Mycotoxigenic Fungi

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Series Editor John M. Walker

School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

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Mycotoxigenic Fungi

Methods and Protocols

Edited by

Antonio Moretti

Institute of Sciences of Food Production, National Research Council, Bari, Italy

Antonia Susca

Institute of Sciences of Food Production,

National Research Council, Bari, Italy

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ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology

ISBN 978-1-4939-6705-6 ISBN 978-1-4939-6707-0 (eBook) DOI 10.1007/978-1-4939-6707-0

Library of Congress Control Number: 2016958563

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The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

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Editors

Antonio Moretti

Institute of Sciences of Food Production National Research Council

Bari, Italy

Antonia Susca

Institute of Sciences of Food Production National Research Council

Bari, Italy

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Mycotoxins are toxic fungal metabolites that cause severe health problems in humans and animals after exposure to contaminated food and feed, having a broad range of toxic effects, including carcinogenicity, neurotoxicity, and reproductive and developmental toxicity. The United Nations Commission on Sustainable Development approved in 1996 a work program on indicators of sustainable development that included mycotoxins in food as one of the components related to protection and promotion of human health.

From that program, the concern due to mycotoxin contamination of agro-food crops is in continuous growth worldwide since the level of their occurrence in final products is still high and the consequent impact on human and animal health significant. Moreover, the economic costs for the whole agricultural sector can be enormous, even in developed countries as shown by the losses in the United States alone that can be around $5 billion per annum. Different approaches have been used in mycotoxin research through years.

First, implications of mycotoxins in humans were investigated in medicine; later agro- ecological aspects and the fundamental mystery of the biological role for production of secondary metabolites are still analyzed. Regulatory limits, imposed in about 80 countries to minimize human and animal exposure to mycotoxins, also have tremendous economic impact on international trading and must be developed using science-based risk assess- ments, such as expensive analytical methods used to detect mycotoxins eventually occurring in food and feed. On the other hand, decontamination strategies for mycotoxins in foods and feeds include treatments that could show inappropriate results because nutritional and organoleptic benefits could be deteriorated by the process. Alternatively, programs of mycotoxin prevention and control could be applied through evaluating the contamination of foodstuffs by the related mycotoxin-producing fungi and therefore screening the poten- tial mycotoxin risk associated.

Because mycotoxins are produced within certain groups of fungi, the understanding of their population biology, speciation, phylogeny, and evolution is a key aspect for establish- ing well-addressed mycotoxin reduction programs. This perspective is of fundamental importance to the correct identification of the mycotoxigenic fungi, since each species/

genus can have a species-specific mycotoxin profile which would change the health risks associated with each fungal species. The previous use of comparative morphology has been quickly replaced in the last two decades by comparative DNA analyses that provide a more objective interpretation of data. Advances in molecular biology techniques and the ability to sequence DNA at very low cost contributed to the development of alternative techniques to assess possible occurrence of mycotoxins in foods and feeds based on fungal genetic variability in conserved functional genes or regions of taxonomical interest, or by focusing on the mycotoxigenic genes and their expression. The possibility of using a highly standardized, rapid, and practical PCR-based protocol that can be easily used both by researchers and by nonexperts for practical uses is currently available for some species/

mycotoxins and hereby proposed. Further progress in transcriptomics, proteomics, and metabolomics will continue to advance the understanding of fungal secondary metabolism

Preface

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and provide insight into possible actions to reduce mycotoxin contamination of crop plants and the food/feed by-products.

Finally, we do hope that readers will find the chapters of Mycotoxigenic Fungi: Methods and Protocols helpful and informative for their own work, and we deeply thank all authors for their enthusiastic and effective work that made the preparation of this book possible.

Bari, Italy Antonio Moretti

Antonia Susca Preface

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1 Mycotoxins: An Underhand Food Problem. . . 3 Antonio Moretti, Antonio F. Logrieco, and Antonia Susca

2 Alternaria Species and Their Associated Mycotoxins. . . 13 Virginia Elena Fernández Pinto and Andrea Patriarca

3 Aspergillus Species and Their Associated Mycotoxins . . . 33 Giancarlo Perrone and Antonia Gallo

4 Fusarium Species and Their Associated Mycotoxins. . . 51 Gary P. Munkvold

5 Penicillium Species and Their Associated Mycotoxins . . . 107 Giancarlo Perrone and Antonia Susca

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6 Targeting Conserved Genes in Alternaria Species . . . 123 Miguel Ángel Pavón, Inés María López-Calleja, Isabel González,

Rosario Martín, and Teresa García

7 Targeting Conserved Genes in Aspergillus Species . . . 131 Sándor Kocsubé and János Varga

8 Targeting Conserved Genes in Fusarium Species. . . 141 Jéssica Gil-Serna, Belén Patiño, Miguel Jurado, Salvador Mirete,

Covadonga Vázquez, and M. Teresa González-Jaén

9 Targeting Conserved Genes in Penicillium Species . . . 149 Stephen W. Peterson

10 Targeting Aflatoxin Biosynthetic Genes . . . 159 Ali Y. Srour, Ahmad M. Fakhoury, and Robert L. Brown

11 Targeting Trichothecene Biosynthetic Genes . . . 173 Songhong Wei, Theo van der Lee, Els Verstappen, Marga van Gent,

and Cees Waalwijk

12 Targeting Ochratoxin Biosynthetic Genes . . . 191 Antonia Gallo and Giancarlo Perrone

13 Targeting Fumonisin Biosynthetic Genes. . . 201 Robert H. Proctor and Martha M. Vaughan

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14 Targeting Other Mycotoxin Biosynthetic Genes . . . 215 María J. Andrade, Mar Rodríguez, Juan J. Córdoba,

and Alicia Rodríguez

15 Evaluating Aflatoxin Gene Expression in Aspergillus Section Flavi . . . 237 Paula Cristina Azevedo Rodrigues, Jéssica Gil-Serna,

and M. Teresa González-Jaén

16 Evaluating Fumonisin Gene Expression in Fusarium verticillioides. . . 249 Valeria Scala, Ivan Visentin, and Francesca Cardinale

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17 Multiplex Detection of Aspergillus Species. . . 261 Pedro Martínez-Culebras, María Victoria Selma, and Rosa Aznar

18 Multiplex Detection of Fusarium Species . . . 269 Tapani Yli-Mattila, Siddaiah Chandra Nayaka, Mudili Venkataramana,

and Emre Yörük

19 Multiplex Detection of Toxigenic Penicillium Species . . . 293 Alicia Rodríguez, Juan J. Córdoba, Mar Rodríguez,

and María J. Andrade

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20 PCR-RFLP for Aspergillus Species. . . 313 Ali Atoui and André El Khoury

21 PCR ITS-RFLP for Penicillium Species and Other Genera . . . 321 Sandrine Rousseaux and Michèle Guilloux-Bénatier

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22 Identification of Ochratoxin A-Producing Black Aspergilli from Grapes

Using Loop-Mediated Isothermal Amplification (LAMP) Assays . . . 337 Michelangelo Storari and Giovanni A.L. Broggini

23 Detection of Transcriptionally Active Mycotoxin Gene Clusters:

DNA Microarray. . . 345 Tamás Emri, Anna Zalka, and István Pócsi

24 Mycotoxins: A Fungal Genomics Perspective. . . 367 Daren W. Brown and Scott E. Baker

Index . . . 381 Contents

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María j. andrade • Faculty of Veterinary Science, Food Hygiene and Safety, Meat and Meat Products Research Institute, University of Extremadura, Cáceres, Spain

alI atouI • Lebanese Atomic Energy Commission-CNRS, Riad El Solh, Beirut, Lebanon;

Laboratory of Microbiology, Department of Natural Sciences and Earth, Faculty of Sciences I, Lebanese University, Hadath Campus, Beirut, Lebanon

roSa aznar • Department of Biotechnology, Institute of Agrochemistry and Food Technology, IATA-CSIC, Valencia, Spain; Department of Microbiology and Ecology and Spanish Type Culture Collection (CECT), University of Valencia, Valencia, Spain Scott e. Baker • US Department of Energy, Environmental Molecular Sciences

Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA

gIoVannI a.l. BroggInI • Institute for Plant Production Sciences, Agroscope, Wädenswil, Switzerland

daren w. Brown • Mycotoxin Prevention and Applied Microbiology Research, US Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research (USDA–ARS–NCAUR), Peoria, IL, USA roBert l. Brown • Southern Regional Research Center, SDA-ARS New Orleans,

LA, USA

FranceSca cardInale • Department of Agricultural, Forest and Food Sciences, University of Turin, Grugliasco, Italy

juan j. córdoBa • Faculty of Veterinary Science, Food Hygiene and Safety, Meat and Meat Products Research Institute, University of Extremadura, Cáceres, Spain

andré el khoury • Centre D’Analyses Et De Recherches, Faculté des Sciences, Université Saint-Joseph, Beyrouth, Lebanon

taMáS eMrI • Faculty of Science and Technology, Department of Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary

ahMad M. Fakhoury • Department of Plant Soil and Agriculture Systems, Southern Illinois University, Carbondale, IL, USA

antonIa gallo • Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Lecce, Italy

tereSa garcía • Facultad de Veterinaria, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, Madrid, Spain MargaVan gent • Biointeractions and Plant Health, Wageningen UR, Wageningen, The

Netherlands

jéSSIca gIl-Serna • Facultad de Ciencias Biologicas, Departamento de Microbiologia, Universidad Complutense de Madrid, Jose Antonio Novais, Madrid, Spain

ISaBel gonzález • Facultad de Veterinaria, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, Madrid, Spain

M. tereSa gonzález-jaén • Facultad de Ciencias Biologicas, Departamento de Genetica, Universidad Complutense de Madrid, Jose Antonio Novais, Madrid, Spain

MIchèle guIlloux-BénatIer • Institut Universitaire de la Vigne et du Vin “Jules Guyot”, Université de Bourgogne, Dijon Cedex, France

Contributors

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MIguel jurado • Facultad de Ciencias Biologicas, Departamento de Genetica, Universidad Complutense de Madrid, Jose Antonio Novais, Madrid, Spain

Sándor kocSuBé • Faculty of Science and Informatics, Department of Microbiology, University of Szeged, Szeged, Hungary

theoVander lee • Biointeractions and Plant Health, Wageningen UR, Wageningen, The Netherlands

antonIo F. logrIeco • Institute of Sciences of Food Production, National Research Council, Bari, Italy

InéS María lóPez-calleja • Facultad de Veterinaria, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, Madrid, Spain

roSarIo Martín • Facultad de Veterinaria, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, Madrid, Spain Pedro Martínez-culeBraS • Department of Preventive Medicine, Public Health, Food

Science and Technology, Bromatology, Toxicology, and Legal Medicine, University of Valencia, Valencia, Spain; Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Valencia, Spain

SalVador MIrete • Facultad de Ciencias Biologicas, Departamento de Genetica, Universidad Complutense de Madrid, Jose Antonio Novais, Madrid, Spain

antonIo MorettI • Institute of Sciences of Food Production, National Research Council, Bari, Italy

gary P. MunkVold • Department of Plant Pathology and Microbiology, Seed Science Center, Iowa State University, Ames, IA, USA

SIddaIah chandra nayaka • DOS in Biotechnology, University of Mysore, Manasagangotri, Mysuru, India

Belén PatIño • Facultad de Ciencias Biologicas, Departamento de Microbiologia, Universidad Complutense de Madrid, Jose Antonio Novais, Madrid, Spain andrea PatrIarca • Laboratorio de Microbiología de Alimentos, Departamento de

Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

MIguel ángel PaVón • Facultad de Veterinaria, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, Madrid, Spain

gIancarlo Perrone • Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Bari, Italy

StePhen w. PeterSon • Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U .S . Department of Agriculture, Peoria, IL, USA

VIrgInIa elena Fernández PInto • Laboratorio de Microbiología de Alimentos, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

IStVán PócSI • Faculty of Science and Technology, Department of Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary

roBert h. Proctor • USDA ARS NCAUR, Peoria, IL, USA; United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA Paula crIStIna azeVedo rodrIgueS • CIMO/School of Agriculture, The Polytechnic

Institute of Bragança, Bragança, Portugal Contributors

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xi alIcIa rodríguez • Faculty of Veterinary Science, Food Hygiene and Safety, Meat and

Meat Products Research Institute, University of Extremadura, Cáceres, Spain

Mar rodríguez • Faculty of Veterinary Science, Food Hygiene and Safety, Meat and Meat Products Research Institute, University of Extremadura, Cáceres, Spain

SandrIne rouSSeaux • Institut Universitaire de la Vigne et du Vin “Jules Guyot”, Université de Bourgogne, Dijon, France

ValerIa Scala • Department of Environmental Biology, University of Rome “Sapienza”, Rome, Italy

María VIctorIa SelMa • Research Group on Quality Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain

alI y. Srour • Department of Plant Soil and Agriculture Systems, Southern Illinois University, Carbondale, IL, USA

MIchelangelo StorarI • Institute for Food Sciences, Agroscope, Bern, Switzerland antonIa SuSca • Institute of Sciences of Food Production, National Research Council,

Bari, Italy

jánoS Varga • Faculty of Science and Informatics, Department of Microbiology, University of Szeged, Szeged, Hungary

Martha M. Vaughan • United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA

coVadonga Vázquez • Facultad de Ciencias Biologicas, Departamento de Microbiologia, Universidad Complutense de Madrid, Jose Antonio Novais, Madrid, Spain

MudIlI VenkataraMana • Microbiology Division, DRDO-BU-Centre for Life sciences, Bharathiar University Campus, Coimbatore, Tamil Nadu, India

elS VerStaPPen • Biointeractions and Plant Health, Wageningen UR, Wageningen, The Netherlands

IVan VISentIn • Department of Agricultural, Forest and Food Sciences, University of Turin, Grugliasco, Italy

ceeS waalwIjk • Biointeractions and Plant Health, Wageningen UR, Wageningen, The Netherlands

Songhong weI • College of Plant Protection, Shenyang Agricultural University, Shenyang, Liaoning, China

taPanI ylI-MattIla • Molecular Plant Biology, Department of Biochemistry, University of Turku, Turku, Finland

eMre yörük • Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Yeni Yuzyil University, Istanbul, Turkey

anna zalka • Kromat Ltd ., Budapest, Hungary

Contributors