An oligonucleotide barcode for species identification in Trichoderma and Hypocrea.

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 questionable. Here we provide a convenient, on-line method for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) specifically allocated 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 different in closely related fungal genera, were used to define genus-specific hallmarks. The library of species-, clade- and genus-specific hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identification program with the web interface located on . TrichOKey v. 1.0 identifies 75 single species, 5 species pairs and 1 species triplet. Verification 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 phylogenetic identification 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 identification of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.

[1]  C. Kubicek,et al.  Phylogeny of the genus trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster , 1998, Fungal genetics and biology : FG & B.

[2]  Irina S Druzhinina,et al.  Trichoderma brevicompactum sp. nov. , 2004, Mycologia.

[3]  J. Bissett.  A revision of the genus Trichoderma. II: Infrageneric classification , 1991 .

[4]  Jeremy R. deWaard,et al.  Biological identifications through DNA barcodes , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[5]  L. Castlebury,et al.  Hypocrea/Trichoderma: species with conidiophore elongations and green conidia , 2003, Mycologia.

[6]  R. Nazar,et al.  Interdependence in the processing of ribosomal RNAs in Schizosaccharomyces pombe. , 1997, Journal of molecular biology.

[7]  G. Samuels.  Trichoderma: a review of biology and systematics of the genus , 1996 .

[8]  C. Gradinger,et al.  Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum and T. polysporum and their newly discovered relatives , 2004, Mycologia.

[9]  O. Petrini,et al.  Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Stalpers,et al.  CBS course of mycology. , 1975 .

[11]  A. Tronsmo,et al.  Trichoderma and Gliocladium in biological control An overview , 1998 .

[12]  A. Pandey,et al.  Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation , 2004, Applied biochemistry and biotechnology.

[13]  J. Bissett.  A revision of the genus Trichoderma. III. Section Pachybasium , 1991 .

[14]  Irina S Druzhinina,et al.  Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters? , 2005, Journal of Zhejiang University. Science. B.

[15]  L. Kredics,et al.  Clinical importance of the genus Trichoderma. A review. , 2003, Acta microbiologica et immunologica Hungarica.

[16]  B. Rannala,et al.  Bayesian phylogenetic inference using DNA sequences: a Markov Chain Monte Carlo Method. , 1997, Molecular biology and evolution.

[17]  E. Buckler,et al.  The evolution of ribosomal DNA: divergent paralogues and phylogenetic implications. , 1997, Genetics.

[18]  S. Rehner,et al.  Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences , 1994 .

[19]  Mark Blaxter,et al.  Molecular barcodes for soil nematode identification , 2002, Molecular ecology.

[20]  L. Kredics,et al.  Clinical importance of the genus Trichoderma , 2003 .

[21]  D. Geiser,et al.  Multilocus phylogenetic structure within the Trichoderma harzianum/Hypocrea lixii complex. , 2003, Molecular phylogenetics and evolution.

[22]  Irina S Druzhinina,et al.  Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. , 2003, Microbiological research.

[23]  Dam,et al.  Molecular Systematics of the Eastern Fence Lizard ( Sceloporus undulatus ): A Comparison of Parsimony, Likelihood, and Bayesian Approaches , 2002 .

[24]  Robert L. Mach,et al.  Confusion Abounds Over Identities of Trichoderma Biocontrol Isolates , 2001 .

[25]  R. Bayoumi,et al.  Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates. , 1993, Molecular and biochemical parasitology.

[26]  J. Strauss,et al.  Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species , 1997 .

[27]  P. Widden,et al.  Seasonality of Trichoderma species in a spruce-forest soil. , 1980 .

[28]  C. Kubicek,et al.  Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya , 2000 .

[29]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[30]  K. O’Donnell,et al.  Molecular systematics and phylogeography of Gibberella fujikuroi species complex , 1998 .

[31]  Hugh B. Nicholas,et al.  GeneDoc: a tool for editing and annotating multiple sequence alignments , 1997 .

[32]  G. Samuels,et al.  Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): species with green ascospores. , 2003 .

[33]  Izabela Makalowska,et al.  FUSARIUM-ID v. 1.0: A DNA Sequence Database for Identifying Fusarium , 2004, European Journal of Plant Pathology.

[34]  K. O’Donnell.  Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris) , 1992, Current Genetics.

[35]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[36]  C. Davey,et al.  The abundance of Trichoderma propagules and the distribution of species in forest soils , 1973 .

[37]  A. Lalev,et al.  Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1. , 2000, Journal of molecular biology.

[38]  C. Kubicek,et al.  Phylogeny and evolution of the genus Trichoderma: a multigene approach , 2002 .

[39]  Sonia N Humphris,et al.  The effects of volatile microbial secondary metabolites on protein synthesis in Serpula lacrymans. , 2002, FEMS microbiology letters.

[40]  D. Geiser.  Practical Molecular Taxonomy of Fungi , 2004 .

[41]  A. Lalev,et al.  Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA. , 1998, Journal of molecular biology.

[42]  A. Rodrigo,et al.  Examination of Trichoderma phylogenies derived from ribosomal DNA sequence data , 2000 .

[43]  E. E. Nelson.  OCCURRENCE OF TRICHODERMA IN A DOUGLAS-FIR SOIL , 1982 .

[44]  C. Kubicek,et al.  REVISION OF TRICHODERMA SECT. LONGIBRACHIATUM INCLUDING RELATED TELEOMORPHS BASED ON ANALYSIS OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES , 1997 .

[45]  Irina S Druzhinina,et al.  Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic clade in Hypocrea/Trichoderma. , 2005, Mycologia.

[46]  U. Thrane,et al.  Identification of Trichoderma strains by image analysis of HPLC chromatograms. , 2001, FEMS microbiology letters.

[47]  H. Evans,et al.  Taxonomy and biocontrol potential of a new species of Trichoderma from the Amazon basin of South America , 2004, Mycological Progress.

[48]  Irina S Druzhinina,et al.  Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates. , 2003, Fungal genetics and biology : FG & B.

[49]  Irina S Druzhinina,et al.  TrichoBLAST: a multilocus database for Trichoderma and Hypocrea identifications. , 2005, Mycological research.

[50]  D. Hibbett,et al.  Phylogenetic species recognition and species concepts in fungi. , 2000, Fungal genetics and biology : FG & B.

[51]  P. Hebert,et al.  Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[52]  M. Penttilä,et al.  Regulation of production of plant polysaccharide degrading enzymes by Trichoderma , 1998 .

[53]  Thomas D. Bruns,et al.  Fungal Molecular Systematics , 1991 .

[54]  T. Hseu,et al.  Molecular cloning and sequence analysis of the cellobiohydrolase i gene fromTrichoderma koningii G-39 , 2005, Current Microbiology.

[55]  Sayed,et al.  Koninginin G, a new metabolite from trichoderma aureoviride , 1999, Journal of natural products.

[56]  F. Dromer,et al.  Trichoderma pseudokoningii as a cause of fatal infection in a bone marrow transplant recipient. , 1995, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[57]  D. Hibbett,et al.  Phylogenetic Species Recognition and Species , 2000 .

[58]  B. Michot,et al.  Ribosomal internal transcribed spacer 2 (ITS2) exhibits a common core of secondary structure in vertebrates and yeast. , 1999, Nucleic acids research.

[59]  F. Martin,et al.  Ribosomal DNA internal transcribed spacers to estimate the proportion of Pisolithus tinctorius and Eucalyptus RNAs in ectomycorrhiza , 1997, Applied and environmental microbiology.

[60]  Irina S Druzhinina,et al.  Trichoderma populations from alkaline agricultural soil in the Nile valley, Egypt, consist of only two species , 2004, Mycological Progress.

[61]  B. Rannala,et al.  Probability distribution of molecular evolutionary trees: A new method of phylogenetic inference , 1996, Journal of Molecular Evolution.