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An Integrated Taxonomic Study of Fusarium Langsethiae, Fusarium Poae and Fusarium Sporotrichioides Based on the Use of Composite Datasets

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An Integrated Taxonomic Study of Fusarium Langsethiae, Fusarium Poae and Fusarium Sporotrichioides Based on the Use of Composite Datasets International Journal of Food Microbiology 95 (2004) 341–349 www.elsevier.com/locate/ijfoodmicro An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets H. Schmidta, A. Adlerb, A. Holst-Jensenc, S.S. Klemsdald, A. Logriecoe, R.L. Machf, H.I. Nirenbergg, U. Thraneh, M. Torpc, R.F. Vogela, T. Yli-Mattilai, L. Niessena,* a Technische Universitat-Mu¨nchen, Lehrstuhl fu¨r Technische Mikrobiologie, Weihenstephaner Steig 16, D-85350 Freising, Germany b Bundesamt fu¨r Agrarbiologie, Wieningerstrabe 8, A-4040 Linz, Austria c National Veterinary Institute, Section of Food and Feed Microbiology, P.O. Box 8156 Dep., N-0033 Oslo, Norway d The Norwegian Crop Research Institute, Plant Protection Centre, Høgskoleveien 7, N-1432 A˚ s, Norway e Institute of Science of Food Production, National Research Council, CNR, Viale Enaudi 51, I-70125 Bari, Italy f Institute for Chemical Engineering, Microbial Biochemistry and Gene Technology Group, Technical University of Vienna, Getreidemarkt 9/166, A-1060 Vienna, Austria g Institute of Plant Virology, Microbiology, and Biological Safety, Federal Biological Research Centre for Agriculture and Forestry, Ko¨nigin-Luise-Str. 19, D-14195 Berlin, Germany h Technical University of Denmark, BioCentrum-DTU, Søltofts Plads 221, DK-2800 Kgs. Lyngby, Denmark i Laboratory of Plant Physiology and Molecular Biology, Department of Biology, University of Turku, FIN-20014 Turku, Finland Abstract An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the h-tubulin and translation elongation factor-1 alpha (EF-1a) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1a and h-tubulin sequence—as well as chromatography—and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent. D 2004 Elsevier B.V. All rights reserved. Keywords: Fusarium; Fungi; Polyphasic; Taxonomy; Numerical; Composite dataset 1. Introduction * Corresponding author. Tel.: +49-8161-715496; fax: +49- 8161-713327. For the elucidation of taxonomic problems, E-mail address: [email protected] (L. Niessen). mycologists are still used to relying on the exami- 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.12.012 342 H. Schmidt et al. / International Journal of Food Microbiology 95 (2004) 341–349 nation of a more or less limited set of characters. On form a group separated from F. poae. Also, PCR- the one hand, morphological and other phenotypical based methods for the rapid detection and distinction observations are still essential for the valid descrip- of the species were developed (Konstantinova and tion of a fungal species, whereas on the other hand, Yli-Mattila, 2004; Mach et al., 2004; Niessen et al., molecular data such as DNA sequences and finger- 2004). Here we present a collaborative study aimed prints are about to dominate fungal systematics. at clarification of the taxonomic position of F. However, only by combining approaches can the langsethiae within the Fusarium section Sporotri- true relationship among different fungal groups be chiella. The objective of the present study was to fully elucidated, and multidisciplinary studies are the consider all available data, morphological, chromato- logical answer to meet this challenge. It has been graphic, and molecular (both fingerprints and nucle- discussed how mycologists could effectively com- otide sequences). By choosing this strategy, we hope bine these different data to produce reliable classifi- to contribute to the understanding of the true rela- cation and identification regimes (Seifert et al., tionships between the different species and put 2000). The prerequisites for this task are now met forward a clearer perception of the whole fungus due to the rapid development in computer technolo- (Kendrick, 1979). gy and software engineering and the availability of diverse datasets. Because of their importance in agriculture and 2. Materials and methods human health, Fusarium species belong to the best studied group of fungal organisms. Their impact is 2.1. Fungal strains mainly based on the ability to cause a number of plant diseases and to synthesise a variety of myco- Information on the examined strains can be toxins which can contaminate food and feed. The retrieved from Torp and Adler (2004).Inthe trichothecenes are considered to be the most hazard- current study, only those strains for which a ous compounds produced by Fusarium species. Type complete set of data comprising all analytical A trichothecenes, especially T-2 toxin and HT-2 methods applied were selected. In particular, the toxin, are among the most toxic substances regularly number of strains included in the EF-1a dataset detected in cereal samples (Marasas et al., 1984).A (Knutsen et al., 2004) was considerably lower than recently discovered species, Fusarium langsethiae is the number of strains included in each of the other believed to be an important producer of these two datasets rendering the number of strains studied toxins in cereals. This species resembles Fusarium quite low. poae in several morphological features, but the latter species has only occasionally been shown to produce 2.2. Sequence data T-2 and HT-2 toxins. Thus, F. langsethiae has some affinity to Fusarium sporotrichioides and F. poae The DNA sequences of parts of the IGS, com- (Torp and Langseth, 1999; Torp and Nirenberg, plete sequences of the ITS region, and partial 2004). However, when toxin patterns are compared, sequences of the h-tubulin gene used are those the species seems to be more similar to F. sporo- described by Yli-Mattila et al. (2004). The partial trichioides than to F. poae, but segregated into a EF-1a sequences of the examined strains are those distinct cluster by statistical analysis of full spectra determined by Knutsen et al. (2004). An alignment chromatographic data (Thrane et al., 2004). Phylo- of the sequences was obtained by subjecting the genetic analyses of combined ribosomal internal sequences to BioNumerics software Version 2.50 transcribed spacer (ITS) regions, and partial sequen- (Applied Maths, Sint-Martens-Latems, Belgium). ces of the intergenic spacer (IGS) region and the h- For alignment and similarity calculation, no conver- tubulin (Yli-Mattila et al., 2003) and EF-1a genes sion costs were used and no gap penalty was (Knutsen et al., 2003), as well as cluster analysis of assigned. The similarity between the sequences AFLP fingerprints (Schmidt et al., 2004), strongly was calculated using the correction of Jukes and suggest that F. sporotrichioides and F. langsethiae Cantor (1969). H. Schmidt et al. / International Journal of Food Microbiology 95 (2004) 341–349 343 2.3. Amplified fragment length polymorphism From these combined data, a consensus matrix was calculated by taking the mean of all pair wise dis- AFLP data were obtained and processed as de- tances between single isolates over all separate data- scribed by Schmidt et al. (2004). Resemblance be- sets. No weights were assigned to the individual tween the fingerprints was calculated using the experiments. To visualise the obtained consensus Pearson correlation. matrix a dendrogram was calculated by the unweight- ed pair-group method using arithmetic means 2.4. Chromatographic data (UPGMA, Sokal and Michener, 1958). From the same matrix, a nonmetric multidimensional scaling (MDS, The chromatographic data on secondary metabo- Shepard, 1962) graph was computed. To check the lites were those obtained and processed by Thrane et robustness of the dendrogram and groups, the cophe- al. (2004) with the exception of data on beauvericin netic correlation and error flags for each node of the and enniatins which were not considered in the current dendrogram (standard deviation) were determined. study. The cubed correlation coefficient calculated by Pairwise resemblance between the data matrices was the COWTool software (Nielsen et al., 1998) was computed using the Pearson correlation. All calcula- directly imported into the BioNumerics software. tions were carried out on complete datasets using the BioNumerics software. 2.5. Other phenotypical data Selected morphological data and growth character- 3. Results istics were recorded as described by Torp and Niren- berg (2004) into a binary character table (legal states 0 A set of strains was chosen for which data of each and 1). The characters used are throughout the text individual character set were available. From these referred to as phenotypical characters and described data a consensus matrix was calculated and trans- and summarized
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