Assessment of genetic diversity of Mediterranean Citrus Tristeza Virus (CTV) isolates and genomic RNA variability associated to their vector transmission

Citrus tristeza virus (CTV), is the causal agent of devastating epidemics that changed the course of citrus industry worldwide. To date, global CTV infection caused the loss of almost 100 million trees, of which 40 millions were from countries of the Mediterranean basin, where potential risks for further serious devastations are foreseen, since the CTV-sensitive sour orange is still the major rootstock adopted over large extends. CTV is adapted to replicate in the phloem cells of Citrus spp., where latent infections are quiet common and may last symptomless for a long time. Meanwhile, dramatic losses have been readily induced by the interaction of severe CTV variants with different scion-rootstock combinations, enabling to discriminate between three distinct syndromes known as quick decline, stem pitting and seedling yellows. Furthermore, CTV is a stylet-borne virus adopted to be transmitted in nature by several aphid species in a semi-persistent manner. The most efficient vector Toxoptera citricidus (Kirkaldy) became recently established in northern Spain and Portugal, which raised the risk for the rapid spread of severe CTV isolates, representing a serious threat to the Mediterranean citrus industry. Moreover, Aphis gossypii (Glover) is a pandemic aphids species that has been reported to be the origin of major CTV epidemics in the Mediterranean basin. Additionally, minor CTV vectors including A. spiraecola (Pagenstecher) and Toxoptera. aurantii (Boyer de Fonscolombe) are prevalent and their extensive populations may have a significant impact in the local virus spread. CTV populations can occur as a mixture of genotypes, which have been reported to be separated by aphid transmission bottlenecks and/or graft propagation, but the mechanism involved during these changes is still largely unknown. The main aim of present research was to conduct a molecular characterization and to evaluate the genetic diversity of the CTV populations within the Mediterranean basin. Furthermore, the experimental vector-transmissibility of some isolates by local aphid biotypes has been evaluated and studies on eventual genomic variations after aphid passage have also been investigated. To reach the above mentioned goals, a total number of 75 CTV sources originated from 15 Mediterranean countries (Albania, Algeria, China, Croatia, Cyprus, Egypt, Italy, Iran, Lebanon, Montenegro, Morocco, Palestine, Portugal, Syria and Trinidad), maintained within CTV collection of the Mediterranean Agronomic Institute of Bari (MAI-B, Italy), have been assigned a genotype profile according to their specific signals using a set of linked molecular markers. Within each exhibited genotype profile, CTV-CP gene targets have been amplified from a representative isolate and subjected to partial genome sequence analysis. In order to evaluate the vector transmission kinetics of CTV within the Mediterranean area, a number of CTV isolates showing diversified genomic traits and pathogenecity have been subjected to experimental transmission trials using the most prevailing local aphid biotypes, including A. gossypii (Glover), A. spiraecola and T. aurantii. Further serological and molecular assays flanking multiple genomic regions have been applied to the aphid-derived sub-isolates, in order to assess eventual alterations in the parental virus populations by aphid passage. The 5àƒ àƒ à,¢ terminal ORF typing and the phyllogenetic relationship of CTV-CPg analysis of CTV isolates, highlightes the high genetic diversity of the Mediterranean CTV populations between and within countries, and evidences the predominant occurrence of CTV isolates as a mixture of genomic variants. The major outcome of this research is that T30 mild strain genotype (Florida) is a stable viral component on the studied area, while, severe VT (Israel) and T36 (Florida) genotypes appeared as relevant variants in the Middle East and East Adriatic regions, respectively. Nevertheless, some p25 haplotype populations clustered close to standard isolates associated with high pathogenic potential, such as T3 (Florida) and SY568 (New Zealand). Vector transmission experiments of the studied isolates showed diverse transmission efficiencies of and evidenced a great spread potential of either mild or severe seedling yellows -A. gossypii combinations. Conversely, the T36-like isolate (MAIB_Q3). was poorly transmissible. In correlation with the consistent prevalence of A. gossypii species on citrus plantings, these findings reflect the high involvement of the citrus entomofauna in the rapid spread and the major outbreaks of tristeza in the Mediterranean area. Within the aphid-generated CTV sub-isolates, minor nucleotide mutations occurred on p18, p20 and p23 genomic regions, but also over the highly conserved CPg sequence, exhibiting new haplotypes. When translated to amino-acid sequences, specific variations on the p23 and p25 regions resulted in slight protein divergences, which in some instances may be accompanied by changes in the pathogenic behaviour of the parental isolate.