ORIGINAL ARTICLE Host-derived media used as a predictor for low abundant, in planta metabolite production from necrotrophic fungi D.P. Overy 1,2 , J. Smedsgaard 2 , J.C. Frisvad 2 , R.K. Phipps 2 and U. Thrane 2 1 Institute of Biological Sciences, Edward Llwyd Building, University of Wales, Ceredigion, UK 2 Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark Introduction Taxa from the Penicillium ser. Corymbifera Frisvad are fungal pathogens responsible for blue mould storage rot of various flower and vegetable bulbs (Overy et al. 2005a). For successful colonization of a plant following epidermal penetration, the fungal pathogen is required to grow extensively within the plant without evoking host necrosis or be able to establish a colony within necrotic tissue. For biotrophic plant pathogens, tissue necrosis creates an uninhabitable environment for the unsuccessful infector, limiting growth by depriving the fungus of nutrients. An accumulation of toxic chemicals (i.e. phytoalexins and reactive oxygen species) have been demonstrated to occur in Allium cepa L. cell cultures in response to fungal elici- tors (Perkovskaya et al. 2004). Release of reactive oxygen species into the cytosol along with hydrolytic enzymes, promote the peroxidation and hydrolysis of essential membrane lipids in the plasmalemma and/or intracellular organelles, promoting leakage of cellular contents, rapid desiccation and cell death (Scandalios 1993; Mehdy 1994). These reactive agents would also have a similar effect upon the fungal membrane. In the case of blue mould rot caused by Penicillium ser. Corymbifera species, pathogen- icity is primarily necrotrophic; therefore, these pathogens possess an ability to detoxify and utilize necrotic tissue as a nutrient source. In this situation the pathogen objective is controlled necrosis of the plant cell during colony for- mation, detoxification of the necrotic environment and mycelial expansion into uninfected plant cells. Keywords Allium, Corymbifera, corymbiferan lactone, corymbiferone, medium-dependent production, secondary metabolism, stimulation. Correspondence D.P. Overy, Institute of Biological Sciences, Edward Llwyd Building, University of Wales, Aberystwyth, Ceredigion SY23 3DA, UK. E-mail: dao@aber.ac.uk 2005/1431: received 5 December 2005, revised 15 March 2006 and accepted 21 March 2006 doi:10.1111/j.1365-2672.2006.03037.x Abstract Aims: Penicillium ser. Corymbifera strains were assayed on a variety of media and from infected Allium cepa tissues to evaluate the stimulation and in planta prediction of low abundance metabolites. Methods and Results: Stimulated production of corymbiferones and the cor- ymbiferan lactones were observed for Penicillium albocoremium, Penicillium al- lii, Penicillium hirsutum, Penicillium hordei and Penicillium venetum strains cultured on tissue media. Target metabolites were sporadically detected from strains cultured on common laboratory media (CYA, MEA and YES). Up to a 376 times increase in corymbiferone and corymbiferan lactone production was observed when culture extracts from CYA and A. cepa agar were compared by high pressure liquid chromatography with ultraviolet and mass spectrometry (LC-UV-MS). The novel metabolite corymbiferone B was purified and struc- ture elucidated from a P. allii/A. cepa tissue medium extract. In planta expres- sion of low abundance, target metabolites were confirmed from infected A. cepa tissue extracts by LC-UV-MS. Conclusions: Secondary metabolite production was directly dependent and influenced by media conditions, resulting in the stimulated production of low abundance metabolites on host-derived media. Significance and Impact of the Study: The use of macerated host tissue media can be applied in vitro to predict in planta expression of low abundance metab- olites and aid in metabolite origin annotation during in planta metabolomic investigations at the host/pathogen interface. Journal of Applied Microbiology ISSN 1364-5072 1292 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1292–1300 ª 2006 The Authors