Indian Phytopath. 67 (4) : 374-382 (2014)

RESEARCH ARTICLE

Secondary metabolite profiling of pathogenic 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 , confirmed placement of Alternaria in or absence of beak, septation and pattern of catenation the . 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 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 (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 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 — terms of quality and number of peaks, were obtained Am 2 Cotton Andhra Pradesh — when using 14 days-old cultures on PDA agar plates. Am 3 Cotton Tamil Nadu — Am 7 Cotton Karnataka — Then, main analysis was carried out using 14-day-old Am 9 Cotton Andhra Pradesh — cultures on PDA at 25 ± 1°C in the dark. 376 Indian Phytopathology 67 (4) : 374-382 (2014)

Fig. 1. Growth of Alternaria isolates on three different culture media after 14 days of inoculation at 25ºC in dark. a). PDA, (b) PCA, (C) DRYES; (1) A. solani, (2) A. porri, (3) A. alternata

Secondary metabolite extraction The samples were injected using a 20µl loop (Rheodyne, Rohnert Park, CA, USA). The column and guard column The extractions of secondary metabolites was carried were thermostatically controlled at 320C. The flow rate out using 14-day-old cultures by adopting method of was 0.6 ml/min and mobile phase consisted of 0.05% Andersen et al. (2005). Three agar plugs (6mm diam) trifluoroacetic acid (TFA) in water (solvent A) and 0.05% were cut from the centre of each colony and nine plugs TFA in acetanitrile (solvent B). The instrument was run were placed in a 2 ml screw-cap vial. The plugs were in a linear gradient mode. The gradient conditions were: extracted with 1ml ethyl acetate containing 1% formic 0-45 min, 20-80%B, 45-50 min, 100% B. The detection acid (v/v) ultrasonically for 60 min in water bath. The was monitored at 270 nm. extracts were transferred to a clean round bottom flask and evaporated to dryness in a rotary vacuum Matrix assisted laser desorption ionization–time of concentrator (HS-2001NS, Hahnshin Scientific Co., flight (MALDI-TOF) mass spectrometry USA), re-dissolved ultrasonically in 1 ml of HPLC methanol, and filtered through a 13 mm 0.22 µ RanDiscTM The matrix α-cyano-4-hydroxycinnamic acid (CHCA) was nylon Syringe filter into a clean 2ml vial prior to TLC, used for MALDI TOF MS analyses in order to detect the HPLC and MALDI-TOF MS analysis. maximum number of secondary metabolite profiles in Alternaria isolates (Chowdappa et al., 2013). The matrix Thin-Layer chromatography: Thin layer chromato- consisted of 10 mgml”1 saturated solutions of CHCA (70% graphy was performed by using method of Anderson et acetonitrile in Milli-Q grade water containing 0.1% al. (2008). Crude metabolite extract of Alternaria isolates trifluoroacetic acid). Equal volume of sample and matrix were spotted on TLC plates prepared from Silica Gel (1µl each) were mixed on a centrifuge tube cap and G254 by using chloroform and chromatographed with spotted on a MALDI plate (MTP 384 Ground steel Target mixture of toluene: ethyl acetate: formic acid (5:4:1; v:v) plate, Bruker). MS analyses were performed in 25KvA at 250C. The resulting bands were visualized under UV- Reflector mode in the range of 0-2000 mass-to-charge light at 365 nm. Developed TLC plates were air-dried ratio (m/z). All spectra were processed by the MALDI- overnight and calculated RT values. TOF MS Ultraflex TOF/TOF, Flex Analysis 2.0 (Bruker Daltonics Germany). Averaged profile spectra fulfilling High Performance Liquid Chromatography the quality criteria were collected from 600 laser shot cycles (N2 Laser, 337nm, 50Hz). For every sample, 4 to The HPLC analysis was carried out on a Shimadzu 10 averaged profile spectra were stored and used for Series LC-10A system (Shimadzu, Kyoto, Japan) analysis. Data analysis and for visual inspection of the consisting of a liquid chromatograph connected to a UV- mass spectra, Flex Analysis 2.0 software (Bruker Dal VIS detector (10A) and controlled by Shimadzu class tonik GmbH, B remen, Germany) was used. VP Workstation software. A base deactivated 150 mm long x 3mm, 3 µm C18 cartridges (cat. No: 4287, In order to assess the reproducibility of TLC, HPLC Phenomenex, CA, USA) was used for all the analyses. and MALDI-TOFMS identification, every isolate was Indian Phytopathology 67 (4) : 374-382 (2014) 377

Fig. 2. The MALDI-TOF mass spectra of Alternaria isolates obtained with (a) PDA, (b) PCA, (C) DRYES; (1) A. solani, (2) A. porri, (3) A. alternata

S1S212 3 4 5678910S2S1

0.85 0.85

0.71 0.71

Fig. 3. TLC profiles of extracts of Alternaria species grown on PDA for 14 days. Tracks: 1. A. solani, 2. A. porri, 3. A. alternata, 4. A. bracissicola, 5. A. brassicae, 6. A. sesame, 7. A. macrospora, 8. A. ricini, 9. A. burnsii, 10. A. carthami along with standards Alternariol (S1, RF 0.71) and Alternariol Mono Methyl Ether (S2, RF 0.85). Solvent system; toluene: ethylacetate: formic acid (5:4:1; v:v:v) viewed at 365 nm under UV light examined in triplicate, three different runs on three metabolites were detected in isolates belonging to different days from three different batches of culture. For different Alternaria species. For example A. solani share every situation, 4 different spots were loaded on the TLC the RF values of 0.444, 0.474 and 0.651which were not plates, HPLC and MALDI-TOF plate, giving a total of 12 present in closely related species like A. porri (0.111, profiles that were derived from each isolate. 0.133, 0.777, 0.903), A. macrospora (0.681). A. burnsii (0.607), which has been referred to as a special form of RESULTS A. alternata (0.192, 0.34), has specific metabolites that is distinct from A. alternata. The isolates from oil-yielding Culture media crops, namely, A. sesame (0.500), and A. ricini (0.933) had distinct metabolites. A. brassicae (0.207, 0.266, When the secondary metabolite profiling was done for 0.637, 0.755) and A. brassicicola (0.162) from cole crops the isolates belonging to 10 Alternaria species grown in also had characteristic metabolites. However, isolates three different media (PDA, PCA and DRYES), the within a species had identical banding patterns. number and intensity of the metabolites were considerable greater in PDA than those of PCA and Secondary metabolite profiling by High Performance DRYES media (Fig. 1,2). Thus, PDA was chosen as a Liquid Chromatography medium for analysis of secondary metabolite profiles. A total of 110 peaks were observed when HPLC profiles were examined (Fig. 4, Table 3). A unique metabolite Secondary metabolite profiling by Thin Layer peaks obtained for all the 10 species of Alternaria. A. Chromatography solani isolates had specific metabolite peaks at RT of A total of 40 bands were observed when TLC profiles 21.80 and 25.951, which were not present in closely were examined (Fig. 3, Table 2). Species-specific related species like A. porri (16.605), A. macrospora 378 Indian Phytopathology 67 (4) : 374-382 (2014)

(21.928). A. burnsii had specific metabolite peaks at Secondary metabolite profiling by MALDI-TOF MS 31.675, 36.105, and 41.05 and A. alternata had at 24.9. The A. sesami (7.00 and 20.575) and A. ricini (24.008, The MALDI-TOF spectra of the Alternaria isolates using 26.75 and 27.318) had distinct metabolite peaks. A. CHCA as matrix is shown in Fig. 5 and table 4. Patterns brassicae (12.567 and 39.15) and A. brassicicola (19.4) in the region of 0 to 2000 m/z were recorded and a total also had characteristic peaks. The isolates within a 66 different masses were observed. Alternaria species species exhibited identical HPLC profiles. exhibited characteristic spectral masses and the major

Table 2. TLC fingerprint of the mycelial extracts of Alternaria species

Alternaria spp. Retention factor

A. solani (OTA 22) 0.25 0.31 0.37 0.44* 0.47* 0.55 0.60 0.65* 0.81 0.96 A. porri (OOA 2) 0.07 0.11* 0.13* 0.28 0.37 0.41 0.48 0.53 0.55 0.70 0.77* 0.85 0.90* A. alternata (Aa4) 0.19 0.34* 0.53 0.58 0.61 0.68 0.78 0.80 0.85 0.96 A. brassicicola (OCA 1) 0.07 0.16* 0.42 0.74 0.81 0.91 0.96 A. brassicae (OCA 3) 0.07 0.20* 0.26* 0.37 0.42 0.51 0.55 0.60 0.63* 0.70 0.75* 0.80 0.85 A. sesami (OSA 12) 0.31 0.41 0.50* 0.51 0.55 0.58 0.61 0.71 0.85 0.96 A. macrospora (Am 1) 0.41 0.48 0.51 0.58 0.61 0.68* 0.71 0.78 0.85 0.96 A. ricini (OAR2) 0.31 0.41 0.48 0.55 0.61 0.71 0.78 0.85 0.93* 0.96 A. burnsii (OCuAb1) 0.41 0.60* 0.61 0.71 0.78 0.85 0.96 A. carthemi (OACr2) 0.31 0.37 0.41 0.48 0.51 0.55 0.58 0.71 0.78 0.85 0.96 Retention factor was calculated as the distance travelled by the compound divided by distance travelled by the solvent. *Denotes species-specific metabolite

Fig. 4. Secondary metabolite profiles of Alternaria spp. generated by HPLC Indian Phytopathology 67 (4) : 374-382 (2014) 379

Table 3. HPLC fingerprint of the mycelial extracts of Alternaria species

Alternaria spp. Retention time (min)

A. solani 11.95 13.06 13.40 13.92 15.40 16.26 18.00 19.12 21.80* 22.27 25.95* 28.56 34.49 36.64 (OTA22) A. porri 13.29 15.40 16.00 16.60* 17.89 19.12 25.00 26.02 (OOA2) A. alternata 11.15 12.70 13.06 13.92 15.28 18.00 19.12 21.53 22.27 24.90* (Aa4) A. brassicicola 7.22 11.15 12.31 13.06 13.92 15.28 16.90 18.34 19.40* 20.54 21.53 22.46 (OCA1) A. brassicae 7.22 8.47 9.82 11.15 12.56* 13.92 15.28 16.26 16.90 18.00 19.12 21.53 22.27 28.56 35.52 39.15* (OCA3) A. sesami 7.00* 8.21 10.62 11.30 12.31 13.92 14.92 16.26 19.12 20.57* 22.27 23.48 (OSA12) A. macrospora 5.45 9.82 11.30 12.70 13.40 18.00 19.12 21.92* 22.27 (Am1) A. ricini 5.45 9.82 12.01 13.40 16.00 18.00 22.90 24.00* 25.00 26.75* 27.31* (OAR2) A. burnsii 7.66 10.62 12.28 13.28 14.92 15.41 17.08 18.00 19.12 20.07 22.90 23.48 25.00 28.56 29.18 30.55 (OCuAb1) 31.67* 36.10* 38.21 39.15 41.05* A. carthami 7.52 8.21 10.28 11.15 11.95 12.70 13.06 13.92 14.70 15.28 16.00 18.34 19.12 20.54 22.27 23.48 (OACr2) *Denotes species-specific metabolite

Fig. 5. Secondary metabolite profiles of Alternaria spp. generated by MALDI-TOF MS 380 Indian Phytopathology 67 (4) : 374-382 (2014)

Table 4. Secondary metabolite masses of Alternaria species as revealed by MALDI-TOF MS

Alternaria Mass (m/z)s spp.

A.solani 420.19 454.35 477.31* 491.30* 459.55 553.79 585.58* 603.67 621.75* 663.22 681.93 703.66 719.61 (OTA 22) 743.53* 827.74* 908.30 926.25* 946.16 1135.45 1153.28 A.porri 454.18 542.19* 556.25* 584.33* 603.39 619.84 680.92 702.92* 718.95 907.56* 929.51 945.57 1152.45 (OOA2) A.alternata 454.53 476.44* 553.91 592.09 620.17 663.15 681.73 703.35 719.48 908.08 930.03 945.16* 1153.11 (Aa4) A.brassicicola 521.58 592.01 620.11 663.06 681.12 703.20 719.22 749.05* 784.46* 875.90* 907.88 630.87 945.89 (OCA1) 969.96* 1134.43* A.brassicae 454.79 482.65* 514.78* 532.92* 554.42 623.21* 664.04 682.19 704.17* 720.23* 908.99 930.79 945.76 (OCA3) A.sesami 420.44 442.42 554.18 664.40* 681.62 703.66 719.81 908.74 930.62 946.67 (OSA12) A.macrospora 442.20* 553.92 592.06 620.16 681.21 703.23 719.36 908.39 930.01 946.06 1135.5 (Am 1) A.ricini 420.26 454.52 553.91 592.12 620.20 663.23 681.28 703.32 719.39 908.03 930.05 947.08 1152.70 (OAR2) A.burnsii 454.66 529.75* 554.09 663.66 681.58 703.37 719.72 767.06* 908.60 930.46 1153.59 1547.50 (OCuAb1) A.carthami 454.64 521.78 554.06 681.42 703.46 719.56 778.84* 908.56 930.47 946.73 1135.61 1547.12 (OACr2) *Denotes species-specific metabolite characteristic and dominating peaks were situated Alternaria species are known to produce a wide range between the ranges of m/z 500 to 800 and m/z 800-1,000. of low molecular weight secondary metabolites or These two regions make the MALDI-TOF MS spectra mycotoxins belonging to different chemical groups quite characteristic for Alternaria species. Some of the including dibenzopyrones, tetramic acids, lactones, known metabolites such as alterporriol C (m/z 603.396) quinones and cyclic peptides (King and Schade, 1984; and alterporriol A (m/z 620.116) were identified, but most Chekowski and Visconti, 1992; Bottalico and Logrieco, of the species-specific metabolites are unknown. A 1998; Patriarca et al., 2007; Ostry, 2008) but are not unique MALDI-TOF MS profile was obtained for isolates necessary for growth or development (Fox and Howlett, of Alternaria. A. solani share the masses m/z 477.317, 2008). In this study, we have shown that all the 10 species 491.301, 585.588, 621.759, 743.531, 827.746 and of Alternaria exhibited characteristic species-specific 926.256 which were not present in closely related species metabolite profiles by TLC, HPLC and MALDI-TOF MS, like A. porri (m/z 542.196, 556.25, 584.338, 702.925 and which could be used as specific biomarkers for 907.566) and A. macrospora (m/z 442.202). A. burnsii differentiation and identification. The culture medium on had specific metabolite masses at m/z 529.757 and which the fungi cultured is critical for satisfactory 767.068. A. alternata share the masses at m/z 476.447 production of secondary metabolites (Frisvad et al., and 945.161. The A. sesami (m/z 664.408) and A. 2008). Media, like DRYES, malt extract agar, yeast extract sucrose agar, and PDA have used for metabolite carthami (m/z 778.84) had distinct metabolite mass. A. production in filamentous fungi (Thrane, 1993; Andersen brassicae (m/z 482.656, 514.781, 532.925, 623.212, et al., 2001, 2003).In this study, PDA has been found to 704.179 and 720.236) and A. brassicicola (m/z 749.057, be highly useful for better production of secondary 784.469, 875.906, 969.969 and 1134.438) also had metabolites, as revealed by MALDI TOF MS, compared characteristic masses. These masses serve as to PCA and DRYES. biomarkers for these species. However, isolates within a species had identical MALDI-TOF profiles. MALDI-TOF MS has emerged as a reliable tool for fast identification and classification of microorganisms. DISCUSSION This technique based on protein finger printing from intact cells or extracted has been shown a powerful tool for the The precise identification of Alternaria species is very identification of Alternaria (Brun et al., 2013; Chowdappa crucial for implementing effective disease management et al., 2013). The present study is the first strategies. The identification of Alternaria species should chemotaxonomic analysis of 10 species of Alternaria be simple and rapid as it has important implications in using MALDI-TOF MS. The most important mass peaks bio-security, disease management and disease were situated between the ranges of m/z 500 to 800 and resistance programmes. Chemical diversity as taxonomic m/z 800-1,000. These two regions make the MALDI-TOF tool has been utilized traditionally based on fatty acids, MS spectra quite characteristic for Alternaria species. proteins, carbohydrates, or secondary metabolites as a Most importantly, MALDI-TOF MS allows simple, reliable, supplement to traditional morphologically based and quick species identification, thus representing a valid (Tyrrell, 1969). Many plant pathogenic alternative to gene sequencing for species diagnosis of Indian Phytopathology 67 (4) : 374-382 (2014) 381

Alternaria. The MALDI-TOF MS analysis can be Andersen, B., Sorensen, J.L., Nielsen, K.F., Gerrits van den completed in a few minutes as opposed to two or more Ende, B. and de Hoog, G.S. (2009). A polyphasic days required for DNA sequence analysis. approach to the taxonomy of the Alternaria infectoria species-group. Fungal Gen. Bio. 46: 642-656. The use of secondary metabolite profiling, as Anderson, B., Hansen, M.E. and Smedsgaard, J. (2005). chemotaxonomic markers, has been used successfully Automated and unbiased image analysis as tool in in the classification and identification of species of large phenotypic classification of small spored Alternaria spp. ascomycete genera including Alternaria, Aspergillus, Phytopathology 95: 1021-1029. Fusarium, Hypoxylon, Penicillium, Stachybotrys and Anderson, B., Kroger, E. and Roberts, G.R. (2002). Chemical Xylaria and in a few genera of basidiomycetes (Frisvad and morphological segregation of Alternariaarborescens, et al., 2008). Very few studies have utilized metabolite A. infectoriaand A. tenuissimaspecies-groups. Mycol. Res. 106: 170-182. production for identification and classification of Alternaria species and these studies were limited to only Anderson, B. and Thrane, U. (1996). Differentiation of Alternaria few species of Alternaria such as A. dauci, A. porri, A. infectoria and Alternaria alternata based on morphology, metabolite profiles, and cultural characteristics. Can. J. solani and A. tomatophila (Andersen et al., 2008), A. Microbiol. 42: 685-689. alternata, A. Arborescens, A. gaisen, A. infectoria, A. longipes, A. tenuissima (Andersen and Thrane, 1996; Andrew, M., Peever, T. and Pryor, B.M. (2009). An expanded multilocus phylogeny does not resolve species within the Andersen et al., 2001, 2002, 2005). All these studies small-spored Alternaria species complex. Mycologia 101: utilized thin layer chromatography and ultra-violet light 95-109. or high performance liquid chromatography and diode Bottalico, A. and Logrieco, A. (1998). Toxigenic array detection (HPLC-DAD) for visualization of both Alternariaspecies of economic importance. In: Mycotoxins known and unknown secondary metabolites. Different in agriculture and food safety. Marcel Dekker, Sinha, K.K. species of plant pathogenic Alternaria are known to and Bhatnagar, D. (Eds.) Inc, New York. USA, 65-108. produce a large number of secondary metabolites or Brugger, E.M., Wagner, J., Schumacher, D.M., Koch, K., combinations of metabolites (Montemurro and Visconti, PodlechJ, Metzler, M. and Lehman, L. (2006). 1992; Rotem, 1994; Andersen and Thrane, 1996; Mutagenicity of the mycotoxin alternariol in cultured Andersen et al., 2001, 2002, 2005, 2008). A. alternata mammalian cells. Toxicology Letters 164: 221-230. has been found to produce altenuene, alternariol, Brun, S., Madrid, H., Gerrits van den Ende, B., Andersen, alternariol monomethyl ether, altertoxin I, tentoxin, and B., Marinach-Patrice, C. and Mazier D. (2013). Multilocus tenuazonic acid (Bottalico and Logrieco, 1998). A. dauci, phylogeny and MALDI-TOF analysis of the plant A. porri and A. solani have common metabolite, zinniol pathogenic species Alternaria dauci and relatives. Fungal (Montemurro and Visconti, 1992; Horiuchi et al., 2003). Bio. 117: 32-40. Alterporriols, altersolanols, macrosporin, and tentoxin Chelkowski, J. and Visconti, A. (1992). Alternaria. Biology, have been shown to occur in A. porri and A. solani plant diseases and metabolites. Elsevier, Amsterdam, the (Suemitsu et al., 1990a, b, 1992; Montemurro and Netherlands. 573. Visconti, 1992). Alternariol monomethyl ether has been Chen, H.Y. and Chen, Y. C. (2005). Characterization of intact reported from A. dauci, erythroglaucin and other Penicillium spores by matrix-assisted laser desorption/ anthraquinones from A. porri and alternaric acid, ionization mass spectrometry. Rapid Comm. Mass Spectr. 19: 3564-3568. alternariol, solanapyrones and zinnolide from A. solani (Montemurro and Visconti, 1992). The present study Chou, H.H. and Wu, W.S. (2002). Phylogenetic analysis of reveals species-specific metabolites of all the 10 species internal transcribed spacer regions of the genus Alternaria and the significance of filament-beaked conidia. Mycol. of Alternaria, but due to lack of standards, many peaks Res. 106: 164-169. in the chromatogram are marked as unknown compounds. As supported by HPLC and MALDI TOF MS, Chowdappa, P., Jyothi Lakshmi, M. and Madhura, S. (2013). 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RAPD Received for publication: July 25, 2014 fragment pattern analysis and morphological segregation Accepted for publication: October 16, 2014