Indian Phytopath. 67 (4) : 374-382 (2014) Secondary metabolite profiling of plant pathogenic Alternaria species by matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry M. JYOTHI LAKSHMI 1 , P. CHOWDAPPA 1 * and RIAZ MAHMOOD 2 1 Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore 560 089, Karnataka, India 2 Department of Post-Graduate Studies and Research in Biotechnology, Kuvempu University, Jnanasahyadri, Shankaraghatta 577 451, Karnataka, India ABSTRACT: Profiling of secondary metabolite production (both known and unknown metabolites) on standardized culture media has proven to be useful for classification and identification of certain morphologically similar species of Alternaria. In this study, secondary metabolite profiling of 50 fungal isolates belonging to 10 plant pathogenic Alternaria species such as A. solani, A. porri, A. brassicicola, A. brassicae, A. sesame, A. alternata, A. macrospora, A. ricini, A. carthami and A. brunsii isolated from vegetable, oil yielding and seed spice crops were examined. Secondary metabolites were extracted from 14 day old cultures, grown on potato dextrose agar, with ethyl acetate containing formic acid. After extraction, the secondary metabolite profiles of all fungal isolates were analyzed using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. These analyses indicated characteristic ‘species-specific metabolite finger prints’. Thus, chemotaxonomic approach is a simple and rapid technique to determine the chemical diversity of the different Alternaria species and to identify species- specific metabolites that could be adopted as chemotaxonomic markers in species identification. This study can be integrated in a polyphasic approach. Key words: Alternaria, HPLC, MALDI –TOF MS, secondary metabolites, TLC RESEARCH ARTICLE *Corresponding author: pallem22@gmail.com Alternaria Nees is a cosmopolitan, anamorphic hyphomyceteous genus encompassing many species of economic importance, including saprophytes, plant pathogens, animal pathogens and as producers of mycotoxins and allergens (Simmons, 2007; Zhang et al., 2009). A multi-locus phylogenetic analysis of the Dothideomycetes, confirmed placement of Alternaria in the Pleosporales. As saprotrophs, they can cause deterioration of food products and animal feeds though production of mycotoxins and other biological active compounds (King and Schade, 1984; Bottalico and Logrieco, 1998). Many species are plant pathogens that cause considerable economic losses every year in a wide range of agriculturally important plants including cereals, vegetables, oil yielding, seed spice, ornamentals and fruit crops worldwide (Thomma, 2003; Rotem, 1994; Ciancio and Mukerji, 2007). In addition, several species cause post harvest diseases that cause spoilage of agricultural output and contamination of food and animal feed by toxins or allergens (Montemuro and Visconti, 1992; Rotem, 1994). They are the producers of powerful toxic secondary metabolites (Ostry, 2008) that have been implicated in the development of cancer in mammals (Brugger et al., 2006). As human pathogens, they incite diseases in immune compromised patients (Anaissie et al., 1989, Rossmann et al., 1996). Moreover, Alternaria spores are one of the most common and potent airborne allergens (Wilken-Jensen and Gravesen, 1984; Karlsson- Borga et al., 1989). A precise and correct identification is essential to understand the relationship between species and behaviour and to formulate effective disease management strategies. Traditionally, Alternaria species have been identified based on conidium shape, size, ornamentation, presence or absence of beak, septation and pattern of catenation (Neergaard, 1945; Joly, 1964; Ellis, 1971, 1976; Simmons, 1992). Simmons (2007), in his monograph on Alternaria, accepted 276 species and distinguished these species based on three-dimensional sporulation patterns and conidial morphology. Identification of Alternaria species based on morphological criteria is always confusing and unreliable. Traditional identification of Alternaria species based on morphological characters has limitations by sterility in cultures or formation of species-complexes of morphologically similar taxa (Brun et al., 2013). Various molecular methods have been used to identify or segregate Alternaria species, but with variable results. Molecular approaches based on RAPD (Cooke et al., 1998; Weir et al., 1998; Roberts et al., 2000) and sequence analyses of ITS, mt SSU, glyceraldehyde 3-phosphate dehydrogenase (gpd) sequences, mt LSU, ß-tubulin, endo-polygalacturonase (endo-PG) and anonymous opening reading frames (Kusaba and Tsuge, 1995; Chou and Wu, 2002; de Hoog and Horre, 2002; Pryor and Bigelow, 2003; Pryor and Gilbertson, 2000; Peever et al., 2004; Andew et al., 2009); and IGS restriction mapping (Hong et al., 2005) showed species- group specificity. However, relationships among species within the same species group were not clearly