Engineering d-limonene synthase down-regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence.

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop 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 have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d-Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d-limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d-limonene content, an over-accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up-regulation of different pathogenesis-related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.