Fungal Genetics and Biology 42 (2005) 813–828 www.elsevier.com/locate/yfgbi 1087-1845/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2005.06.007 Technological advancement An oligonucleotide barcode for species identiWcation in Trichoderma and Hypocrea Irina S. Druzhinina a,¤ , Alexei G. Kopchinskiy a , Monika Komoj a , John Bissett b,1 , George Szakacs c , Christian P. Kubicek a a Division of Gene Technology and Applied Biochemistry (DGTAB), Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/1665, A-1060 Vienna, Austria b Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Center (ECORC), Central Experimental Farm, Ottawa, Ont., Canada K1A 0C6 c Department of Agricultural Chemical Technology, Technical University of Budapest, 1111 Budapest, Gellert ter 4, Hungary Received 14 February 2005; accepted 19 June 2005 Available online 9 September 2005 Abstract One of the biggest obstructions to studies on Trichoderma has been the incorrect and confused application of species names to isolates used in industry, biocontrol of plant pathogens and ecological surveys, thereby making the comparison of results question- able. Here we provide a convenient, on-line method for the quick molecular identiWcation of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) speciWcally allo- cated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rDNA repeat. The barcode was developed on the basis of 979 sequences of 88 vouchered species which displayed in total 135 ITS1 and 2 haplotypes. Oligonucleotide sequences which are constant in all known ITS1 and 2 of Hypocrea/Trichoderma but diVerent in closely related fungal genera, were used to deWne genus-speciWc hallmarks. The library of species-, clade- and genus-speciWc hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identiWcation program with the web interface located on www.isth.info. TrichOKey v. 1.0 identiWes 75 single species, 5 species pairs and 1 species triplet. VeriWcation of the DNA-barcode was done by a blind test on 53 unknown isolates of Trichoderma, collected in Central and South America. The obtained results were in a total agreement with phy- logenetic identiWcation based on tef1 (large intron), NCBI BLAST of vouchered records and postum morphological analysis. We conclude that oligonucleotide barcode is a powerful tool for the routine identiWcation of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.  2005 Elsevier Inc. All rights reserved. Keywords: Oligonucleotide barcode; Internal transcribed spacer; Trichoderma; Hypocrea; Molecular identiWcation; Phylogeny; PHP; MySQL; ISTH 1. Introduction The anamorphic fungal genus Trichoderma (Hypocre- ales, Ascomycota) contains fungi frequently found on decaying wood and in soil (Klein and Eveleigh, 1998; Sam- uels, 1996), where its individual species can comprise a major portion of the total fungal biomass (Danielson and Davey, 1973; Nelson, 1982; Widden and Abitbol, 1980). Some species of the genus are economically important pro- ducers of industrial enzymes (Trichoderma reesei , D Hypocrea jecorina; Kubicek and Penttilä, 1998) and antibiotics (Sivasithamparam and Ghisalberti, 1998), or are applied as biocontrol agents against plant pathogens (e.g., T. harzianum, D H. lixii; T. atroviride, D H. atroviri- dis; T. asperellum; Hjeljord and Tronsmo, 1998). More * Corresponding author. Fax: +4315880117299. E-mail address: druzhini@mail.zserv.tuwien.ac.at (I.S. Druzhinina). URLs: www.vt.tuwien.ac.at/druzhinina, www.isth.info (I.S. Druzhinina). 1 Contribution number 05-557 from the eastern Cereal and Oilseed Research Center.