Preface Fig.3 Cro section of a leaf h Pustula les oomy cota) causing white rust on er.The for the sexual reproductiono oogonium.at-antheridium ed 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 technigues into what has beer traditionally considered as mo ophology-fhe us in the of fungal species and varieties.Data s of ger omes rs used as sources for m ecula 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 cultiva- tion.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 Schizophyllun m commune.(b)basidi carp of Daedale na.(c) a lacrymans on timber.（g ane vine downy mildew,(h)-Pestalotiopsi clavispora(Photo:C.Kesselboth).(i)- ewcombe,JD m -Dichotomocladium robustum,(p)-Absidia psychrophilia, zveospores from parricida.(r)-Mucor rouxii.(s)-Absidia cylin ospo ra.(t)-Caecomyces sp.isolated from sheep es sp.Isorated non of nuclei).()islated from cow():(y)-thick walled resting spores from Sorosphaera veronicae,(z)-sporosori from Sorosphaera veronicae
unambiguous detection and identification of fungi to explore the fungal diversity as a coherent whole. Molecular techniques, particularly the technology of the polymerase 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 grapevine 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) – Caecomycessp. 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 < 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) x Preface
Preface 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 hidden in the genetic information and allow to control the degree of resolution by choosing the appropriate genes. his book,we highlight the advances of the past decade,both in and in the und ng of approac entific tion an on and ation of fungi using m ecular mark ers and compa those with classical procedures traditionally used for species designation.The limitations in the availability of type material,reference strains,and reference nucleotide sequences set boundaries in the molecular identification.For example the image displaying multicellular,melanin-pigmented conidia (size:90 um) from strain CID1670(Fig.2i),which was kindly provided by George Newcombe (University of Idaho.Center for Research on Invasive Species and Small Popula riata na e fo der of this book.The was rcc as an en dophy comycete from perennial herb Cen the aste rea stoeb b uted by conventional ITS barcoding to the pleosporalean genus Drechslera and in a narrower sense to Bipolaris sorokiniana.Since species of Bipolaris had never beer reported from any species of Centaurea in earlier reports,neither its effects on its host nor the final taxonomic delimitation are known.Nucleotide sequences of additional genes and a more in-depth phylogenetic study may even suggest that this strain was a new species.Therefore.it would make sense to distinguish between refined identification f fung only found in ex ceptional bi es in orde to explore ne sp en nytes and high-thro denti died fungi in orde orily naive th e ind of fungi hypothesis about the plant invasion-mediated progression of novel phytopathogens will be discussed in the first chapter.The second and third chapter concerns with the diagnostics and the challenge to identify"fungus-like plant pathogens from the oomycetes and the plasmodiophorids.respectively.The fourth chapter leads over the applications of molecular markers and DNA sequences in the identification of funga】 pathogens in grain legu es and ce eals followed by various aspects of ive det of Eu spp. and Mo ho seolina, ze a r com crops omic plants.Du the detection of ochr nic fur penicilli.and oth er postharvest pathogens like Mucor and Rhizopus is elucidate The molecular identification of wood rotting and endophytic fungi as well as anaerobic rumen fungi finish the first part on plant pathological and environmenta biological aspects.The second part deals with human pathological and clinical aspects.The introduction gives a contribution about new approaches in fungal DNA preparation from whole blood following multiplex PCR detection.Novel techni ques in the depetion of the background host DNA n favour r of enrichment of the
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 inconsistencies. Genetic markers constantly reflect the identification treasure hidden in the genetic information and allow to control the degree of resolution by choosing the appropriate genes. In this book, we highlight the advances of the past decade, both in methodology and in the understanding of genomic organization and approach problems of the identification and differentiation of fungi using molecular markers and compare those with classical procedures traditionally used for species designation. The limitations in the availability of type material, reference strains, and reference nucleotide sequences set boundaries in the molecular identification. For example, the image displaying multicellular, melanin-pigmented conidia (size: 90 mm) from strain CID1670 (Fig. 2i), which was kindly provided by George Newcombe (University of Idaho, Center for Research on Invasive Species and Small Populations, Moscow, ID, USA), may serve as an appropriate cautionary note for readers of this book. The strain was recovered as an endophytic ascomycete from the asterid perennial herb Centaurea stoebe (spotted knapweed). The fungus could be attributed by conventional ITS barcoding to the pleosporalean genus Drechslera and in a narrower sense to Bipolaris sorokiniana. Since species of Bipolaris had never been reported from any species of Centaurea in earlier reports, neither its effects on its host nor the final taxonomic delimitation are known. Nucleotide sequences of additional genes and a more in-depth phylogenetic study may even suggest that this strain was a new species. Therefore, it would make sense to distinguish between refined identification of fungi uncommonly found in exceptional biotopes in order to explore new species, e.g., as endophytes, and high-throughput molecular identi- fication of well-studied fungi in order to serve the needs of industrial application. The role of fungi as pathogens of evolutionarily naive plants including a hypothesis about the plant invasion-mediated progression of novel phytopathogens will be discussed in the first chapter. The second and third chapter concerns with the diagnostics and the challenge to identify “fungus-like” plant pathogens from the oomycetes and the plasmodiophorids, respectively. The fourth chapter leads over the applications of molecular markers and DNA sequences in the identification of fungal pathogens in grain legumes and cereals followed by various aspects of qualitative and quantitative detection of Fusarium spp. and Macrophomina phaseolina, pathogenic on maize and other corn crops or economic plants. During the course of the book, the detection of ochratoxigenic fungi, mainly aspergilli and penicilli, and other postharvest pathogens like Mucor and Rhizopus is elucidated. The molecular identification of wood rotting and endophytic fungi as well as anaerobic rumen fungi finish the first part on plant pathological and environmental biological aspects. The second part deals with human pathological and clinical aspects. The introduction gives a contribution about new approaches in fungal DNA preparation from whole blood following multiplex PCR detection. Novel techniques in the depletion of the background host DNA in favour of enrichment of the Preface xi
xii Preface fungal contaminant DNA following different modifications of PCR approaches represent powerful tools in the detection of a wide variety of human pathogenic fungi causing sepsis and other life-threatening diseases that result from excessive host rest onses to fungal infections.The survey continues with conventional strate gies for the molec detection of Malass dermatophyte unistic f un caus tive agents of deep mycoses as wel myco Ochroconis gallopava infect amplification method (LAMP).The book closes with reviews about prospects and perspectives of molecular markers for the identification of Absidia-like fungi and other zygomycetes. The editors thank all contributors for their valuable reviews and comments, which were crucial for the accomplishment of this book.Furthermore,we express our gratitude to all authors who contributed figures and images for the cover and the sadding cover of dlypreinofthis boo.The January 2010 Youssuf Gherbawy Kerstin Voigt
fungal contaminant DNA following different modifications of PCR approaches represent powerful tools in the detection of a wide variety of human pathogenic fungi causing sepsis and other life-threatening diseases that result from excessive host responses to fungal infections. The survey continues with conventional strategies for the molecular detection of Malassezia, dermatophytes, opportunistic fungi, and causative agents of deep mycoses as well as paracoccidioidomycosis and Ochroconis gallopava infection via a novel tool, the loop-mediated isothermal amplification method (LAMP). The book closes with reviews about prospects and perspectives of molecular markers for the identification of Absidia-like fungi and other zygomycetes. The editors thank all contributors for their valuable reviews and comments, which were crucial for the accomplishment of this book. Furthermore, we express our gratitude to all authors who contributed figures and images for the cover and miscellaneous parts adding a great deal to the illustration of this book. The cover of the book was kindly supported by “leography.com.” January 2010 Youssuf Gherbawy Kerstin Voigt xii Preface
Contents Part I Plant Pathological and Environmental Biological Aspects 1 Fungal Pathogens of Plants in the George Newcombe and Frank M.Dugan 2 Molecular Techniques for Classification and Diagnosis of Plant Pathogenic Oomycota35 Otmar Spring and Marco Thines 3 Plasmodiophorids:The Challenge to Understand Soil-Borne, Obligate Biotrophs with a Multiphasic Life Cycle .51 Sigrid Neuhauser,Simon Bulman,and Martin Kirchmair 4 Application s of Molecular Markers and DNA Sequences in Identifying Fungal Pathogens of Cool Season Grain Legumes . .79 Evans N.Njambere,Renuka N.Attanayake,and Weidong Chen 5 Quantitative Detection of Fungi by Molecular Methods: A Case Study on Fusarium 93 Kurt Brunner and Robert L.Mach 6 DNA-Based Tools for the Detection of Fusarium spp. Pathogenic on Maize107 Ivan Visentin,Danila Valentino,Francesca Cardinale, and Giacomo Tamietti 7 Molecular Detection and Identification of Fusarium oxysporum.131 Ratul Saikia and Narendra Kadoo
Contents Part I Plant Pathological and Environmental Biological Aspects 1 Fungal Pathogens of Plants in the Homogocene . 3 George Newcombe and Frank M. Dugan 2 Molecular Techniques for Classification and Diagnosis of Plant Pathogenic Oomycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Otmar Spring and Marco Thines 3 Plasmodiophorids: The Challenge to Understand Soil-Borne, Obligate Biotrophs with a Multiphasic Life Cycle . . . . . . . . . . . . . . . . . . . . 51 Sigrid Neuhauser, Simon Bulman, and Martin Kirchmair 4 Applications of Molecular Markers and DNA Sequences in Identifying Fungal Pathogens of Cool Season Grain Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Evans N. Njambere, Renuka N. Attanayake, and Weidong Chen 5 Quantitative Detection of Fungi by Molecular Methods: A Case Study on Fusarium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Kurt Brunner and Robert L. Mach 6 DNA-Based Tools for the Detection of Fusarium spp. Pathogenic on Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Ivan Visentin, Danila Valentino, Francesca Cardinale, and Giacomo Tamietti 7 Molecular Detection and Identification of Fusarium oxysporum . 131 Ratul Saikia and Narendra Kadoo xiii
xiv Contents 8 Molecular Chemotyping of Fusarium graminearum, F.culmorum,and F.cerealis Isolates From Finland 159 9 Molecular Characterization and Diagnosis of Macrophomina phaseolina:A Charcoal Rot Fungus .179 Bandamaravuri Kishore Babu,Ratul Saikia,and Dilip K Arora 10 Molecular Diagnosis of Ochratoxigenic Fungi.195 Daniele Sartori,Marta Hiromi Taniwaki,Beatriz Iamanaka, and Maria Helena Pelegrinelli Fungaro 11 Molecular r Barcoding of Microscopic Fungi with Emphasis alean .213 and Kerstin Hoffmann 12 Advances in Detection and Identification of Wood Rotting Fungi in Timber and Standing Trees .251 Giovanni Nicolotti.Paolo Gonthier,and Fabio Guglielmo 13 Molecular Div Liang-Dong Guo versity and Identificaion f Endophytic 14 Molecular Identification of Anaerobic Rumen Fungi.297 Martin Eckart,Katerina Fliegerova,Kerstin Hoffmann, and Kerstin Voigt Part II Human Pathological and Clinical Aspects aches in Fungal DNA Pr ep aration fre m Whole d Su athogen Detecti Via Multiplex PCR .317 16 Classification of Yeasts of the Genus Malassezia by Sequencing of the ITS and D1/D2 Regions of DNA .337 Lidia Perez-Perez,Manuel Pereiro,and Jaime Toribio 17 DNA-Based Detection of Human Pathogenic Fungi: Dermatophytes,Opportunists,and Causative Agents of Deep Lo Putignani.Silvia D'Arezzo,Maria Grazia Paglia. .357 and Paolo Visca
8 Molecular Chemotyping of Fusarium graminearum, F. culmorum, and F. cerealis Isolates From Finland and Russia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Tapani Yli-Mattila and Tatiana Gagkaeva 9 Molecular Characterization and Diagnosis of Macrophomina phaseolina: A Charcoal Rot Fungus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Bandamaravuri Kishore Babu, Ratul Saikia, and Dilip K Arora 10 Molecular Diagnosis of Ochratoxigenic Fungi . . . . . . . . . . . . . . . . . . . . . . . 195 Daniele Sartori, Marta Hiromi Taniwaki, Beatriz Iamanaka, and Maria Helena Pelegrinelli Fungaro 11 Molecular Barcoding of Microscopic Fungi with Emphasis on the Mucoralean Genera Mucor and Rhizopus . . . . . . . . . . . . . . . . . . . . . 213 Youssuf Gherbawy, Claudia Kesselboth, Hesham Elhariry, and Kerstin Hoffmann 12 Advances in Detection and Identification of Wood Rotting Fungi in Timber and Standing Trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Giovanni Nicolotti, Paolo Gonthier, and Fabio Guglielmo 13 Molecular Diversity and Identification of Endophytic Fungi . . . . . . . 277 Liang-Dong Guo 14 Molecular Identification of Anaerobic Rumen Fungi . . . . . . . . . . . . . . . . 297 Martin Eckart, Katerina Fliegerova´, Kerstin Hoffmann, and Kerstin Voigt Part II Human Pathological and Clinical Aspects 15 New Approaches in Fungal DNA Preparation from Whole Blood and Subsequent Pathogen Detection Via Multiplex PCR . . . . 317 Roland P. H. Schmitz, Raimund Eck, and Marc Lehmann 16 Classification of Yeasts of the Genus Malassezia by Sequencing of the ITS and D1/D2 Regions of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Lidia Pe´rez-Pe´rez, Manuel Pereiro, and Jaime Toribio 17 DNA-Based Detection of Human Pathogenic Fungi: Dermatophytes, Opportunists, and Causative Agents of Deep Mycoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 Lorenza Putignani, Silvia D’Arezzo, Maria Grazia Paglia, and Paolo Visca xiv Contents