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Mal secco, caused by the fungus Plenodomus tracheiphilus, is a xylem disease that is a limiting factor for lemon production in the Mediterranean. Resistance or field tolerance are major goals for lemon breeders; however, there is scant information regarding the heritability of mal secco resistance in breeding populations. As with other vascular diseases, phenotyping is the bottleneck for ascertaining resistance and susceptibility, and a validated protocol for greenhouse phenotyping would be valuable to accelerate the selection of tolerant trees before field evaluation. We report phenotyping of 148 hybrids of Khasi papeda (Citrus latipes; tolerant to mal secco) × lemon (susceptible to the disease) in field and greenhouse conditions. Field evaluation was performed on all hybrids for 2 to 3 consecutive years on trees subjected to high natural-pathogen pressure. Detection of the fungal infection was performed by visual observation and real-time polymerase chain reaction (PCR). The first infections occurred ≈6 months after planting, but 2 years of observations were needed for a reliable estimation of susceptibility. The spread of the disease did not occur uniformly throughout the plot, with patterns of spread within rows, probably resulting from infections from plant to plant. The possible errors in the estimation of susceptibility as a result of the uneven distribution of infections in the plot were reduced by using more than one replicate tree per hybrid. The correlation between phenotyping scores and cycle threshold values was weak (r = –0.48, P < 0.001). Three years after planting, hybrids clustered into three groups—susceptible, tolerant, and intermediate—based on symptom progression. A subset of 65 self-rooted hybrids was also subjected to stem inoculation in an unheated greenhouse, with two to seven biological replicates per hybrid. Three months after inoculation, the samples were monitored for symptoms appearance and subjected to real-time PCR pathogen quantification. We observed a weak (r = 0.41) but significant (P < 0.001) correlation between phenotypes in the field and the greenhouse, indicating that, in our conditions, field evaluation remains the best method for phenotyping. However, artificial inoculations might help to discard the highly susceptible hybrids before field evaluation.

Iron chlorosis is one of the most serious abiotic stresses affecting citrus (Citrus sp.) culture in the Mediterranean Basin. A trial was performed with potted tolerant and sensitive rootstocks that were grown in volcanic and calcareous soils. Microarray analysis allowed for the identification of differentially expressed genes putatively involved in iron (Fe) deficiency. Most of the differentially expressed genes isolated from the root tips were of unknown function; the remaining genes were related to the oxidative stress response (e.g., glutathione peroxidase), hormone metabolism and signaling (e.g., small auxin up RNA family protein genes), biological regulation, protein turnover, and the tricarboxylic acid cycle (e.g., aconitase). Additionally, the majority of the Fe stress-related genes expressed in the sensitive Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) and tolerant Carrizo citrange (Citrus sinensis × P. trifoliata) rootstocks identified using real-time reverse transcription–polymerase chain reaction (RT-PCR) were related to regulation, the oxidative stress response, and hormone metabolism and signaling, thereby confirming the array data. Furthermore, validation of the differentially expressed genes in seven tolerant and sensitive rootstocks grown in a field trial under chlorotic conditions was performed. In general, the gene expression profiles reflect the different responses of rootstocks, possibly as a result of the various genetic mechanisms involved in the response to Fe deficiency. Moreover, the expression of aconitase was analyzed in the roots and juice to evaluate the implication of the different aconitase isoforms (Aco), which are derived from specific cellular compartments, in the different tissues. The involvement of the mitochondrial isoform (Aco2) was directly correlated with the acidity of the juice, whereas the cytosolic one (Aco3), which corresponds to the aconitase isolated during the microarray analysis, was found specifically in the roots.