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Genetic Diversity and Phylogenetic Analysis of Citrus tristeza virus Isolates from Turkey.

ABSTRACT: The presence of Citrus tristeza virus (CTV) in Turkey has been known since the 1960s and the virus was detected in all citrus growing regions of the country. Even though serological and biological characteristics of CTV have been studied since the 1980s, molecular characteristics of CTV isolates have not been studied to date in Turkey. In this study, molecular characteristics of 15 CTV isolates collected from different citrus growing regions of Turkey were determined by amplification, cloning, and sequencing of their major coat protein (CP) genes. The sequence analysis showed that the CP genes were highly conserved among Turkish isolates. However, isolates from different regions showed more genetic variation than isolates from the same region. Turkish isolates were clustered into three phylogenetic groups showing no association with geographical origins, host, or symptoms induced in indicator plants. Phylogenetic analysis of Turkish isolates with isolates from different citrus growing regions of the world including well-characterized type isolates of previously established strain specific groups revealed that some Turkish isolates were closely related to severe quick decline or stem pitting isolates. The results demonstrated that although CTV isolates from Turkey are considered biologically mild, majority of them contain severe components potentially causing quick decline or stem pitting.

SUBMITTER: Erkis-Gungor G

PROVIDER: S-EPMC6393893 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:The presence of Citrus tristeza virus (CTV) has previously been reported in citrus growing regions of Turkey. All serologically and biologically characterized isolates including Iğdır, which was the first identified CTV isolates from Turkey, were considered mild isolates. In this study, molecular characteristics of the Iğdır isolate were determined by different methods. Analysis of the Iğdır isolate by western blot and BD-RT-PCR assays showed the presence of MCA13 epitope, predominantly found in severe isolates, in the Iğdır isolate revealing that it contains a severe component. For further characterization, the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp) genes representing the 3' and 5' half of CTV genome, respectively, were amplified from dsRNA by RT-PCR. Both genes were cloned separately and two clones for each gene were sequenced. Comparisons of nucleotide and deduced amino acid sequences showed that while two CP gene sequences were identical, two RdRp clones showed only 90% and 91% sequence identity in their nucleotide and amino acid sequences, respectively, suggesting a mixed infection with different strains. Phylogenetic analyses of the CP and RdRp genes of Iğdır isolate with previously characterized CTV isolates from different citrus growing regions showed that the CP gene was clustered with NZRB-TH30, a resistance breaking isolate from New Zealand, clearly showing the presence of severe component. Furthermore, two different clones of the RdRp gene were clustered separately with different CTV isolates with a diverse biological activity. While the RdRp-1 was clustered with T30 and T385, two well-characterized mild isolates from Florida and Spain, respectively, the RdRp-2 was most closely related to NZRB-G90 and NZRB-TH30, two well-characterized resistance breaking and stem pitting (SP) isolates from New Zealand confirming the mixed infection. These results clearly demonstrated that the Iğdır isolate, which was previously described as biologically a mild isolate, actually contains a mixture of mild and severe strains.

Project description:Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures.

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Genetic Diversity and Phylogenetic Analysis of Citrus tristeza virus Isolates from Turkey.

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