Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives Sophie BRUN a,b,c, * ,1 , Hugo MADRID d,1 , Bert GERRITS VAN DEN ENDE d , Birgitte ANDERSEN e , Carine MARINACH-PATRICE b,c , Dominique MAZIER a,b,c , G. Sybren DE HOOG d,f,g,h a AP-HP, Groupe Hospitalier Piti e-Salp^ etri ere, Service Parasitologie-Mycologie, 47-83 Boulevard de l’H^ opital, 75013 Paris, France b INSERM, UMRS 945, 91 bd de l’H^ opital, 75013 Paris, France c Universit e Pierre et Marie Curie-Paris 6, Paris, France d CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands e Center for Microbial Biotechnology (CMB), Department of Systems Biology, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark f Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 94248, 1090 GE Amsterdam, The Netherlands g Peking University Health Science Center, Research Center for Medical Mycology, Peking University First Hospital, 8 Xishiku Street, Xicheng District, Beijing, China h Sun Yat-sen Memorial Hospital, Sun-yat Sen University, 107 Yanjiang Xilu, Guangzhou, China article info Article history: Received 26 June 2012 Received in revised form 9 November 2012 Accepted 16 November 2012 Available online 12 December 2012 Corresponding Editor: Stephen W. Peterson Keywords: Alternaria MALDI-TOF Molecular identification Phylogeny Plant pathology Pleosporales abstract The genus Alternaria includes numerous phytopathogenic species, many of which are eco- nomically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the exis- tence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase ( gpd ) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. sol- ani, and the third included all strains of A. tomatophila, as well as all but one strain of A. sol- ani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens. ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. * Corresponding author. Present address: AP-HP, H^ opitaux Universitaires Paris Seine-Saint-Denis, H^ opital Avicenne, 125 rue de Stalin- grad, 93009 Bobigny Cedex, France. Tel.: þ33 (0) 1 48 95 56 51; fax: þ33 (0) 1 48 95 56 57. E-mail address: sophie.brun@avc.aphp.fr (S. Brun). 1 These authors equally contributed to the work. journal homepage: www.elsevier.com/locate/funbio fungal biology 117 (2013) 32 e40 1878-6146/$ e see front matter ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2012.11.003