Abbreviations: CG, Colletotrichum gloeosporioides; HR, hypersensitive response; HP, Hypericum perforatum; MeJ, methyl jasmonate; ROS, reactive oxygen species; SA, salicylic acid; 1,3,6,7-xanthone, xanthone with the 1,3,6,7 oxygenation pattern 49 Hypericum perforatum Cultures as a Tool to Study Plant Defence Mechanisms Against Anthracnose (Colletotrichum gloeosporioides) Luis F.R. Conceição • Gregory Franklin • Catarina Ribeiro • Alberto C.P. Dias * University of Minho, Department of Biology, 4710-057 Braga, Portugal Corresponding author: * acpdias@bio.uminho.pt Keywords: Colletotrichum gloeosporioides, HPLC-DAD-MS/MS, Hypericum perforatum, methyl jasmonate, oxidative burst, phenolics, reactive oxygen species, salicylic acid, xanthones ABSTRACT To elucidate the defence mechanisms of Hypericum perforatum L. against Colletotrichum gloeosporioides, we utilized cell suspension cultures. When primed with methyl jasmonate, H. perforatum cells showed a double oxidative burst upon Colletotrichum gloeosporioides elicitation typical of a hypersensitive response. Phenolic profile of the cells was modified upon various stimuli such as salicylic acid, MeJ and C. gloeosporioides. The possible importance of reactive oxygen species production and phenolics (xanthones) as components of defence mechanism of H. perforatum against biotic stress is discussed. 1. INTRODUCTION St. John’s wort (Hypericum perforatum; HP) is a medicinal plant widely used in folk medicine in several countries. Its properties have been mentioned since ancient times, by physicians like Dioscorides, Galen and Plinio. In the Medieval age, St. John’s oil was a very famous unguent utilised as a remedy for several ailments. The earlier use of St. John’s wort was mainly based on traditional knowledge. In the past decades, systematic approaches in phytochemical, pharmacological and clinical studies gave a better knowledge of the medicinal properties of this plant. Recently, the potential of HP extracts or compounds to act as antiviral (Miskovsky 2002), antitumour (Schempp et al. 2002), neuroprotective (Silva et al. 2005), and antioxidant (Silva et al. 2004) agents have been investigated. Hypericin has been considered as a promissory compound for the photodynamic therapy of cancer (Agostinis et al. 2002, Blank et al. 2004). Nowadays, the utilization of HP extracts for the treatment of depression has become prominent and some recent reviews focusing this issue can be pointed out (Erdelmeyer et al. 2000, Di Carlo et al. 2001, Butterweck 2003). Several studies have successfully demonstrated the clinical effectiveness of HP preparations in properly controlled clinical trials (Trautmann-Sponsela and Dienel 2004, Szegedi et al. 2005). As a consequence, the demand for St. John’s wort derived pharmaceuticals and dietary supplements have drastically increased in recent years (Wills et al. 2000). Presently, St. John’s wort is one of the best-selling herbal medicines worldwide with an important share in the phyto-pharmaceutical and OTC market of Europe and USA. The commercially available HP derived products include sophisticated phyto-pharmaceuticals, tinctures, teas, juices and oily macerates. Also, several brands of common food products, such as beverages and yoghurts, include St John’s wort extracts as additives. A major dilemma faced by consumers, manufacturers, and regulators is the high degree of variability in the product quality. Efforts are being made by the phyto- pharmaceutical industry in order to improve the quality and constancy of the supply of Hypericum products. Nowadays, most of the commercial products are derived from selected HP accessions cultivated in the field instead of wild plant collections as raw material. Today, HP cultivation covers several hundred hectares in Europe (Gaudin et al. 2003). Anthracnose caused by the fungus Colletotrichum gloeosporioides (CG) is a major problem in field cultivation of HP. This fungus is responsible for significant losses in HP plantations by lowering the yield and eventually modifying chemical composition of the biomass produced. Extensive efforts have been taken to obtain HP plants resistant to anthracnose but, until now, this goal has not been achieved. Understanding the biochemical and molecular basis of resistance is a prerequisite to obtain disease-resistant varieties, when natural resistance is inadequate. Nevertheless, little is known about the defence responses of HP against pathogen attack. Recently, differential accumulation of hyperforin and hypericin in HP plantlets was observed as a response to CG elicitation (Gibson and Sirvent 2002).