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Improving ITS sequence data for identification of plant pathogenic fungi

Summary

Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

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Acknowledgments

RHN acknowledges financial support from Swedish Research Council of Environment, Agricultural Sciences, and Spatial Planning (FORMAS, 215-2011-498). ArA acknowledges financial support from European Funds through COMPETE and by National Funds through the Portuguese Foundation for Science and Technology (FCT) within projects PTDC/AGR-FOR/3807/2012 - FCOMP-01-0124-FEDER-027979 and PEst-C/MAR/LA0017/2013. SB is supported by National Science Foundation Grant DBI 1046115. The Austrian Centre of Industrial Biotechnology (ACIB) contribution (BH) was supported by FFG, BMWFJ, BMVIT, ZIT, Zukunftsstiftung Tirol, and Land Steiermark within the Austrian COMET program FFG Grant 824186. Financial support to JP was partially provided by the Polish Ministry of Science and Higher Education (MNiSW), grant no. NN303_548839. OLP acknowledges financial support from FAPEMIG and CNPq. TMP was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute through the Biomonitoring 2.0 project (OGI-050). The GenBank staff is acknowledged for helpful discussions and data sharing. The NEFOM network is acknowledged for infrastructural support. The authors have no conflict of interests to report.

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Correspondence to Kessy Abarenkov.

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Anders Bjørnsgard Aas, Siti A. Alias, Artur Alves, Cajsa Lisa Anderson, Alexandre Antonelli, A. Elizabeth Arnold, Barbara Bahnmann, Mohammad Bahram, Johan Bengtsson-Palme, Anna Berlin, Sara Branco, Putarak Chomnunti, Asha Dissanayake, Rein Drenkhan, Hanna Friberg, Tobias Guldberg Frøslev, Bettina Halwachs, Martin Hartmann, Beatrice Henricot, Ruvishika Jayawardena, Ari Jumpponen, Håvard Kauserud, Sonja Koskela, Tomasz Kulik, Kare Liimatainen, Björn D. Lindahl, Daniel Lindner, Jian-Kui Liu, Sajeewa Maharachchikumbura, Dimuthu Manamgoda, Svante Martinsson, Maria Alice Neves, Tuula Niskanen, Stephan Nylinder, Olinto Liparini Pereira, Danilo Batista Pinho, Teresita M. Porter, Valentin Queloz, Taavi Riit, Marisol Sánchez-García, Filipe de Sousa, Emil Stefańczyk, Mariusz Tadych, Susumu Takamatsu, Qing Tian, Dhanushka Udayanga, Martin Unterseher, Zheng Wang, Saowanee Wikee and Jy Yan contributed equally to the project and are listed in alphabetical order.

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Supplementary Item 1

The 39 journals screened for ITS sequences from type specimens/ex-type cultures of fungi described (or re-typified) during the period 2000–2012 (variously and as applicable). The time coverage of the journal is indicated. Only papers whose title suggested that one or more new species were described (or that one or more species were re-typified) were examined. In some few cases, individual articles were not clear on whether a sequence actually came from the type specimen or not, and we hope that ongoing efforts to standardize what data to provide (and how to provide them) when describing new species in a molecular context will be successful. (PDF 32 kb)

Supplementary Item 2

Screenshots showing examples of how the user can interact with, and modify, the public fungal ITS sequences in UNITE. The processes illustrated include taxonomic re-annotation, addition of ecological and geographical metadata, annotation of basic sequence properties, and association of sequences with voucher specimens/cultures. Structured INSDC searches as well as browsing among species hypotheses are also shown. A manual for the PlutoF sequence management environment is available at http://unite.ut.ee/temp/plutof2/files/PlutoF_2.5_Manual_small.pdf (PDF 2956 kb)

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Nilsson, R.H., Hyde, K.D., Pawłowska, J. et al. Improving ITS sequence data for identification of plant pathogenic fungi. Fungal Diversity 67, 11–19 (2014). https://doi.org/10.1007/s13225-014-0291-8

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Keywords

  • Phytopathogenic fungi
  • Molecular identification
  • ITS
  • Taxonomy
  • Annotation