Full genome characterization of 12 citrus tatter leaf virus isolates for the development of a detection assay.

Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.

Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry.

The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus × Poncirus) nucleus and C. reticulata or C. aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.

Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain.

The economically important rootstock species Poncirus trifoliata is resistant to most isolates of Citrus tristeza virus (CTV), but not to members of the CTV resistance-breaking (RB) strain presently found in New Zealand. In this study, five known and suspected RB isolates were separated from field mixtures, and their genomes were sequenced in full. It was found that the RB isolates are members of a single phylogenetically distinct clade with an average of 90.3% genomic nucleotide sequence identity to the closest extant isolate, T36. These isolates also show evidence of multiple recombination events throughout their evolutionary history, with T36, T30 and VT-like isolates, and with each other. Finally, the genomic sequences of these isolates show that several genes contain unique polymorphisms that may or may not be involved in overcoming resistance. These data will aid in the understanding of host-virus interactions, and the mechanism of resistance in P. trifoliata.

Molecular characterization of CEVd strains that induce different phenotypes in Gynura aurantiaca: structure-pathogenicity relationships.

Two Citrus exocortis viroid isolates (CEVd-s and CEVd-129) that induce severe and mild symptoms in Gynura aurantiaca, respectively, have been characterized. They present nucleotide sequences in the pathogenicity motifs P(L), C and P(R) similar to those of "Class A" and "Class B". Infectivity and symptom expression in G. aurantica and tomato were evaluated with a selection of sequence variants recovered from both isolates. As expected, the two variants selected from CEVd-s induced severe symptoms. The variants selected from CEVd-129 induced mild symptoms, except one of them, named MJ, that presented an unusual genotype and induced severe symptoms in G. aurantiaca. The biological properties of MJ show that the two nucleotide changes of the C domain normally associated with the P(L) and P(R) motifs of "Class B" strains are not implicated in symptom expression. The relationship between "Class A" and "Class B" strains with the symptoms induced in clementine trees grafted on trifoliate orange is discussed.

Expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance.

Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata ( Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). To be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. Several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of progeny. Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange ( Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees.

Regulation of gibberellin 20-oxidase gene expression and gibberellin content in citrus by temperature and citrus exocortis viroid.

A cDNA clone coding for a gibberellin (GA) 20-oxidase ( CcGA20ox1), an enzyme of GA biosynthesis, which when expressed in vitro catalyzed the conversion of GA(12) to GA(9) and of GA(53) to GA(20), was isolated from the citrus hybrid Carrizo citrange (C itrus sinensis x Poncirus trifoliata). Transcripts of CcGA20ox1 were abundant in the apex and leaves and much less abundant in internodes, nodes and roots. Seedlings of Carrizo citrange cultured under a 32 degrees C/27 degrees C (day/night) regime elongated more than seedlings growing under 17 degrees C/12 degrees C conditions. The effect of higher temperature was associated with more CcGA20ox1 transcripts and with higher content of GA(1), the main active GA in citrus, in the shoot. The infection of Etrog citron ( Citrus medica) plants with citrus exocortis viroid (CEVd), which produces a stunted phenotype, reduced the levels of transcripts in the apical shoot hybridizing to the gene CcGA20ox1 of Carrizo citrange and the content of GA(1). Thus GA(1) content correlated with CcGA20ox1 transcript levels. In contrast, results for gibberellic acid (GA(3)) and paclobutrazol applications to Carrizo citrange showed that CcGA20ox1 expression was subject to feed-back regulation. These observations indicate that the feed-back regulation of GA20ox operates mostly when the levels of active GAs have been dramatically altered. The results also show that the growth reduction induced by environmental (temperature) and biotic (CEVd) factors may be partially due to the modulation of the expression of GA20ox genes.

Sequence analysis of a 282-kilobase region surrounding the citrus Tristeza virus resistance gene (Ctv) locus in Poncirus trifoliata L. Raf.

Citrus tristeza virus (CTV) is the major virus pathogen causing significant economic damage to citrus worldwide, and a single dominant gene, Ctv, provides broad spectrum resistance to CTV in Poncirus trifoliata L. Raf. Ctv was physically mapped to a 282-kb region using a P. trifoliata bacterial artificial chromosome library. This region was completely sequenced to about 8x coverage using a shotgun sequencing strategy and primer walking for gap closure. Sequence analysis predicts 22 putative genes, two mutator-like transposons and eight retrotransposons. This sequence analysis also revealed some interesting features of this region of the P. trifoliata genome: a disease resistance gene cluster with seven members and eight retrotransposons clustered in a 125-kb gene-poor region. Comparative sequence analysis suggests that six genes in the Ctv region have significant sequence similarity with their orthologs in bacterial artificial chromosome clones F7H2 and F21T11 from Arabidopsis chromosome I. However, the analysis of gene colinearity between P. trifoliata and Arabidopsis indicates that Arabidopsis genome sequence information may be of limited use for positional gene cloning in P. trifoliata and citrus. Analysis of candidate genes for Ctv is also discussed.