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.

We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.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 transmitted by aphids in a semipersistent manner. CTV virions are filamentous flexuous particles about 2,000 nm long, with two coat proteins (CP and CPm) covering 95 and 5% of the particle length, respectively (8). The CTV genome is a single-stranded, positive-sense RNA of 19,226 to 19,296 nucleotides (nt) (18,27,48,51) organized in 12 open reading frames encoding at least 19 proteins. These include two papain-like proteases, replication-associated proteins (RNA polymerase, helicase, and methyltransferase), a homologue of the HSP70 protein, two coat proteins (CP and CPm), RNA-binding protein p23 (23), a p20 protein that accumulates in the amorphous inclusion bodies (14), and other proteins of so far unknown function (p61, p13, and p18) (Fig. 1). CTV-infected plants contain, in addition to the genomic RNA, 3Ј-coterminal subgenomic RNAs (15) and defective RNAs (D RNAs), the latter resulting from extensive internal deletions of the genomic RNA (2,26,28,50).CTV isolates differing in the type and intensity of symptoms induced in different citrus species and cultivars and in their aphid transmissibility have been reported worldwide (38). In the last two decades, efforts have been taken to develop molecular techniques for rapid differentiation of CTV isolates and identification of molecular markers related to CTV-induced symptoms. Variation in serological reactivity, peptide maps of the coat protein, double-stranded RNA (dsRNA) patterns, hybridization with cDNA probes, restriction fragment length polymorphism, and single-strand conformation polymorphism (SSCP) have been described in attempts to differentiate CTV isolates (29).Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular or genetic variation. To date, the complete g...

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

Summary.Citrus tristeza closterovirus (CTV) isolates of several geographical origins were compared for variations in their coat protein (CP) gene by analysis of single‐strand conformation polymorphism (SSCP). The CP gene of 17 isolates was reverse transcribed, amplified by polymerase chain reaction (PCR), and 22 clones were inserted into a plasmid vector. These clones were sequenced and found to have between 91.7% and 99.8% sequence homology. Clones were amplified and the PCR products denatured and compared by SSCP analysis in 8% polyacrylamide gels. Using two different electrophoretic conditions, the patterns were different for 16 or 17 clones. Four pairs of clones (T36/T66, P1/Q2, 03/8Q, and E1/E2) differing by 10, 2, 1 and 1 nucleotides, respectively, could not be distinguished using either condition. When these clones were compared by SSCP after digestion with Eco91I (BstEII) three of the pairs (T36/T66, P1/Q2, and 03/8Q) could be differentiated, whereas the clones E1 and E2 (differing by 1 nucleotide) remained indistinguishable. Thus, SSCP analysis combining two electrophoretic conditions and restriction of eight clones with Eco91I allowed discrimination between 21 of the 22 CP gene clones selected.SSCP analysis may provide a procedure to identify and differentiate CTV isolates based on comparisons of several genes or gene regions. It is rapid and cheap and may drastically reduce the amount of sequencing necessary for accurate comparisons.