Root specific elicitation and antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum

Rosmarinic acid (RA) is a multifunctional caffeic acid ester present in sweet basil (Ocimum basilicum L.). Untransformed normal roots of O. basilicum harbored the maximum titers (0.98% g fresh weight basis) of RA compared to leaves and shoots. Hairy root cultures of O. basilicum transformed with Agrobacterium rhizogenes (ATCC-15834) showed three-fold increases in growth and RA production compared to the untransformed normal roots. Upon elicitation with fungal cell wall elicitors (CWE) from Phytophthora cinnamoni, the production of RA was enhanced 2.67-fold compared with the untreated control. Roots were induced to exude RA by fungal in situ challenge with Pythium ultimum, to our knowledge an undocumented observation. Absence of RA in the root exudates of unchallenged root cultures proves that RA under normal circumstances is not exuded. RA showed antimicrobial activity against a range of soil-borne microorganisms, with its most deleterious effects against Pseudomonas aeruginosa, an opportunistic soil bacterium and human pathogen. Confocal and scanning imaging of Aspergillus niger hyphae treated with RA (250 μM) exhibited damaged cytoskeletons with broken interseptas and convoluted cell surfaces resulting in a multinucleated stage compared to the untreated control. Both strains of P. aeruginosa tested, PAO1 and PA14, showed increased spatial division and condensation of DNA upon RA treatment compared to the untreated control. Our findings suggest that in nature RA is a constitutive antimicrobial compound that may be released into the surrounding rhizosphere upon microbe challenge.