Molecular Identification of Fungi

Molecular Identification of Fungi Youssuf Gherbawy l Kerstin Voigt Editors Molecular Identification of Fungi Editors Prof. Dr. Youssuf Gherbawy Dr. Kerstin Voigt South Valley University University of Jena Faculty of Science School of Biology and Pharmacy Department of Botany Institute of Microbiology 83523 Qena, Egypt Neugasse 25 [email protected] 07743 Jena, Germany [email protected] ISBN 978-3-642-05041-1 e-ISBN 978-3-642-05042-8 DOI 10.1007/978-3-642-05042-8 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009938949 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: WMXDesign GmbH, Heidelberg, Germany, kindly supported by ‘leopardy.com’ Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Dedicated to Prof. Lajos Ferenczy (1930–2004) microbiologist, mycologist and member of the Hungarian Academy of Sciences, one of the most outstanding Hungarian biologists of the twentieth century Preface Fungi comprise a vast variety of microorganisms and are numerically among the most abundant eukaryotes on Earth’s biosphere. They enjoy great popularity in pharmaceutical, agricultural, and biotechnological applications. Recent advances in the decipherment of whole fungal genomes promise a continuation and accelera- tion of these trends. New techniques become available to facilitate the genetic manipulation of an increasing number of fungal organisms to satisfy the demand of industrial purposes. The increasing importance-driven search of novel detection techniques and new fungal species initiated the idea for a book about the molecular identification of fungi. The kingdom of the fungi (Mycota) appears as the sister group of the multicellular animals (Metazoa) as an independent, apparently monophyletic group within the domain Eukarya, equal in rank to green plants (Viridiplantae) and animals (Metazoa). Fungi are originally heterotrophic eukaryotic microorganisms harboring chitin in their cell walls and lacking plastids in their cytoplasm. Formerly, the oomycetes, slime moulds and plasmodiophorids were considered as fungi based on their ability to produce fungus-like hyphae or resting spores. Whereas the Oomycota are classified to the stramenopile algae (Chromista or Heterokonta), and the plasmodial and cellular slime moulds (Mycetozoa) belong to the Amoebozoa. The Plasmodiophoromycota are among the cercozoan Rhi- zaria closely related to the foraminifers. A three-protein phylogeny of the fungi and their allies confirms that the nucleariids, phagotrophic amoebae with filose pseudopods in soil and freshwater, may represent descendants of a common ancestor at the animal–fungal boundary (Fig. 1). The fungal kingdom encom- passes the Asco-, Basidio-, Glomero-, Zygo- and Chytridiomycota. The former four phyla are terrestrial fungi developing nonflagellated spores (aplanosporic), whereas the Chytridiomycota represent aquatic and zoosporic (planosporic) fungi, which split into three individual taxon groups, the aerobic Blastocladio- and Chytridiomycota sensu stricto and the anaerobic Neocallimastigomycota. The Zygomycota are among the most basal terrestrial fungi, which evolved in a paraphyletic manner. Hence, the phylum was divided into different subphyla, vii viii Preface Fig. 1 The evolution of the fungi and allied fungi-like microorganisms based on a concatenated neighbor-joining analysis using mean character differences as distance measure on 1,262 aligned amino acid characters comprising translation elongation factor 1 alpha, actin, and beta-tubulin (500, 323 and 439 characters, respectively) from 80 taxa. The prokaryotic elongation factor Tu, MreB (TM1544), and FtsZ (both homologous to actin and tubulin, respectively) from Thermotoga maritima were used as out group taxon representing the bacterial domain the Mucoro-, Kickxello-, Zoopago- and Entomophthoromycotina, whose phylogenetic relationships are not fully understood yet. In the phylogenetic tree shown in Fig. 1, the Entomophthoromycotina group together with the Ichthyosporea, a relationship, is not well supported by clade stability proportions. Fungi develop a wide diversity of morphological features, which are shared with many fungi-like microorganisms (Fig. 2), among those the white rust and downy mildew “fungi” (Fig. 2g) are obligate parasites of plants and develop fungus-like hyphae with haustoria (ht) in asexual and thick-walled, ornamented oospores (os) from fertilized oospheres after fusion of an oogonium (og) with an antheridium (at) during sexual reproduction (Fig. 3). The distribution of fungi among the various ecological niches of the biosphere seems to be infinite. Estimates suggest a total of 1.5 million fungal species, only less than a half has been merely described yet. This implies a backlog demand, which comes along with a rising importance of novel techniques for a rapid and Preface ix Fig. 2 The morphological diversity of fungi and fungi-like microorganisms. (a–f ): basidiomy- cetes (Agaricomycotina; Photos: M. Kirchmair); (g) oomycetes (Peronosporales; Photo: O. Spring); (h–j): multicellular conidia from imperfect stages of ascomycetes (Pezizomycotina); (k–s): zygomycetes (Mucoromycotina; Photos: K. Hoffmann, scanning electron microphoto- graphs o & q: M. Eckart & K. Hoffmann): (k, l, p, r, s) – different types of multispored sporangia, (m, n, o): different types of uni‐fewspored sporangiola; (t–x): reproductive structures (zoospor- angia) from anaerobic chytridiomycetes (Neocallimastigomycota; Photos: K. Fliegerova); (y, z): plasmodiophorids (Plasmodiophoromycota; Photos: S. Neuhauser & M. Kirchmair). x Preface Fig. 3 Cross-section of a leaf infected with Pustula tragopogonis (Peronosporales, Oomycota) causing white rust on sunflower. The microphotograph shows structures, which are typical for the sexual reproduction of oomycetes: ht – haustorium, ld – lipid droplet inside an oospore, os – oospore, og – oogonium, at – antheridium fused to an oogonium (Photo: A. Heller) unambiguous detection and identification of fungi to explore the fungal diversity as a coherent whole. Molecular techniques, particularly the technology of the poly- merase chain reaction, have revolutionized the molecular biology and the molecular diagnosis of fungi. The incorporation of molecular techniques into what has been traditionally considered as morphology-based taxonomy of fungi helps us in the differentiation of fungal species and varieties. Databases of genomes and genetic markers used as sources for molecular barcodes are being created and the fungal world is in progress to be unveiled with the help of bioinformatics tools. Genome projects provide evidence for ancient insertion elements, proviral or prophage remnants, and many other patches of unusual composition. Consequently, it becomes increasingly important to pinpoint genes, which characterize fungal organisms at different taxonomic levels without the necessity of previous cultivation. Unfortunately, the initiative of an excessive use of molecular barcoding has been hampered by a lack of sufficient and novel synapomorphic nucleotide < Fig. 2 (continued) (a) – basidiocarp of Schizophyllum commune,(b) – basidiocarp of Daedalea quercina,(c) – hymenophor from basidiocarp of Daedalea quercina,(d) – basidiocarp of Trametes sp., (e) – mycelium of Antrodia sp spreading over a trunk of a tree, (f ) – dry rot caused by Serpula lacrymans on timber, (g) –symptomatology from Plasmopara viticola, the causal agent of grape- vine downy mildew, (h)–Pestalotiopsis clavispora (Photo: C. Kesselboth), (i)–Bipolaris cf. sorokiniana (Photo: G. Newcombe), ( j)–Fusarium sp. (Photo: C. Kesselboth), (k)–Mucor indicus,(l)–Helicostylum elegans,(m)–Thamnidium elegans,(n)–Dichotomocladium sp., (o) – Dichotomocladium robustum,(p)–Absidia psychrophilia,(q) – zygospores from Lentamyces parricida,(r)–Mucor rouxii,(s)–Absidia cylindrospora,(t)–Caecomyces sp. isolated from sheep (lugol staining), (u)–Caecomyces sp. isolated from sheep, (v)–Neocallimastix frontalis (bisbenzimide staining of nuclei), (w)–Anaeromyces mucronatus isolated from cow (bisbenzimide staining of nuclei), (x)–Neocallimastix frontalis isolated from cow (lugol staining); (y) – thick walled resting spores from Sorosphaera veronicae,(z) – sporosori from Sorosphaera veronicae Preface xi characters and signature sequences. Moreover, high intraspecific variability of conventional molecular characters makes it difficult to identify species borders. However, DNA sequences and other genetic markers provide large amounts of data which are cultivation-independent and do not depend on physiological inconsis- tencies. Genetic markers constantly reflect the identification treasure

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