Indian Phytopath. 67 (4) : 374-382 (2014) RESEARCH ARTICLE Secondary metabolite profiling of plant pathogenic Alternaria species by matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry M. JYOTHI LAKSHMI1, P. CHOWDAPPA1* and RIAZ MAHMOOD2 1Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore 560 089, Karnataka, India 2Department 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 Alternaria Nees is a cosmopolitan, anamorphic is essential to understand the relationship between hyphomyceteous genus encompassing many species of species and behaviour and to formulate effective disease economic importance, including saprophytes, plant management strategies. pathogens, animal pathogens and as producers of mycotoxins and allergens (Simmons, 2007; Zhang et al., Traditionally, Alternaria species have been identified 2009). A multi-locus phylogenetic analysis of the based on conidium shape, size, ornamentation, presence Dothideomycetes, confirmed placement of Alternaria in or absence of beak, septation and pattern of catenation the Pleosporales. As saprotrophs, they can cause (Neergaard, 1945; Joly, 1964; Ellis, 1971, 1976; deterioration of food products and animal feeds though Simmons, 1992). Simmons (2007), in his monograph on production of mycotoxins and other biological active Alternaria, accepted 276 species and distinguished these compounds (King and Schade, 1984; Bottalico and species based on three-dimensional sporulation patterns Logrieco, 1998). Many species are plant pathogens that and conidial morphology. Identification of Alternaria cause considerable economic losses every year in a wide species based on morphological criteria is always range of agriculturally important plants including cereals, confusing and unreliable. Traditional identification of vegetables, oil yielding, seed spice, ornamentals and fruit Alternaria species based on morphological characters crops worldwide (Thomma, 2003; Rotem, 1994; Ciancio has limitations by sterility in cultures or formation of and Mukerji, 2007). In addition, several species cause species-complexes of morphologically similar taxa (Brun post harvest diseases that cause spoilage of agricultural et al., 2013). Various molecular methods have been used output and contamination of food and animal feed by to identify or segregate Alternaria species, but with toxins or allergens (Montemuro and Visconti, 1992; variable results. Molecular approaches based on RAPD Rotem, 1994). They are the producers of powerful toxic (Cooke et al., 1998; Weir et al., 1998; Roberts et al., secondary metabolites (Ostry, 2008) that have been 2000) and sequence analyses of ITS, mt SSU, implicated in the development of cancer in mammals glyceraldehyde 3-phosphate dehydrogenase (gpd) (Brugger et al., 2006). As human pathogens, they incite sequences, mt LSU, ß-tubulin, endo-polygalacturonase diseases in immune compromised patients (Anaissie et (endo-PG) and anonymous opening reading frames al., 1989, Rossmann et al., 1996). Moreover, Alternaria (Kusaba and Tsuge, 1995; Chou and Wu, 2002; de Hoog spores are one of the most common and potent airborne and Horre, 2002; Pryor and Bigelow, 2003; Pryor and allergens (Wilken-Jensen and Gravesen, 1984; Karlsson- Gilbertson, 2000; Peever et al., 2004; Andew et al., 2009); Borga et al., 1989). A precise and correct identification and IGS restriction mapping (Hong et al., 2005) showed species- group specificity. However, relationships among *Corresponding author: [email protected] species within the same species group were not clearly Indian Phytopathology 67 (4) : 374-382 (2014) 375 resolved because of insufficient genetic information Table 1. Alternaria isolates used in the present study contained within the loci chosen for genetic analysis. To Fungal Host State NCBI develop a more robust technique for discrimination of isolates accession No Alternaria, additional analyses other than these genetic loci are required. Alternaria solani OTA 22 Tomato Karnataka HQ270459 In addition to morphology and molecular analysis, OTA 66 Tomato Uttara Pradesh JF796063 secondary metabolite profiling (small organic compounds OTA 73 Tomato Karnataka JF491204 that are not directly useful for growth, development, or OTA 78 Tomato Karnataka JF491209 reproduction of an organism) has been utilized to OTA 81 Tomato Karnataka JF491212 differentiate morphologically similar species within a Alternaria porri genera in Ascomycota (Smedsgaard and Frisvad, 1996; OOA 2 Onion Karnataka JF710495 Frisvad, 1987; Frisvad et al., 2008). This chemotaxonomic OOA 6 Onion Karnataka JF710488 approach has also found useful in distinguishing species, OOA 8 Onion Karnataka JF710494 species-groups, and closely-related taxa in Alternaria OOA 12 Onion Karnataka JF710497 (Andersen and Thrane, 1996; Andersen et al., 2001, OOA 13 Onion Karnataka JF710491 2002, 2005, 2008), even in isolates that have failed to Alternaria brassicicola OCA 7 Cauliflower Karnataka JF710522 sporulate in cultures (Andersen et al., 2009). These OCA 8 Cauliflower Meghalaya JF710521 studies showed that secondary metabolite profiling can OCA 11 Cauliflower Sikkim JF710517 be a reliable tool for characterization and differentiation OCA 10 Cauliflower Sikkim JF710518 of plant pathogenic fungi. Matrix-assisted laser OCA 12 Cauliflower Assam JF710519 desorption/ionization time-of-flight mass spectrometry Alternaria brassicae (MALDI TOF MS) has been successfully used as a useful OCA 2 Rape seed New Delhi JF710515 diagnostic tool alternative to available immunodiagnostic OCA 3 Rape seed Himachal Pradesh — and molecular methods for the objective identification of OCA 4 Rape seed Assam JF710516 fungi (Chen and Chen, 2005; Schmidt and Kallow, 2005; OCA 5 Rape seed Meghalaya — Qian et al., 2008; Brun et al., 2013; Chowdappa et al., OCA 15 Rape seed Delhi — 2013). The method was used to measure metabolites Alternaria sesame on the surface of growing cultures and extracted OSA 11 Sesame Karnataka JF710582 secondary metabolites. The objective of the present study OSA 12 Sesame Andhra Pradesh JF710583 was to assess whether profiling of secondary metabolites OSA 09 Sesame Tamil Nadu JF710584 through TLC, HPLC and MALDI-TOF MS could be used OSA 08 Sesame Karnataka JF710585 as chemotaxonomic markers for rapid identification and OSA18 Sesame Karnataka — classification of Alternaria species isolated from Alternaria ricini vegetable, fruits, oil-yielding and seed spice crops. OAR 1 Castor Andhra Pradesh JF710543 OAR 2 Castor Karnataka JF710544 OAR 3 Castor Tamil Nadu — MATERIALS AND METHODS OAR 4 Castor Andhra Pradesh — OAR 5 Castor Andhra Pradesh — Fungal isolates Alternaria carthami OAcr 1 Safflower Andhra Pradesh JF 710541 Fifty morphologically and genetically well characterized OAcr2 Safflower Andhra Pradesh JF710542 isolates belong to 10 species of Alternaria collected from OAcr3 Safflower Karnataka — different geographical locations in India were used for OAcr 4 Safflower Maharashtra — analysis. Table 1 provides details of the isolates, OAcr 5 Safflower Karnataka — geographical origin, host source and GenBank accession Alternaria alternata numbers for ITS region of r DNA. Isolates were OTA29 Tomato Karnataka JF710500 maintained on slopes of potato dextrose agar (PDA) at OTA 11 Tomato Andhra Pradesh HQ270456 4°C. OTA48 Tomato Jammu and Kshmir JF796070 OTA56 Tomato Himachal Pradesh JF796071 Culture conditions OTA 65 Tomato Uttara Pradesh JF796077 Alternaria burnsii Results of preliminary MALDI-TOF MS analyses of OCuAb1 Cumin Rajasthan — Alternaria isolates grown on potato dextrose agar (PDA), OCuAb4 Cumin Gujarat — potato carrot agar (PCA) and Dichloran rose bengal yeast OCuAb3 Cumin Rajasthan — extract sucrose agar (DRYES) (Fig. 1) at two different OCuAb4 Cumin Rajasthan — temperatures (25 and 30°C), at varying incubation OCuAb5 Cumin Rajasthan — periods (from 10 to 20 days) and two photoperiods (light Alternaria macrospora and dark), indicated that the best spectral profiles, in Am 1 Cotton Karnataka —