Downregulated d-limonene transgenic orange trees with increased linalool content showed resistance to Phyllosticta citricarpa

Terpene volatiles play an important role in the interactions among specialized pathogens and fruits. Citrus Black Spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with losses in different citrus-growing areas worldwide. The pathogen may infect the fruit for 20-24 weeks after petal fall but the typical hard spot symptoms appear when the fruit become almost mature, as consequence of fungal colonization and induction of cell lyses around essential oil cavities. D-limonene represents about 95% of total oil gland content in mature orange fruit. We have used here d-limonene synthase downregulated orange fruit generated via an antisense (AS) approach to investigate whether d-limonene decrease content in peel oil glands may affect fruit interaction with P. citricarpa compared to that of empty-vector (EV) controls. AS fruit showed enhanced resistance to the fungus compared to EV ones. Because of d-limonene decreased content, an over-accumulation of linalool and other monoterpene alcohols was found in AS compared to EV fruit. A global gene expression analysis at 2h and 8d after inoculation with P. citricarpa revealed activation of defence responses in AS fruit, via the upregulation of different PR genes, likely due to the enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, the main altered terpene volatile compounds including linalool at concentrations emulating those present in AS fruit, showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for developing new strategies to obtain resistance to fruit diseases. Microarray assays were performed with the flavedo of two independent antisense (AS1 and AS7) and empty-vector (EV) control transgenic ‘Navelina’ mature orange fruit. AS and EV samples were compared randomly on the same slide with three biological replicates for each genotype (AS1, AS7 and EV) at two time-points (2 hours and 8 days after inoculation with the fungus Phyllosticta citricarpa)