock. SP isolates induces stem pitting syndrome of grapefruits and some sweet orange varieties, which reduces fruit sizes and the loss of yield and quality. The viral disease is a serious threat to citrus industry. CTV is a member of genus Closterovirus in family Closteroviridae. It has a single-stranded positive-sense genomic RNA about 19.3kb, which ranges the largest genome among the plant RNA viruses. Untill now, the genomes of 15 CTV isolates have been complertely sequenced in the world. Five CTV genotypes T30, VT, T36, T3 and B165 have been identified based on the characteristics of their genomic sequences. In order to have a deep understanding of the population structures and molecular constitutions of CTV in China, we molecularly characterized a highly divergent CTV isolate S45, analysed the effect of aphid transmission on CTV molecular variants composition. The obtained results are as follows.1. Primers were designed based on the reported CTV genomic structures and sequences of reported CTV isolates.15 segments covering the full genome of CTV were amplified from isolates S45 by RT-PCR. All PCR products were cloned. Nine to sixteen clones were analyzed by PCR-SSCP. Results revealed that S45 was composed of multiple molecular variants, and each of the amplified fragments contained 2-6 haplotypes. The clones showed polymorphic SSCP patterns were sequenced. Results of sequence alignments showed that there were molecular variations in all analyzed regions. The clones of fragment M2 showed the largest genetic distance of 0.255±0.019. The variation in P27 region was relatively lower, and the inter-clonal genetic distance clones is 0.033±0.007. Whereas, CP gene showed much less variation, and only 2-4 base differences were identified among six sequenced clones with a genetic distance 0.003±0.002. Sequence comparison revealed that some regions contained moleculars showing a high sequence similaritiy with both mild strains T30 and severe isolate NZRB-G90, and some cloned sequences had relatively low sequence similarities with both T30 and NZRB-G90, suggesting that there might be recombination between the two isolates.2. A full length genomic sequence 19243 nt (named S45-R) was obtained by jioning the sequences of molecular variants of the adjacent segments. S45-R had 80.0%-93.5% with genomic sequences of 15 repoted isolates. S45-R showed the highest similarity 93.5% with the mild isolate T30, but its CP gene is mostly close to a severe strain NZRB-G90 with a similarity 97.2% at nucleotide level. However, P23 protein of S45-R has the same amino acids with that of mild isolate T30 at sites which used for the differentiation of severe and mild strains. Those results furthermore incicated that there were recombination happened in the CTV isolate S45. RDP software was used to analysis recombinant events in S45-R. Eight potential recombination events were identified, and each recombination event was detected by at least three methods in RDP software. The recombination event at 11483-13744 nt covering genes P33, P6 and P65 was detected by seven procedures, with the P values from 1.216×10-148 to 8.315×10-27. The identified major and minor parents of S45-R were mild isolate T30 and severe isolate NZRB-G90, respectively.3. CP gene of the seven CTV isolates conserved at our laboratory was amplified by RT-PCR. PCR-SSCP analysis was performed on clones of RT-PCR products. Results showed that the amplified products of isolates N4, N9 and N25 had a single SSCP haplotype, the amplified products of isolates N3, N21, Z30 consisted of three haplotypes, and Z5 was a mixed group of seven haplotypes.The result indicated that the isolate had a complex population structure containing several molecular variants. Futhermore, the isolate Z5 was transmitted onto 20 seedlings of sweet orange by aphis gossypii. The constituon of molecular variants in aphid-inolulated plants were detected by PCR-SSCP analysis for CP gene. Results showed that only one seedling was infected with the virus, and aphid transmission had molecular variant selectivity. SSCP bands of the 13 clones were identical to the predominant bands of clones from isolate Z5.