12th European Fusarium Seminar

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Bordeaux

EFS12 Programme & Abstracts

12th European Fusarium Seminar

FUSARIUM

MYCOTOXINS, TAXONOMY, GENOMICS, BIOSYNTHESIS, PATHOGENICITY, RESISTANCE, DISEASE CONTROL

12th-16th MAY 2013 PALAIS DE LA BOURSE, BORDEAUX, FRANCE

Edited by INRA UR1264 MycSA, 71 avenue E. Bourlaux, CS20032, 33882 Villenave d’Ornon, France Printed in 2013 by www.copy-media.net, CS20023, 33693 Mérignac Cedex, France The 12th European Fusarium Seminar logo was created by Laetitia Pinson-Gadais, INRA UR1264 MycSA, Bordeaux, France Photo credits Christian Barreau, Françoise Turtaut, Christine Ducos, & Laetitia Pinson-Gadais

TABLE OF CONTENTS Welcome from the Organising Committee

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Sponsorship

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Scientific Programme

7

Lecture and Poster Index

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Oral Presentations

25

Poster Presentations

91

Author Index

237

List of Participants

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ORGANISING COMMITTEE PROGRAM CHAIR: Christian BARREAU (FR) INTERNATIONAL SCIENTIFIC COMMITTEE: Gerhard ADAM (AT) Elzbieta CZEMBOR (PL) Etienne DUVEILLER (MX) Miriam ETCHEVERRY (AR) Geert HAESAERT (BE) Linda HARRIS (CA) Mariana ITTU (RO) Corby KISTLER (USA) Antonio LOGRIECO (IT) John MANNERS (AU)

Akos MESTERHAZY (HU) Thomas MIEDANER (DE) Paul NICHOLSON (UK) Isabelle OSWALD (FR) Florence RICHARD-FORGET (FR) Ulf THRANE (DK) David VAN SANFORD (USA) Altus VILJOEN (ZA) Cees WAALWIJK (NL)

LOCAL SCIENTIFIC COMMITTEE: Marie FOULONGNE-ORIOL Francis FLEURAT-LESSARD Nadia PONTS

Olivier PUEL Jean-Michel SAVOIE

LOCAL ORGANISERS: Vessela ATANASOVA-PENICHON Christian BARREAU Francis FLEURAT-LESSARD Marie FOULONGNE-ORIOL

Laetitia PINSON-GADAIS Nadia PONTS Florence RICHARD-FORGET Jean-Michel SAVOIE

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WELCOME FROM THE ORGANISING COMMITTEE More than twenty-five years have passed since the first European Fusarium Seminar started in 1987 at the initiative of Jerzy CheBkowski at the Warsaw Agricultural University (Poland). Since then it has been organized every two to three years in a different country, involving scientists, breeders, stakeholders and policy makers acting worldwide together to fight again the “cereal killer” Fusarium. This year, it is the first time France hosts this prestigious seminar, and the city of Bordeaux is very proud to gather the Fusarium community together in the historic building of “Le Palais de la Bourse”. The first description of the disease caused by Fusarium, named then “Fusarium Head Blight”, was made by G.W. Smith in 1884. Twenty years later, the experimental reproduction of the disease allowed the discovery of the diversity in susceptibility of cereals to the pathogen and opened the era of breeding for resistance to Fusarium. Years of development of modern agriculture and the use of fungicides allowed the control of Fusarium Head blight. However, when climatic conditions are unfavorable, there still are episodes of blight epidemics impossible to contain. In addition, the discovery that Aspergillus species in the early 1960 produced the highly toxic and carcinogenic aflatoxins led to the investigation of other molds for production of such mycotoxins in crops. It was discovered that many Fusarium species could produce harmful mycotoxins such as trichothecenes, zearalenone and fumonisines. To protect their consumers, developed countries elaborated strict regulations that constrain the trade of cereals worldwide. This has been the case in Europe since 2007. These regulations gave tremendous impulse to the research on Fusarium and its allies, as can be judged by the exponentially increasing number of publications dealing with Fusarium species and their mycotoxins. Since the first European Fusarium Meeting in 1987, the research evolved, and a decade ago, we entered in the era of “omics”. The publication of the genome of various Fusarium species and the study of their transcriptome, proteome and metabolome, now allow the considering of the “Systems Biology” of mycotoxin production. The deciphering of the genomes of different model hosts and cereals is progressing rapidly today. We are all working with the great hope that understanding the fungal/plant interactions at the molecular level will allow us to win the battle. It is this hope that motivates our struggle and brings people not only from Europe, but also from many countries worldwide to Bordeaux to exchange their latest findings and ideas during this meeting. I wish to express here my thanks to all my colleagues who greatly helped me to th make this 12 European Fusarium Seminar as engaging, informative, and fruitful as possible. On behalf of the scientific committee, I trust everybody will have a very successful and exiting seminar. On behalf of the organising committee, we are glad to welcome you to Bordeaux and hope you will appreciate our charming historic city. Christian BARREAU Chair of EFS12 “Welcome to Bordeaux” 3

SPONSORSHIP

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The 12 European Fusarium Seminar gratefully acknowledges the following companies and organisations for their generous contributions.

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SCIENTIFIC PROGRAMME th

12 European Fusarium Seminar Palais de la Bourse Bordeaux, France, 12-16 May 2013

Sunday, May 12th 15:00 – 19:00 19:00 – 21:00

Registration & Poster fixing Welcome Cocktail

Monday, May 13th 09:00 – 10:00 10:00 – 10:30

10:30 – 11:00

Welcome and Opening Introductory Lecture “Fusarium pathogenomics: how to become a cereal killer?” John Manners (CSIRO Plant Industry, Australia) Coffee Break

Session 1: Fusarium – Genetics, Genomics and Systems Biology Chairpersons: Nadia Ponts (FR) & Theo van der Lee (NL) 11:00 – 11:30 Keynote lecture “Systems biology of the yeast Saccharomyces cerevisiae: a model for all fungi?” Steve Oliver (University of Cambridge, UK) 11:30 – 11:45 “Comparative genomics between 3- and 15-ADON F. graminearum chemotypes” Gopal Subramaniam (Agriculture and Agri-Food Canada) 11:45 – 12:00 “EBR1, a master regulator shaping the transcriptional landscape of Fusarium graminearum” Theo van der Lee (Wageningen-UR, The Netherlands) 12:00 – 12:15 “The GABA shunt of Fusarium graminearum is essential for successful colonization of wheat spikes” Jakob Bönnighausen (University Hambourg, Germany) 12:15 – 12:30 “Fgap1-mediated response to oxidative stress in trichothecene-producing Fusarium graminearum” Mathilde Montibus (INRA Bordeaux-Aquitaine, France) 12:30 – 12:45 “Using redox-proteomics to identify targets of NADPH oxidase-generated reactive oxygen species in Fusarium graminearum” Christof Rampitsch (Agriculture and Agri-Food Canada) 12:45 – 13:00 “Eukaryotic translation initiation factor 5A regulates reactive oxygen species, DON, and virulence of Fusarium graminearum and its activation is essential for cell viability” Wilhelm Shäfer (University Hamburg, Germany) 7

13:00 – 14:00 14:00 – 15:00 15:00 – 15:15

Lunch Poster session I (sessions 1 to 3; odd numbers) “Transcriptomic profiling of fumonisin B biosynthesis by Fusarium verticillioides” Nadia Ponts (INRA Bordeaux-Aquitaine, France) 15:15 – 15:30 “Molecular network of nitrate sensing and assimilation in Fusarium fujikuroi” Andreas Pfannmüller (Universität Münster, Germany) 15:30 – 15:45 “The linoleate synthase gene lds1 thoroughly affects conidiogenesis and fumonisin synthesis in Fusarium verticillioides” Valeria Scala (Università La Sapienza, Italy) Session 2: Secondary metabolites – Biochemistry, Biosynthesis, Feed and Food Safety Chairpersons: Isabelle Oswald (FR) & Corby Kistler (USA) 15:45 – 16:15 Keynote lecture “Metabolisation of deoxynivalenol in planta: old and new compounds and their role in food safety” Frantz Berthiller (BOKU, Austria) 16:15 – 16:45 Coffee Break 16:45 – 17:00 “Cellular adaptations for trichothecene biosynthesis in Fusarium graminearum” H. Corby Kistler (University of Minnesota, USA) 17:00 – 17:15 “Ecological role of mycotoxins produced by Fusarium graminearum” Christian Steinberg (INRA Dijon, France) 17:15 – 17:30 “Elucidation of the F. graminearum butenolide biosynthetic gene cluster” Linda Harris (Agriculture and Agri-Food Canada) 17:30 – 17:45 “Fusarium graminearum in depth: a novel method to identify new metabolites by isotopic labeling and high resolution mass spectrometry” Patricia Cano (INRA Toulouse, France) 17:45 – 18:00 “Evidence for birth-and-death evolution and horizontal transfer of a mycotoxin biosynthetic gene cluster in Fusarium” François van Hove (Université Catholique du Louvain, Belgium) 18:00 – 18:15 “Awaking' silent gene clusters in the rice pathogen Fusarium fujikuroi” Sarah Rösler (Universität Münster, Germany) 18:15 – 18:30 “The type of interaction between type B Trichothecenes on the intestine varies with the dose” Imourana Alassane-Kpembi (INRA Toulouse, France) 18:30 – 18:45 “Dose response study based in vitro selection of an adsorbent capable to alleviate the negative in vivo effects of zearalenone in female weaned piglets” Monique Baeke (Kemin, Belgium) 8

Tuesday, May 14th Session 3: Pathogenesis – Epidemiology and Population Genetics Chairpersons: Christian Steinberg (FR) & Quirico Migheli (IT) 08:30 – 9:00 Keynote lecture “The life cycle of a head blight pathogen, Fusarium graminearum, and its importance to agriculture” Frances Trail (Michigan State University, USA) 09:00 – 09:15 “Fusarium graminearum: Species or Clade?” John F. Leslie (Kansas State University, USA) 09:15 – 09:30 “Dynamic of production and maturation of Gibberella zeae perithecia on crop debris” Valentina Manstretta (Università Cattolica del Sacro Cuore, Italy) 09:30 – 09:45 “Insights into the Fusarium-wheat root pathosystem uncover a hidden danger to wheat production” Sven Gottwald (Justus-Liebig University Giessen, Germany) 09:45 – 10:00 “Characterization of the Fusarium root rot complex in soybean” Gary Munkvold (Iowa State University, USA) 10:00 – 10:15 “Genetic and phenotypic diversity of Fusarium graminearum, and interactions between Fusarium species in oats” Heidi U. Aamot (Bioforsk, Norway) 10:15 – 10:30 “New emerging trichothecene-producing Fusarium species in northern Europe and Asia” Tapani Yli-Mattila (University of Turku, Finland) 10:30 – 11:00 Coffee Break 11:00 – 11:15 “Fusarium Head Blight of Wheat in Algeria: level of grains contamination and evaluation of wheat cultivars for resistance to F. culmorum and F. graminearum” Zouaoui Bouznad (Université Tlijène Laghouat, Algeria) 11:15 – 11:30 “Comparisons of Fusarium species obtained from healthy and diseased wheat plants in three agroecological regions of Turkey” Berna Tunali (Ondokuz Mayis University, Turkey) 11:30 – 11:45 “Race Scenario of Fusarium oxysporum f sp. ciceris, wilt pathogen of chickpea (Cicer arietinum L.)” Mamta Sharma (ICRISAT, India) 11:45 – 12:00 “Synopsis of microscopic and molecular studies in F. langsethiae pathogenicity” Hege H. Divon (Norwegian Veterinary Institute, Norway) 12:00 – 13:00 Poster session I (sessions 1 to 3; even numbers) 13:00 – 14:00 Lunch

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Session 4: Genetics of Hosts – Plant Resistance to Fusarium, Variety Development Chairpersons: Thierry Langin (FR) & Paul Nicholson (UK) 14:00 – 14:30 Keynote lecture 1 “Resistance improvement of wheat to Fusarium head blight: challenges and possibilities” Hermann Buerstmayr (BOKU, Austria) 14:30 – 14:45 “Progress in breeding FHB-resistant winter wheat in Ontario, Canada” Ljiljana Tamburic-Ilincic (University of Guelph, Canada) 14:45 – 15:00 “Identification of Frontana derived QTL linked to Fusarium head blight, Fusarium damaged kernel and deoxynivalenol content” Agnes Szabo-Hever (Cereal Research Nonprofit Ltd., Hungary) 15:00 – 15:15 “FHB resistance in soft red winter wheat: breeding and genomic selection” Clay Sneller (Ohio State University, USA) 15:15 – 15:30 “Screening for new sources of FHB and DON resistance in Chinese germplasm collected at CIMMYT genebank” Xinyao He (International Maize and Wheat Improvement Center, Mexico) 15:30 – 15:45 “Expression QTL mapping for Fusarium Head Blight resistance in Wheat” Mina Samad Zamini (BOKU, Austria) 15:45 – 16:00 “A systemic approach in wheat breeding for high yield and resistance to Fusarium graminearum” Pedro Luiz Scheeren (Embrapa, Brazil) 16:00 – 16:30 Coffee Break 16:30 – 16:45 “Molecular and genetic analysis of Fusarium head blight resistance in triticale (xTriticosecale)” Thomas Miedaner (University of Hohenheim, Germany) 16:45 – 17:15 Keynote lecture 2 “Metabolo-proteomics approach to identify candidate genes for wheat resistance to Fusarium head blight” Ajjamada Kushalappa (McGill University, Canada) 17:15 – 17:30 “Semi-dwarf ‘uzu’ barley carries enhanced resistance to a range of pathogens including Fusarium culmorum” Fiona Doohan (University College Dublin, Ireland) 17:30 – 17:45 “Meta-analysis of resistance to Fusarium head blight among tetraploid wheat genetic resources – implications for resistance breeding of durum wheat” Maria Buerstmayr (BOKU, Austria) 17:45 – 18:00 “Wheat gene network dynamics in response to Fusarium graminearum and functional validation of candidate resistance genes” Gerald Siegwart (BOKU, Austria) 10

18:00 – 18:15

18:15 – 18:30

“Identification of wheat susceptibility factors Fusarium graminearum” Chérif Chetouhi (INRA Clermont-Ferrand, France) “In planta inactivation of Fusarium mycotoxins” Gerhard Adam (BOKU, Austria)

to

Wednesday, May 15th Session 5: Disease Control and Forecasting Models Chairpersons: Francis Fleurat-Lessard (FR) & Susanne Vogelgsang (CH) 08:30 – 09:00 Keynote lecture

09:00 – 09:15

09:15 – 09:30

09:30 – 09:45 09:45 – 10:00

10:00 – 10:15

10:15 – 10:30

10:30 – 11:00 11:00 – 11:15

11:15 – 11:30

11:30 – 12:30 12:30 – 13:30 13:30 – 19:00 19:00 – 22:00

“Plant disease prediction using data mining and machine learning: a case study on Fusarium head blight and deoxynivalenol content in winter wheat” Sofie Landschoot (University College Ghent, Belgium) “Wheat monitoring in Switzerland: Which cropping factors influence occurrence of Fusarium species and mycotoxins?” Susanne Vogelgsang (Agroscope Reckenholz-Tänikon Research Station ART, Switzerland) “Influence of agricultural practices on Fusarium spp. and mycotoxin contamination of Norwegian cereal” Ingerd S. Hofgaard (Bioforsk, Norway) “Forecasting helps to target DON toxin testing” Timo Kaukoranta (MTT Agrifood Research Finland) “Forecasting of Fusarium Head Blight and deoxynivalenol in wheat with FusaProg to support growers and industry” Hans-Rudolf Forrer (Research Station Agroscope Reckenholz-Tänikon ART, Switzerland) “Mycotoxins risk assessment in cereals and corn, from monitoring to predictive models” Alain Froment (Syngenta, France) “Biological strategy applied to maize preharvest agroecosystem in Argentina to prevent fumonisin contamination” Miriam Etcheverry (Universidad Nacional de Río Cuarto, Argentina) Coffee Break “Maize kernel antioxidants and their potential involvement in Gibberella and Fusarium Ear Rot resistance” Vessela Atanasova-Penichon (INRA Bordeaux, France) “Trichoderma gamsii 6085 as a tool for the biological control of FHB on wheat” Sabrina Sarrocco (University of Pisa, Italy) Poster session II (sessions 4 to 6; odd numbers) Lunch Architecture & Wine Discovering Tour of Saint Emilion Gala Dinner 11

Thursday, May 16th Session 6: Future Challenges for Europe and Worlwide Chairpersons: Christian Barreau (FR) & Antonio Logrieco (IT) 09:00 – 09:30 Keynote lecture “The risks related to Fusarium mycotoxins at global level: emerging problems and possible solutions” Antonio Logrieco (Institute of Sciences of Food Production, Italy) 09:30 – 09:45 “Future challenges of Fusarium and mycotoxins on cereals in Northern Europe” Päivi Parikka (MTT Agrifood Research, Finland) 09:45 – 10:00 “Climate change impacts on mycotoxins in cereal grain production” Monique de Nijs (RIKILT Wageningen UR, The Netherlands) 10:00 – 10:15 “Uneven and surprising colonization of water pipes of hospitals and non-hospital sites by Fusarium oxysporum and F. dimerum” Véronique Edel-Hermann (INRA Dijon, France) 10:15 – 10:30 “Dermatologic infections by Fusarium species in a tropical clinic” Anne D. van Diepeningen (CBS-KNAW Fungal Biodiversity Centre, The Netherlands) 10:30 – 11:00 Coffee Break 11:00 – 12:00 Poster session II (sessions 4 to 6; even numbers) 12:00 – 12:30 Closing Remarks Important information: Lunches and coffee breaks will be served on site. Oral presentations: all speakers must bring their presentations to the uploading area (near the front desk) no later than 20 minutes prior the half-day of their scheduled talk. Poster presentations: each poster will be up for 2 days, according to their scientific session (see table below). They must be mounted in the morning of the first day, and dismounted in the evening of the second day. Speakers are expected to attend their posters during the designated time periods and according to their attributed numbers (even or odd). Session 1: Fusarium – Genomics, Genomics and Systems Biology Poster Session I Session 2: Secondary Metabolites – Biochemistry, th (Monday 13 through Biosynthesis, Feed and Food Safety th Tuesday 14 ) Session 3: Pathogenesis – Epidemiology and Population Genetics Session 4: Genetics of Hosts – Resistance to Poster Session II Fusarium, Variety Development th (Wednesday 15 Session 5: Disease Control and Forecasting Models through Thursday 16th) Session 6: Future Challenges for Europe and Worlwide 12

LECTURE AND POSTER INDEX Oral Presentations Abstracts Opening Fusarium pathogenomics: How to become a cereal killer?

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Session 1: Fusarium - Genetics, Genomics and Systems Biology Systems biology in the yeast Saccharomyces cerevisiae: a model for all fungi? 29 Comparative chemotypes

genomics

between

3-

and

15-ADON

F.

graminearum 30

EBR1, a master regulator shaping the transcriptional landscape of Fusarium graminearum 31 The GABA shunt of Fusarium graminearum is essential for successful colonization of wheat spikes 32 Fgap1-mediated response to oxidative stress in trichothecene-producing Fusarium graminearum. 33 Using redox-proteomics to identify targets of NADPH oxidase-generated reactive oxygen species in Fusarium graminearum. 34 Eukaryotic translation initiation factor 5A regulates reactive oxygen species, DON, and virulence of Fusarium graminearum and its activation is essential for cell viability. 35 Transcriptomic profiling of fumonisin B biosynthesis by Fusarium verticillioides 36 Molecular network of nitrate sensing and assimilation in Fusarium fujikuroi

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The linoleate synthase gene lds1 thoroughly affects conidiogenesis and fumonisin synthesis in Fusarium verticillioides 38 Session 2: Secondary Metabolites - Biochemistry, Biosynthesis, Feed and Food Safety Metabolisation of deoxynivalenol in planta: Old and new compounds and their role in food safety 39 Cellular adaptations for trichothecene biosynthesis in Fusarium graminearum

40

Ecological role of mycotoxins produced by Fusarium graminearum

41

Elucidation of the F. graminearum butenolide biosynthetic gene cluster

42

Fusarium graminearum in depth: a novel method to identify new metabolites by isotopic labelling and high resolution mass spectrometry 43 13

Evidence for birth-and-death evolution and horizontal transfer of a mycotoxin biosynthetic gene cluster in Fusarium 44 'Awaking' silent gene clusters in the rice pathogen Fusarium fujikuroi

45

The type of interaction between type B Trichothecenes on the intestine varies with the dose 46 Dose response study based in vitro selection of an adsorbent capable to alleviate the negative in vivo effects of zearalenone in female weaned piglets. 47 Session 3: Pathogenesis - Epidemiology and Population Genetics The life cycle of a head blight pathogen, Fusarium graminearum, and its importance to agriculture 49 Fusarium graminearum: Species or Clade?

50

Dynamic of production and maturation of Gibberella zeae perithecia on crop debris 51 Insights into the Fusarium-wheat root pathosystem uncover a hidden danger to wheat production 52 Characterization of the Fusarium root rot complex in soybean

53

Genetic and phenotypic diversity of Fusarium graminearum, and interactions between Fusarium species in oats 54 New emerging trichothecene-producing Fusarium species in northern Europe and Asia 55 Fusarium head blight of wheat in Algeria: preliminary investigations into the relationship with some isolates and cultivars resistance 56 Comparisons of Fusarium species obtained from healthy and diseased wheat plants in three agro-ecological regions of Turkey 57 Race Scenario of Fusarium oxysporum f sp. ciceris, wilt pathogen of chickpea (Cicer arietinum L.) 58 Synopsis of microscopic pathogenicity.

and

molecular

studies

in

F.

langsethiae 59

Session 4: Genetics of Hosts - Plant Resistance to Fusarium, Variety Development Resistance improvement of wheat to Fusarium head blight: challenges and possibilities 61 Progress in breeding FHB-resistant winter wheat in Ontario, Canada

62

Identification of Frontana derived QTL linked to Fusarium head blight, Fusarium damaged kernel and deoxynivalenol content 63 FHB Resistance in Soft Red Winter Wheat: Breeding and Genomic Selection

14

64

Screening for new sources of FHB and DON resistance in Chinese germplasm collected at CIMMYT genebank 65 Expression QTL mapping for Fusarium Head Blight resistance in Wheat

66

A systemic approach in wheat breeding for high yield and resistance to Fusarium graminearum 67 Molecular and genetic analysis of Fusarium head blight resistance in triticale (xTriticosecale) 68 Metabolo-proteomics approach to identify candidate genes for wheat resistance to fusarium head blight 69 Semi-dwarf ‘uzu’ barley carries enhanced resistance to a range of pathogens including Fusarium culmorum 70 Meta-analysis of resistance to Fusarium head blight among tetraploid wheat genetic resources – implications for resistance breeding of durum wheat 71 Wheat gene network dynamics in response to Fusarium graminearum and functional validation of candidate resistance genes 72 Identification of wheat susceptibility factors to Fusarium graminearum

73

In planta inactivation of Fusarium mycotoxins

74

Session 5: Disease Control and Forecasting Models Plant disease prediction using data mining and machine learning: a case study on Fusarium head blight and deoxynivalenol content in winter wheat 75 Wheat monitoring in Switzerland: Which cropping factors influence occurrence of Fusarium species and mycotoxins? 76 Influence of agricultural practices on Fusarium spp. and mycotoxin contamination of Norwegian cereals 77 Forecasting helps to target DON toxin testing

78

Forecasting of Fusarium Head Blight and Deoxynivalenol in Wheat with FusaProg to support Growers and Industry 79 Mycotoxins risk assessment in cereals and corn, from monitoring to predictive models 80 Biological strategy applied to maize preharvest agroecosystem in Argentina to prevent fumonisin contamination 81 Maize kernel antioxidants and their potential involvement in Gibberella and Fusarium Ear Rot resistance 82 Trichoderma gamsii 6085 as a tool for the biological control of FHB on wheat

83

Session 6: Future Challenges for Europe and Worlwide The risks related to Fusarium mycotoxins at global level: emerging problems and possible solutions 85 15

Future challenges of Fusarium and mycotoxins on cereals in Northern Europe 86 Climate change impacts on mycotoxins in cereal grain production

87

Uneven and surprising colonization of water pipes of hospitals and nonhospital sites by Fusarium oxysporum and F. dimerum 88 Dermatologic infections by Fusarium species in a tropical clinic

89

Poster Presentations Abstracts Session 1: Fusarium – Genetics, Genomics and Systems Biology P1 - RNA-Seq analysis reveals new gene models and alternative splicing in the Fusarium graminearum 93 P2 - Genome-wide transcriptional response to ambient pH and Pac1 regulatory factor in Fusarium graminearum 94 P3 - Significance of the hydrophobin FgHyd5p in Fusarium graminearum

95

P4 - Studying gene expression in fungus and planta to understand the interaction Fusarium verticillioides-maize 96 P5 - Comprehensive inventory on coding and non-coding features of the genome of Fusarium fujikuroi. 97 P6 - A retrotransposon based approach for the detection of intraspecific variation among Fusarium oxysporum formae speciales 98 P7 - FCSTUA controls pathogenicity and morpho-physiological traits in Fusarium culmorum 99 P8 - Interactions between different Fusarium species to uncover multi-toxin synergistic mechanisms 100 P9 - Agrobacterium-mediated insertional mutagenesis of Fusarium oxysporum f. sp. cubense for identification of key genes in the infection cycle of the pathogen 101 P10 - Interspecific Hybrids between Fusarium fujikuroi and Fusarium proliferatum 102 P11 - Identifying indicators of soil suppressiveness to fungal diseases

103

P12 - Disentangling mycotoxin regulatory pathways in Fusarium graminearum by quantitative genetics 104 Session 2: Secondary Metabolites - Biochemistry, Biosynthesis, Feed and Food Safety P13 - Masked mycotoxins in durum wheat: a greenhouse experiment

105

P14 - Metabolomics of growth and type B trichothecenes production in Fusarium graminearum 106 16

P15 - Effect of pH and temperature on Fusarium langsethiae growth and on T2 and HT2 toxins production in liquid medium 107 P16 - Volatile compounds in grain of various wheat cultivars naturally infected and inoculated with Fusarium culmorum 108 P17 - Semiochemical interactions between toxigenic Fusarium fungi and insects 109 P18 - Effect of cry 1Ab toxins on FUM gene cluster expression and on fumonisin production by Fusarium verticillioides 110 P19 - Natural and natural-like inhibitors of trichothecene biosynthesis by Fusarium 111 P20 - Changes in fungal biomass and fumonisin production by Fusarium proliferatum strains in the presence of host plant extracts 112 P21 - Correlation between Fusarium DNA and mycotoxin levels in Finnish oats samples 113 P22 - Geographic differences in trichothecene chemotypes of Fusarium graminearum in the Northwest and North of Iran 114 P23 - Co-occurrence of mycoflora, aflatoxins and fumonisins in maize and rice seeds from markets of different districts in Cairo, Egypt 115 P24 - The impact of Fusarium and Microdochium species on the safety and quality of UK malting barley 116 P25 - Influence of pre-harvest moisture and harvest time on fusarium mycotoxin concentrations in winter wheat 117 P26 - Contamination of wheat grain with microscopic fungi and their metabolites in Poland in the years 2006–2009 118 P27 - Fumonisins occurrence in maize cobs infected with opposite mating type strains of F. verticillioides 119 P28 - Phylogenetic diversity of and fumonisin gene cluster distribution within Fusarium isolates from wild banana in China 120 P29 - Different levels of fumonisin production and FUM gene cluster expression on 2B710Hx corn hybrid 121 P30 - Monitoring fumonisin levels in maize samples from Italy during 2006– 2012 122 P31 - Quantification of Fusarium fungi and their mycotoxins in common buckwheat grain 123 P32 - Chemotype diversity and pathogenicity of Fusarium graminearum species complex originating from Serbian cereals grain 124 P33 - Fusarium toxin in forage rice grown in a paddy field in Japan

125

P34 - A survey on pre- and post-harvest garlic bulbs: Fusarium proliferatum occurrence and fumonisins (B1, B2) accumulation 126

17

P35 - Screening deoxynivalenol in oat using a quick-method with comparison to a quantitative GC-MS analysis 127 P36 - Comparison of Veratox® for T-2/HT-2 ELISA test with GC-MS and LCMS methods 128 P37 - Validation of an ELISA for the determination of fumonisin in maize samples for human consumption 129 P38 - Prediction of deoxynivalenol content in wheat by Near Infrared Reflectance Spectroscopy 130 P39 - PCR chemotyping of Fusarium graminearum, F. culmorum and F. cerealis isolated from winter wheat in Wallonia, Belgium 131 P40 - Induction of cytotoxicity and apoptosis in mouse blastocysts by enniatin 132 P41 - Evaluation of antifungal activity of ethanol and methanol extracts from Punica granatum peels on fungal strains producing mycotoxins 133 Session 3: Pathogenesis - Epidemiology and Population Genetics P42 - Fusarium graminearum/Gibberellea zeae perithecia formation on winter wheat straw and maize stalks in Swedish climate 135 P43 - Heterochromatin protein 1 (Hep1) deletion in F. graminearum causes hypervirulence on wheat heads. 136 P44 - A rapid in vitro assay to select mutants of Fusarium impaired in pathogenicity. 137 P45 - Fusarium poae: chemotype, plant-pathogen interaction and response to oxidative stress triggers 138 P46 - Study of the in vitro growth and pathogenicity of a collection of Fusarium spp. and Microdochium nivale obtained from the ear and the collar of wheat collected in the central region of Algeria 139 P47 - Trichothecene production by Fusarium graminearum isolates from Argentina and its relationship with aggressiveness and fungal colonization of the wheat spike 140 P48 - Study of in vitro growth and pathogenicity of some isolates of Fusarium spp. causal agent of Fusarium head scab (FBH) of wheat in Algeria 141 P49 - Pathogenicity of Fusarium temperatum and Fusarium subglutinans on maize stalk and ear under artificial inoculation under field conditions 142 P50 - Effect of timing of inoculation and Fusarium species on the development of Fusarium head blight and deoxynivalenol contamination in oat 143 P51 - Selective pathogenicity and virulence of Fusarium graminearum species complex members on maize, wheat and sorghum 144 P52 - Aggressiveness and deoxinivalenol production graminearum isolates from different inoculum sources

of

Fusarium 145

P53 - Fusarium crown rot of wheat: a survey of Minnesota wheat fields 18

146

P54 - Fusarium Head Blight agents and mycotoxin contamination in barley kernels in Italy 147 P55 - Occurrence of Fusarium species isolated from Barley and Bread wheat grain and detection of Deoxynivalenol in Northern Tunisia 148 P56 - Fusarium species associated to durum wheat during 2011-2012

149

P57 - Analysis of the Fusarium graminearum species complex in Brazil shows high diversity and changes in species prevalence affected by host and geographic region 150 P58 - Fusarium species associated with Head blight and Foot and Root Rot on durum wheat in Sardinia, Italy: Results from a 12- year survey 151 P59 - Head blight of wheat in South Africa is associated with numerous Fusarium species and chemotypes 152 P60 - Fusarium mycotoxin contamination and Fusarium species in Polish wheat in 2010-2012 153 P61 - Fusarium head blight of wheat in Algeria: Preliminary investigations into the relationship with some isolates and cultivars resistance 154 P62 - Biodiversity of Fusarium spp. on cereals in different regions of Russia

155

P63 - Interspecific and intraspecific variability of Fusarium fungi

156

P64 - Plant pathogenic fungal interactions in oats

157

P65 - Molecular and chemical analysis of trichothecene diversity of Gibberella zeae populations from corn, wheat and potatoes in eastern Canada 158 P66 - PCR validation and chemotyping of causal Fusarium species of Fusarium head blight on south african wheat 159 P67 - Genetic and mycotoxigenic diversity of isolates belonging to the Fusarium incarnatum-equiseti species complex, and recovered from maize and banana in China 160 P68 - Population analysis of Fusarium graminearum sensu stricto from wheat and maize in the United Kingdom 161 P69 - The French Fusarium Collection: a living resource for mycotoxin research 162 P70 - ToxiFusaDB: the online catalogue of the MycSA Fusarium strains collection 163 P71 - Real time PCR for FHB quantification: bias analysis?

164

P72 - Fusarium spp. on maize in Belgium, from biodiversity to biocontrol

165

P73 - Creation of the State Collection of Fusarium fungal strains

166

P74 - The molecular characterization and determination of genetic variability in Fusarium verticilloides strains isolated from maize in Turkey 167 P75 - Monitoring of maize contamination by Fusarium mycotoxins in Poland in 2012 168 19

P76 - Fusarium verticillioides and F. subglutinans mating types – distribution and molecular structure 169 P77 - Geographic distribution and multilocus analysis subglutinans and F. temperatum from maize worldwide

of

Fusarium 170

P78 - Molecular characterization of Fusarium species occurring on olive fruits in Apulia 171 P79 - Evaluation of Fusarium wilt resistance among the accessions of eggplant (Solanum melongena L.) 172 Session 4: Genetics of Hosts - Plant Resistance to Fusarium, Variety Development P80 - Breeding resistance for Fusarium head blight in supporting higher efficiency of the integrated plant management in wheat 173 P81 - Breeding of cereal cultivars resistant to Fusarium fungi

174

P82 - Interaction between Quantitative Trait Loci (QTL) for Fusarium head blight (FHB) resistance and Fusarium graminearum 15-ADON and 3-ADON chemotypes in spring wheat 175 P83 - Advantage of using native sources of FHB resistance in breeding winter wheat in Ontario, Canada 176 P84 - Evaluation of German winter wheat cultivars for resistance against Fusarium head blight and mycotoxin reduction 177 P85 - Promising Fusarium head blight resistance in durum wheat

178

P86 - Selection of aggressive Fusarium isolates for breeding

179

P87 - Developing test method to oats and barley for resistance to Fusarium langsethiae 180 P88 - Variation for Fusarium head blight resistance and Fusarium toxins accumulation in winter triticale lines inoculated with Fusarium culmorum 181 P89 - Identification of physiological traits in wheat conferring passive resistance to Fusarium head blight 182 P90 - Chemotype-specific Fusarium isolates applied for phenotyping of type II resistance to FHB in wheat 183 P91 - Mapping QTLs for Fusarium head blight response in a durum wheat elite population 184 P92 - Forthcoming development of diverse FHB and DON resistant wheat in South Africa 185 P93 - Identification and characterization of wheat genes contributing in plant resistance to the mycotoxin deoxynivalenol 186 P94 - Resistance of winter wheat breeding lines to Fusarium head blight and accumulation of Fusarium toxins in grain 187 20

P95 - Evaluation of Fusarium Head Bblight resistance in a panel of durum wheat (Triticum turgidum L.) 188 P96 - Oat resistance to HT2 and T2-producing Fusarium langsethiae

189

P97 - Susceptibility of cereal species to Fusarium langsethiae, a potent producer of HT2 and T2 190 P98 - Impact of co-infection by Microdochium spp. and Fusarium graminearum on the assessment of new wheat varieties’ tolerance to FHB and deoxynivalenol 191 P99 - Testing varieties at GEVES for resistance to Fusarium head blight on cereals: A way to improve genetic progress in the French Catalogue and to reduce the use of pesticides 192 P100 - S-Methyl-DON: Chemical synthesis and toxicity of a novel DON metabolite 193 P101 - Functional characterization of a lipid transfer protein associated with Qfhs.ifa-5A 194 P102 - Transient silencing of a PR-4 gene decreases type I resistance against Fusarium graminearum 195 P103 - RNA-Sequencing as a tool for the analysis of the pathosystem maizeFusarium verticillioides 196 P104 - Maize/Fusarium interaction and ear rot resistance in the CANADAIR project 197 P105 - Fusarium verticillioides ear rot and fumonisin accumulation resistance in Italian maize germplasm 198 P106 - Genetic variation for ear rot resistance and mycotoxin content of Polish maize elite inbreed lines after inoculation with Fusarium graminearum and F. verticillioides 199 P107 - Fusarium wilt and its implications on alfalfa perenniality

200

P108 - Effect of phlobaphene accumulation in maize kernel pericarp on Fusarium ear rot levels in Lombardia 201 P109 - Comparative analysis of resistance in Fusarium wilt-resistant and susceptible watermelons: reinforcement of the structure barrier 202 P110 - Identification and characterization of new resistant accessions to Fusarium oxysporum f. sp. pisi within a Pisum spp. germplasm collection. 203 Session 5: Disease Control and Forecasting Models P111 - Effect of time of application of the fungicide prothioconazole on Fusarium Mycotoxins in maize 205 P112 - Timing and efficacy of fungicides against Fusarium head blight in malting barley 206

21

P113 - Agronomic practices and risk for mycotoxins in northern cereal production 207 P114 - Effect of direct drilling on Fusarium foot and root rot in durum wheat, barley and oat in Tunisia 208 P115 - Effect of previous crops and climatic conditions on Fusarium foot and root rot and yield of Durum wheat in North West Tunisia 209 P116 - The potential risk of grain colonisation by fumonisin-producing Fusarium spp. and fumonisin synthesis in commercial maize in South Africa 210 P117 - Influence of weather conditions and planting dates on deoxynivalenol accumulation in commercial maize hybrids grown in Ontario, Canada 211 P118 - Evaluation of Predictive Models for Wheat Fusarium Head Blight under Growing Conditions of Quebec, Canada 212 P119 - Predicting Deoxynivalenol in Oats under Northern European Conditions 213 P120 - A hierarchical bayesian approach to predict the risk of Fusarium head blight in wheat 214 P121 - Pathogenic fungi associated with Fusarium seedling root rot in winter cereals in 2012 215 P122 - Distribution of the airborne inoculum of Gibberella zeae in Belgium

216

P123 - The frequency of isolation of Fusarium species from stem bases of grass weeds of field crops in Tunisia 217 P124 - Saprophytic survival of Fusarium graminearum in crop residues

218

P125 - The potential of biofungicides in controlling soil born pathogen on tomato by inducing defense response 219 P126 - Study of the antagonistic effect of Trichoderma spp. against Fusarium spp. and M. nivale involved in Fusarium head blight and root rot of wheat. 220 P127 - Biological formulations for control of Fusarium verticillioides and fumonisins in maize at field level 221 P128 - Lactic acid bacteria [LAB]: potential for control of Fusarium growth

222

P129 - Screening of antagonistic activity of indigenous bacteria against two Fusarium species 223 P130 - Effect of CLO-1 biofungicide on perithecial production of Gibberella zeae on crop residues 224 P131 - Identification of Pseudomonas bacteria associated with roots of Piper tuberculatum able to inhibit in vitro growth of Fusarium solani f. sp. piperis 225 P132 - Study of the effect of Pseudomonas spp. fluorescent for the suppression of Fusarium wilt of tomato 226 P133 - Busseola fusca and Fusarium verticillioides interaction on Fusarium ear rot and fumonisin production in Bt and non-Bt maize hybrids in South Africa 227 22

P134 - Screening of Emericella nidulans for biological control of tomato Fusarium wilt in Lao PDR 228 Session 6: Future Challenges for Europe and Worlwide P135 - Current situation with Fusarium head blight on small grain cereals in Russia 229 P136 - Combating of Fusarium oxysporum f. sp. albedinis attacking date palm in the south west of Algeria by natural substances 230 P137 - A role for carboxylesterases in the chemotype shift in F. graminearum populations? 231 P138 - Occurrence of Fusarium spp., black Aspergillus spp., and associated mycotoxins in Italian maize in 2011 232 P139 - Relationship between contamination in wheat kernels

cadmium

and type

B trichothecenes 233

P140 - Degradation of fumonisins by microorganisms in moist corn grain silages 234 P141 - Digestibility and absorption of deoxynivalenol-3-β-glucoside in in vitro models 235 P142 - ISHAM Working group on Clinical Fusarium

236

23

24

ORAL PRESENTATIONS

25

26

OPENING LECTURE

Fusarium pathogenomics: killer?

How to become a cereal

J. Manners, D. Gardiner, K. Kazan, S. Chakraborty, L. Covarelli, J. Sperschneider, J. Taylor CSIRO Plant Industry, Brisbane, Canberra and Perth, Australia E-mail: [email protected]

Many Fusarium pathogens cause devastating diseases on cereals such as wheat and barley. Although Fusarium head blight (FHB) is a well studied disease, these cereal hosts are also susceptible to crown and root rot diseases caused by the same Fusarium pathogens. Several wheat-infecting Fusarium pathogens produce mycotoxins, and in some instances, these may be important for virulence. In addition, in FHB disease, toxin accumulation in infected grain can threaten human and animal health and restrict trade. In the last few years, significant progress has been made towards a better understanding of the processes involved in pathogenesis and toxin biosynthesis in cereal-infecting Fusaria, as well as host resistance mechanisms, often through the use of functional and comparative genomic analyses. Current sequencing technologies make obtaining the basic genome sequences of cereal-infecting Fusaria a relatively trivial exercise and the availability of the gold standard sequence of F. graminearum provides an essential reference. Studies of several newly acquired genomic sequences of Fusarium cereal pathogens have indicated that a significant part of the genome and gene content vary between species and isolates. Comparative genomic analysis, either by using sequence-based BLAST analyses or an alternative novel molecular pattern analysis across the many sequenced genomes of other fungi, has provided new opportunities to identify genes that have roles in virulence on cereal hosts. In several instances, these genes have interesting evolutionary histories, involving multiple horizontal transfer events. These studies are suggestive of a cereal-infecting pathogen pan-genome. Parts of this pan-genome appear to be shared between diverse pathogens. Multiple genic combinations have been added to the pan-genome through evolutionary time to provide new virulence capabilities that allow adaptation to cereal host types. In this presentation, we will review these new advances and also discuss future research gaps that need bridging for the development of sustainable plant protection strategies against Fusarium pathogens. Keywords: Fusarium, pathogenomics

27

28

KEYNOTE LECTURE SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Systems biology in the yeast cerevisiae: a model for all fungi?

Saccharomyces

S. Oliver University of Cambridge, UK E-mail: [email protected]

29

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Comparative genomics between 3- and 15-ADON F. graminearum chemotypes S. Walkowiak, L. Wang, G. Subramaniam Eastern Cereal and Oilseed Research Centre, Ottawa, Canada E-mail: [email protected]

Fusarium graminearum is the principal cause of fusarium head blight in North America. The disease results in severe losses in yield and quality of cereals. Epidemiological studies document that the large proportion of F. graminearum isolates produce 3- or 15-acetyl deoxynivalenol (ADON). Recent studies show a shift from 15-ADON to 3-ADON producers in Canada and north central USA. In greenhouse studies, the 3-ADON isolates are more aggressive and are able to spread quicker than its counterpart. Furthermore, the two-stage culture used to induce 15-ADON production do not show accumulation of 3-ADON. These differences between the two chemotypes prompted us to sequence several 3- and 15-ADON isolates. The analysis of this deep sequencing will be addressed. Keywords: comparative genomics, 3-ADON, 15-ADON

30

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

EBR1, a master regulator shaping the transcriptional landscape of Fusarium graminearum Z. Chunzhao 1,2 Lee

1,2,3,4

1,2

2,5

3

, C. Waalwijk , P. J. G. M. de Wit , D. Tang , T. van der

1

Plant Research International, P.O. Box 6708 PB, Wageningen, The Netherlands; 2Graduate School Experimental Plant Sciences, Wageningen, The Netherlands; 3State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; 4Graduate University of Chinese Academy of Sciences, Beijing 100049, China; 5Wageningen University, Laboratory of Phytopathology, P.O. Box 6708 PB, Wageningen, The Netherlands E-mail: [email protected]

Mycotoxins are secondary metabolites that are produced by fungi. The expression of genes involved in the production of secondary metabolites is often repressed under nutrient-rich conditions, when the available resources seem to be primarily used to promote fungal growth. When confronted with stress conditions, fungi can significantly increase the production of secondary metabolites. The relationship between primary and secondary metabolism, and the components that regulate the switch between both life-forms in Fusarium graminearum was studied. Previously, we have identified that a gene knock-out of ebr1 results in pleiotropic effects, including reduced pathogenicity, reduced radial growth, enhanced hyphal branching and overproduction of pigment. EBR1 (Enhanced Branching 1) encodes a Gal4-like Zn2Cys6 transcription factor, which is constitutively expressed. RNA-seq analyses of the wild-type and Δebr1 demonstrated that this gene is a master regulator involved in the switch between primary and secondary metabolism. The regulation of secondary metabolism in the wild type and the ebr1 knock-out mutant has been studied at different developmental stages and under various growth conditions. We compared the expression of genes that hallmark primary metabolism such as the ribosomal genes, to the expression of genes implicated in the production of secondary metabolites such as polyketide synthases and non-ribosomal peptide synthetases. Whereas the wild-type strain produces limited amounts of secondary metabolites under nutrient-rich conditions, a significant increase in the expression of genes implicated in the production of secondary metabolites was observed in the knock-out strain Δebr1. These results may provide new targets to reduce the production of secondary metabolites and the identification of new fungicides that may reduce the amount of mycotoxins. Keywords: transcription, RNA-Seq, regulation, secondary metabolism

31

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

The GABA shunt of Fusarium graminearum is essential for successful colonization of wheat spikes J. Bönnighausen, J. Bormann, W. Schäfer Molecular Phytopathology and Genetics, University Hamburg, Germany E-mail: [email protected]

We evaluated the roles of the non protein amino acid y-amino butyric acid (GABA) in the Triticum aestivum - Fusarium graminearum interactions. The GABA shunt is a conserved stress releated pathway which can be used as an alternative route to bypass two steps of the tricaboxylic acid (TCA) cycle. Its importance for the life cycle of a fungal pathogen has not been described yet. We constructed single and double deletion mutants for both GABA transaminases. Growth of F graminearum wild type with GABA as the sole N-source strongly induced deoxynivalenol (DON) production. The ΔΔGAT1;2 mutants were unable to produce DON after GABA induction in culture, instead we observed by HPLC a massive accumulation of GABA in the mycelium of the double mutant. During vegetative growth, the ΔΔGAT1;2 mutant showed a decreased resistance against hydrogen peroxide mediated stress. During wheat infection, the individual single disruptants were only slightly reduced, whereas the double mutant exhibited a strong reduction in virulence. During initial infection, ΔΔGAT1;2 mutants were able to infect the inoculated spikelet, crossed the rachis node and grew into the rachis. Later, they failed to colonize more than one spikelet above and below the inoculated one, maintaining only app. 25% of wild type virulence. Interestingly, no significant differences were found regarding the expression of the trichothecene biosynthesis genes or the amount of DON during infection. However, the expression levels of the SSADH genes and a GABA permease were highly up-regulated in the ΔΔGAT1;2 mutants, suggesting a disturbed internal GABA level. Concomitantly, the expression level of the citrate synthase gene, a marker gene for the activity of the TCA cycle, decreased 50%. We conclude that the GABA shunt modulates the fungal stress response, possibly to ROS, during wheat colonization and is necessary to maintain the primary energy metabolism under energy demanding conditions. Keywords: Fusarium, stress metabolism, GABA

32

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Fgap1-mediated response to oxidative stress in trichothecene-producing Fusarium graminearum. 1

1

1

1

1

M. Montibus , N. Ponts , E. Zehraoui , C. Ducos , F. Richard-Forget , C. 1 Barreau . 1

INRA, UR1264-MycSA, 71 avenue Edouard Bourlaux, CS20032, F-33883 Villenave d’Ornon Cedex. E-mail: [email protected]

The filamentous fungus Fusarium graminearum infects cereals and corn. It is one of the main causal agent of “Fusarium Head Blight” and “Maize Ear Rot”. During infection, it produces mycotoxins belonging to the trichothecenes family that accumulate in the grains. Although the biosynthetic pathway involving specific Tri genes has been elucidated, the global regulation of toxin biosynthesis remains enigmatic. It is now established that oxidative stress modulates the production of toxins by F. graminearum. H2O2 added in liquid cultures increases the expression of Tri genes involved in the biosynthesis of type B trichothecenes as well as downstream trichothecene accumulation. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1p mediates response to oxidative stress via nuclear localization and activation of genes coding for detoxification enzymes. In this study, we investigate the role of Yap1p homolog in F. graminearum, Fgap1, in response to oxidative stress and its eventual role in the regulation of trichothecene production. A deleted mutant and a strain expressing a constitutively activated form of the Fgap1 factor in F. graminearum were constructed. In the presence of oxidative stress by H2O2, Tri genes expression levels and trichothecene production by the strain lacking the gene Fgap1 are enhanced, whereas Tri genes expression and toxin accumulation are severely diminished in the mutant strain expressing Fgap1 constitutively. Expression profiles of genes encoding detoxification enzymes potentially controlled by Fgap1 were further analyzed by qRT-PCR. These results are currently being deepened by a transcriptomic approach. The involvement of Fgap1 in other types of stress responses has also been investigated. In particular, cadmium and osmotic stress affect growth in the deleted strain. Keywords: Fusarium graminearum, secondary metabolites, oxidative stress, Fgap1

33

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Using redox-proteomics to identify targets of NADPH oxidase-generated reactive oxygen species in Fusarium graminearum. 1

2

1,2

C. Rampitsch, G. Subramaniam , M. Joshi , T. Fan

1

1

Agriculture and Agri-Food Canada, Winnipeg MB; 2Agriculture and Agri-Food Canada, Ottawa ON. E-mail: [email protected]

The regulated production of reactive oxygen species by NADPH oxidases NoxA and NoxB in Fusarium graminearum (Fgr) is essential for the establishment of Fusarium head blight in wheat. Knock-out mutants, FgrΔNoxAB, are nonpathogenic and produce no perithecia in vitro, although normal levels of DON are – secreted. Nox A and B oxidize NADPH to generate O 2 and thence H2O2 during cellular differentiation, creating an oxidizing environment intracellularly, in which susceptible cysteine residues on target proteins are oxidized. This can profoundly affect the activity of these proteins, and they are candidate participants in redoxmediated control of cellular processes, including cellular differentiation and pathology. Two strategies were used to identify targeted proteins in the redox proteome: 1) 2-D electrophoresis, using monobromo-bimane to label reduced Cys residues, followed by MS-based protein identification, and 2) an affinityenrichment strategy based upon biotinylation of targeted Cys residues, with relative quantification by spectral counting. Using these strategies, we have identified several proteins which are potentially targeted, i.e. those which were oxidized in WT but not in FgrΔNoxAB, under mycotoxin-inducing conditions in vitro. Confirmation of biological activity through mutagenesis is underway, with the aim of further understanding the role of redox regulation in Fgr pathogenesis. Keywords: Nox, proteomics, redox

34

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Eukaryotic translation initiation factor 5A regulates reactive oxygen species, DON, and virulence of Fusarium graminearum and its activation is essential for cell viability. 1

2

1

A. L. Martinez-Rocha , M. Woriedh , W. Schäfer . 1

Biocenter Klein Flottbek, Molecular Phytopathology and Genetics, University of Hamburg, Biology and Plant Biochemistry, University of Regensburg E-mail: [email protected]

2

Cell

The eukaryotic translation initiation factor 5A (EIF5A) is a highly conserved protein from archeabacteria to higher eukaryotes with the exception of the bacteria. EIF5A acts as a nucleo-cytoplasmatic shuttle protein of mRNAs to the ribosomes and is the only known protein containing the unique aminoacid hypusine. Hypusin is formed by a posttranslational modification of lysine in two enzymatic steps, catalysed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). EIF5A has a role in diseases as diverse as HIV infection, malaria, cancer, and diabetes. The involvement of EIF5A in fungal phyto-pathogenesis is unknown. Until now, only inhibition or silencing has been tested to control hypusination of eIF5A and its consequent results. We over-expressed the enzymes that control hypusination of eIF5A in the fungal pathogen Fusarium graminearum. Overexpression of DHS leads to an increase in virulence and a decrease of reactive oxygen species (ROS). DON levels during wheat infection are slightly increased. However, over-expression of DOHH abolishes infection of F. graminearum in wheat and leads to an over-production of ROS and a strong decrease in DON. Over-expression of both genes together produces similar levels of infection, ROS and DON as the wild type strain. These results suggest that hypusinated eukaryotic translation initiation factor 5A regulates reactive oxygen species, DON, and virulence of Fusarium graminearum. We showed previously, that CNI-1493 inhibits F. graminearum DHS and thereby virulence to wheat and maize. Now, we fused mCherry with EIF5A and labeled the nucleus with a histone H1-GFP fusion. During germination EIF5A is evenly distributed in the cytoplasm and the nucleus. After addition of 10 µM of the DHS inhibitor CNI-1493, EIF5A vanished from the cytoplasm into the nucleus. Now the cells died and lysed, visualizing why gene disruption of the DHS gene is lethal. Keywords: Fusarium, EIF5A, DON, ROS

35

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Transcriptomic profiling of fumonisin B biosynthesis by Fusarium verticillioides N. Ponts, E. Zehraoui, L. Pinson-Gadais, F. Richard-Forget, C. Barreau INRA UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS20032, 33882 Villenave d'Ornon Cedex – France E-mail: [email protected]

The plant fungal pathogen Fusarium verticillioides can infect various plants worldwide, including maize, and contaminate kernels with mycotoxins of the fumonisin family. Fumonisins B are stable polyketides that resist agrifood processing and are classified as potentially carcinogenic. As such, contamination of food and feeds with these toxic secondary metabolites must be avoided. Numerous factors influence fumonisins B accumulation on maize, including the composition of the grains on which Fusarium develops. In particular, several phenolic compounds were shown to inhibit fumonisin B biosynthesis. Preliminary analyses showed that free phenolic acids are particularly abundant in immature grains, i.e., at the onset of toxin production, from cereal cultivars on which mycotoxins tend to accumulate less. We tested in vitro the effect of chlorogenic, caffeic, and ferulic acid on fumonisin B production in F. verticillioides. All three compounds inhibit fumonisin B accumulation, caffeic acid being the most efficient with that regard. We investigated the mechanisms by which these phenolic acids may exert their inhibitory properties and analyzed whole genome expression levels by RNA-seq. Sequenced reads were mapped to the reference genome of F. verticillioides and results were analyzed according to the current annotation available at the Fusarium Comparative Database. Doing so, we identified 175 and 1133 potential new genes and transcripts, respectively. We also found that the genes involved in the fumonisins biosynthetic pathway are all inhibited in the presence of any of the three tested phenolic acids. Finally, we identified sets of genes that are regulated specifically by a given phenolic acid, and others that follow similar patterns in all tested conditions. As a whole, our results show a large re-organization of Fusarium’s transcriptome upon phenolic acid treatment. Keywords: Fusarium, secondary metabolite, RNA-seq, antioxidant

36

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

Molecular network of nitrate sensing and assimilation in Fusarium fujikuroi 1

1,2

A. Pfannmüller , P. Wiemann , B. Tudzynski

1

1

Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, Münster, Germany ; 2Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, WI, United States E-mail: [email protected]

The phytopathogenic ascomycete Fusarium fujikuroi produces a broad spectrum of interesting secondary metabolites, including the agriculturally applied plant hormone gibberellic acid (GA3), the red pigment bikaverin and the fungal toxin fusarin C. The biosynthesis of these three secondary metabolites depends greatly on the amount and quality of the available nitrogen source. At high nitrogen concentrations, the biosynthesis of gibberellins and bikaverin are repressed on the transcriptional level, while fusarin C genes are repressed at nitrogen starvation conditions. Because of this impact on secondary metabolism, the detailed understanding of the nitrogen sensing and regulatory network in F. fujikuroi is of great interest. Previous work has shown that F. fujikuroi is able to sense the availability of ammonium by the ammonium-transporter MepB and the glutamine synthetase GS. However, it is not known if nitrate is sensed as a separate nitrogen source or + after its metabolization to NH4 and glutamine. Therefore, we analyzed orthologues of the Aspergillus nidulans nitrate-specific activator NirA, the nitrate reductase NiaD and the nitrate transporter NrtA, to gain more insights on the nitrate sensing and assimilation process in F. fujikuroi. We show the impact of FfNirA on the expression of certain nitrate-assimilatory genes and its subcellular localization depending on the nitrogen availability and the activity of putative nitrate sensors. Since the deletion of ffnirA, but not ffniaD led to a deregulation effect on secondary metabolism under nitrate sufficient conditions, we are investigating the putative role of FfNrtA as a nitrate transceptor and screen for additional FfNirA-controlled genes that might be involved in the nitrate sensing of F. fujikuroi. Keywords: Fusarium fujikuroi, secondary metabolism, nitrate, sensing

37

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

The linoleate synthase gene lds1 thoroughly affects conidiogenesis and fumonisin synthesis in Fusarium verticillioides 1

1

2

2

3

2

V. Scala , M. Reverberi , E. Camera , M. Ludovici , C. Dall’Asta , M. Cirlini , 4 4 4 1 P. Giorni , R. Gregori , P. Battilani , C. Fanelli 1

Environmental Biology Department, University of Rome “Sapienza”, Rome, Italy; 2Laboratorio di Fisiopatologia Cutanea e Centro Integrato di Metabolomica, Istituto Dermatologico San Gallicano IRCCS, Roma, Italy; 3Department of Organic and Industrial Chemistry, Food Chemistry & Natural Substances Unit, University of Parma, Viale G.P. Usberti 17/A - 43100 Parma, Italy; 4Istituto di Entomologia e Patologia Vegetale, Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy E-mail: [email protected]

Fusarium verticillioides is one of the most important fungal pathogens to cause ear and stalk rot in maize, even if frequently asymptomatic, producing the harmful series of compounds named fumonisins. Plant and fungal oxylipins have been shown to play a crucial role in determining the outcome of the interaction between the pathogen and its host in some pathosystems. Moreover, oxylipins result as factors able to modulate the secondary metabolism in fungi. In this study we inactivate the resident copy of the linoleate diol synthase 1 gene (lds1-acc. N. FVEG_09294.3) of Fusarium verticillioides strain (ITEM 10027). LDS1 produces mainly 8-HPODE and subsequently different di-HODEs. These specific oxylipins are involved in the control of sexual/asexual reproduction, secondary metabolism and pathogenicity in Aspergillus nidulans (Tsitsigiannis and Keller, 2007). This study is aimed to find the function of this gene in F. verticillioides. The genomic organization of lds1 gene is highly polymorphic. Thus the most affected phenotype of lds1 mutant strains herein obtained presented multiple insertion events. All the mutant strains are profoundly altered in asexual reproduction. Notably, the lds1 strains hyper-sporulate and the one (T) with multiple insertion 9 7 events are the most affected in this feature (8,56 x 10 vs 6,40 x 10 conidia/mL of the WT). Most important, mutant strains present a fumonisin profile (B series and minor analogues) alteration directly proportional to the number of insertions, being, also in this case, the T strain the most affected. This peculiar profile of an oxylipin impaired mutant, i.e. hyper-sporulating and mycotoxin hyper-producer, reminds the behavior of the ppoB mutant strain in A. nidulans (Tsitsigiannis and Keller, 2007), being even ppoB a lds-coding gene. These results indicate that also in F. verticillioides oxylipins play a major role in driving the fungus lifestyle and these active compounds could act as repressor/inducer on asexual reproduction and the secondary metabolism of this fungus. Keywords: oxylipins, Fusarium, ppo, fumonisin

38

KEYNOTE LECTURE SESSION 2: SECONDARY METABOLITES BIOCHEMISTRY, BIOSYNTHESIS, FEED AND FOOD SAFETY



Metabolisation of deoxynivalenol in planta: Old and new compounds and their role in food safety 1

1

1

1

1

1

F. Berthiller , V. Nagl , B. Kluger , E. Varga , A. Malachova , C. Büschl , H. 1 2 1 3 1 Schwartz , M. Lemmens , R. Schuhmacher , G. Adam , R. Krska University of Natural Resources and Life Sciences, Vienna, Austria (BOKU); 1Department IFA-Tulln, Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, 3430 Tulln, Austria; 2Department IFA-Tulln, Biotechnology in Plant Production, 3430 Tulln, Austria; 3 Department of Applied Genetics and Cell Biology, 1190 Vienna, Austria E-mail: [email protected]

Fusarium mycotoxins are prone to be metabolised by growing plants on the field. The toxins can be chemically altered as part of the plants´ defense against fungal invasion and harmful xenobiotics, such as deoxynivalenol (DON), are more or less efficiently detoxified by various crops. The occurring metabolites, sometimes referred to as “masked mycotoxins”, can partly explain the mode of action of plant resistance. Furthermore, masked mycotoxins are also transferred into food. The possible hydrolysis of these metabolites back to their toxic parents during mammalian digestion raises concerns, as the total mycotoxin load of consumers might be underestimated. We employed a metabolomics LC-HR-MS based approach using in vivo stable isotopic labelling combined with a newly developed sophisticated software tool (MetExtract) to extract biological features originating from true metabolites. Flowering ears were inoculated with a mixture of non-labelled and 13C labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides DON and the known DON-3-glucoside (D3G), eight further DONbiotransformation products were found by the untargeted screening approach. These metabolites include two DON-glutathione forms and their processing products DON-Scysteine and DON-S-cysteinyl-glycine. In a recent survey, 374 beer samples from 38 countries were analysed for the presence of DON and D3G. In total, 77% of all beers contained DON, while even 93% contained D3G above the limit of detection. Average concentrations of all beers were 8.4 µg DON/L and 6.9 µg D3G/L. The highest contamination was detected in a pale beer from an Austrian microbrewery with 89 µg DON/L and 81 µg D3G/L. While the average contamination of beer is not of toxicological concern for moderate beer drinkers, heavy beer consumption considerably contributes to the overall DON intake. We have been able to describe the fate of DON-3-glucoside during digestion with in vitro and in vivo models. These results suggest that DON-3-glucoside is little bioavailable and readily hydrolysed to DON during digestion. In rats the liberated DON is partially converted to DOM-1 and excreted in faeces. DON is also taken up in the blood stream and metabolised to DON-glucuronide prior to excretion by urine. As the majority of D3G is excreted in faeces after hydrolisation, D3G in food and feed seems to have a significantly lower toxic equivalency compared to DON. Due to the differences regarding the anatomy and gut microbiota, the bioavailability and more importantly the metabolisation may be species dependent. Further studies, including other animal models are warranted. Keywords: masked mycotoxins, deoxynivalenol, deoxynivalenol-3-glucoside, digestion

39

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

Cellular adaptations for trichothecene biosynthesis in Fusarium graminearum 1

1,2

1

1

J. Menke , J. Weber , K. Broz , H. C. Kistler 1

Department of Plant Pathology, University of Minnesota, St. Paul, 55108, USA; Phytopathologie, Universität Hamburg D-22609 Germany E-mail: [email protected]

2

Molekulare

Several species of the filamentous fungus Fusarium colonize plants and produce toxic small molecules that contaminate agricultural products, rendering them unsuitable for consumption. Among the most destructive of these species is F. graminearum, which causes disease in wheat and barley and often infests the grain with harmful trichothecene mycotoxins. Synthesis of these secondary metabolites is induced during plant infection or in culture in response to chemical signals. Our results show that trichothecene biosynthesis involves a complex developmental process that includes dynamic changes in cell morphology and the biogenesis of novel subcellular structures. Two cytochrome P-450 oxygenases (Tri4p and Tri1p) involved in early and late steps in trichothecene biosynthesis were tagged with fluorescent proteins and shown to co-localize to vesicles we named “toxisomes.” Toxisomes, the inferred site of trichothecene biosynthesis, dynamically interact with motile vesicles containing a predicted major facilitator superfamily protein (Tri12p) previously implicated in trichothecene export and tolerance. The immediate isoprenoid precursor of trichothecenes is the primary metabolite farnesyl pyrophosphate. Changes occur in the cellular localization of the isoprenoid biosynthetic enzyme HMG CoA reductase when trichothecene non-induced cultures are transferred either to trichothecene inducing or noninducing media. Initially localized in the cellular endomembrane system, HMG CoA reductase, upon trichothecene induction, increasingly is targeted to toxisomes. Metabolic pathways of primary and secondary metabolism thus may be coordinated and co-localized under conditions when trichothecene synthesis occurs. Keywords: mycotoxin, localization, secondary metabolism, isoprenoids

40

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

Ecological role of mycotoxins produced by Fusarium graminearum 1

1

1

2

2

1

M. Abid , L. Fayolle , C. Héraud , P. Mangin , L. Falchetto , N. Gautheron , J. 1 1 1 1 Laurent , E. Gautheron , V. Edel-Hermann , C. Steinberg 1

INRA, UMR1347 Agroécologie 17 rue Sully, BP 86510, F-21000 Dijon, France; 2INRA, UE Domaine d’Epoisses, F-21110 Bretenières, France E-mail: [email protected]

Fusarium graminearum is a plant pathogenic fungus, producing mycotoxins some of which remain in the crop residues let in the field after harvest. Whether the presence of mycotoxins in the crop residues gives an advantage to F. graminearum to survive and develop a primary inoculum in the presence of the whole soil biota including fungi, bacteria, protozoa, nematodes and earthworms was tested. The impact of deoxynivalenol (DON) on the soil communities was evaluated in the field and in microcosms, in wheat and in maize residues under tillage and no-tillage conditions. The disease development and the yield were noted in the field experiment. Some DON resistant active fungal decomposers and nitrogen fixing bacteria were picked and the dynamics of F. graminearum was observed in their presence, in the presence or absence of DON. DON in crop residues had an impact on the biotic components of the soil but the impact depended on the communities and on the location of the residues. The molecular biomass showed that fungal and bacterial densities were significantly affected by DON. The latter played significant role on the structure of bacterial and protozoan community while the nematodes and fungal communities remained unaffected. However, the minimal inhibitory concentration tests revealed that the susceptibility of some competitive fungal strains to DON was dose-dependent. Earthworms (Lumbricus terrestris) were not affected by the presence of mycotoxin. The degradation of DON in the residues was dependent on the time, the location of residues and the soil biota. Obviously DON gave no advantage for the survival and development of primary inoculum during the decomposition of crop residues in the soil. Moreover fungal decomposers can be selected on their enzymatic potential towards organic matter more than on the DON resistance to increase the degradation of the straw left at the surface and limit the subsequent development of F. graminearum. Keywords: Biological control, habitat, soil ecology, saprophytic phase

41

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY

Elucidation of the F. biosynthetic gene cluster



graminearum

BIOCHEMISTRY,

butenolide

L. J. Harris, A. Johnston, W. Bosnich, A. Leblanc, B. Blackwell, D. Schneiderman Eastern Cereal & Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, Ontario K1A 0C6 Canada E-mail: [email protected]

Fusarium graminearum is a significant pathogen in temperate climes worldwide, causing head blight in small grain cereals like wheat, barley, oats as well as ear and stalk rot in maize. This fungus is capable of producing a wide range of secondary metabolites, including acetyldeoxynivalenol, zearalenone, culmorin and butenolide. We have been exploiting the genomic resources of F. graminearum to characterize genes and gene clusters involved in secondary metabolism. FGSG_08079 (But1) was previously shown to be required for butenolide production (Harris et al., 2007) and resides within a gene cluster (FGSG_08077 – FGSG_08084) that is highly induced under conditions also favorable for trichothecene biosynthesis. The FGSG_08077 – FGSG_08084 cluster is expressed within 24 hours of F. graminearum infection of wheat and barley heads and maize ears. We have disrupted six additional genes within this cluster and conducted metabolite profiling of the mutant strains grown in vitro using HPLC and NMR. FGSG_08080 (But2) encodes a Zn(2)-Cys(6) zinc binuclear cluster protein whose disruption prevents induction of this gene cluster. Two of the candidate structural genes (FGSG_08081 and FGSG_08083) are required for butenolide biosynthesis while disruption of two other genes only reduced butenolide production. The predicted activities of the required proteins correlate well with the proposed butenolide pathway. We also observed that disruption of FGSG_08077 and FGSG_08081 led to reduced 15-ADON production in vitro. Although wheathead inoculation with these gene disruptants also resulted in significantly less DON in planta, there was no significant difference when normalized against the amount of fungal genomic DNA. The role of butenolide in fungal biology is not known; the loss of butenolide biosynthesis does not result in the dramatic loss of virulence observed with trichothecene nonproducers. Keywords: butenolide, graminearum

42

gene

cluster,

secondary

metabolism,

Fusarium

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

Fusarium graminearum in depth: a novel method to identify new metabolites by isotopic labelling and high resolution mass spectrometry 1,2

1

1

1

P. M. Cano , E. Jamin , S. Tadrist , P. Bourdaudhui , M. Péan 1 1,2 6 1,2 Debrauwer , I. P. Oswald , M. Delaforge , O. Puel

3,4,5

, L.

1

INRA, UMR 1331, Toxalim, Research Center in Food Toxicology, F-31027 Toulouse, France; Université de Toulouse, INP, Toxalim, F-31076 Toulouse, France; 3CEA, DSV, IBEB, Groupe de Recherches Appliquées en Phytotechnologie, F-13108 Saint-Paul-les-Durance, France; 4CNRS, UMR Biologie Végétale & Microbiologie Environnementale, F-13108 Saint-Paul-les-Durance, France; 5AixMarseille Université, F-13108 Saint-Paul-les-Durance, France; 6CEA Saclay, iBiTec-S, SB2SM and URA CNRS 8221, F-91191 Gif sur Yvette, France E-mail: [email protected] 2

Characterization of fungal secondary metabolomes has become a challenge of great interest in the last decades due to the emergence of fungal threats to natural ecosystems and public health; and also due to the industrial interest of many of these molecules. In view of this, the aim of the present study was to develop an integrated approach to analyse fungal metabolomes. The method we present hereby combines high resolution mass spectrometry and double isotopic labelling which efficiently enabled the unambiguous determination of exact chemical formulas, getting rid of problems coming from interference of nonbiological molecules. More precisely, the Aspergillus fumigatus strain NRRL 35693, an extremely hazardous human pathogen and the Fusarium graminearum strain PH1, a devastating plant pathogen, were grown on wheat grains (Triticum aestivum) with different isotopic enrichments: (1) naturally enriched grains, (2) 13 13 15 grains enriched 96.88% C, (3) grains enriched with 53.37% C and 96.8% N. Methanol extracts of each culture was then analysed by reversed phase liquid chromatography coupled to LTQ-Orbitrap mass spectrometer. Data of the 3 cultures were cross-analysed with an in-house developed software. Metabolites were characterized with the metabolite database, Antibase 2012, annotated with MS/MS experiments and identified by comparison with standards when possible. The method was firstly successfully validated with the well-known metabolome of Aspergillus fumigatus. Application of the method on the metabolome of Fusarium graminearum allowed the characterization of 37 new compounds including fusaristatin A which had never been isolated from this specie before, bringing a new perspective on the toxicity of this fungus. This kind of analysis will undoubtedly facilitate the study of fungal metabolomes. Keywords: Fusarium graminearum, metabolome, isotopic labelling, HPLC-FT-MS

43

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

Evidence for birth-and-death evolution and horizontal transfer of a mycotoxin biosynthetic gene cluster in Fusarium 1

2

3

3

1

R. H. Proctor , F. Van Hove , A. Susca , G. Stea , M. Busman , T. van der 4 4 1 3 Lee , C. Waalwijk , T. J. Ward , A. Moretti 1

United States Department of Agriculture, Agriculture Research Service, National Center for Agricultural Utilization Research, Peoria, Illinois, USA; 2Université catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology, Mycothèque de l’université catholique de Louvain (BCCMTM /MUCL), Louvain-la-Neuve, Belgium; 3National Research Council, Institute of Sciences of Food Production, Bari, Italy; 4Plant Research International B.V., Wageningen, The Netherlands E-mail: [email protected]

In fungi, genes required for synthesis of secondary metabolites are often clustered. The FUM gene cluster is required for synthesis of fumonisins, a family of toxic secondary metabolites produced by species in the Fusarium (Gibberella) fujikuroi species complex (FFSC). Fumonisins are a health and agricultural concern because their consumption is epidemiologically associated with cancer and neural tube defects in humans and other animals. Among FFSC species, the FUM cluster is uniform in gene order and orientation, but located in different genomic positions. Phylogenetic analyses indicated discord between species phylogenies and FUM gene-based phylogenies. Subsequent constraint analyses confirmed the discord, and analyses of variation in synonymous sites indicated that cluster divergence predated, in some cases, and postdated, in one case, divergence of lineages of Fusarium in which the cluster occurs. The results are not consistent with the discord resulting from trans-species evolution of ancestral cluster alleles, or with interspecies hybridization, but are consistent with duplication of the cluster within an FFSC ancestor and subsequent loss and sorting of paralogous clusters in a manner consistent with the birth-and-death evolution seen in several multigene families. Although the results are also consistent with horizontal transfer of the cluster, such a model is less parsimonious because it requires multiple transfer events from unknown but related donors to multiple FFSC recipients. However, the analyses do provide strong support for horizontal transfer of the cluster from FFSC to another Fusarium lineage. Thus, despite conservation of gene organization within it, the Fusarium FUM cluster has had a complex evolutionary history. Keywords: fumonisin, secondary metabolism, biosynthetic gene cluster

44

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

'Awaking' silent gene clusters in the rice pathogen Fusarium fujikuroi 1,2

1

1

2

S. M. Rösler , E. M. Niehaus , J. J. Espino , H. U. Humpf , B. Tudzynksi

1

1

Institute for Biology and Biotechnology of Plants, Westfälische Wilhelms-University, Schlossplatz 8, D-48149 Münster, Germany; 2 Institute of Food Chemistry, Westfälische Wilhelms-University, Corrensstraße 45, D-48149 Münster, Germany E-mail: [email protected]

The filamentous fungus Fusarium fujikuroi is a phytopathogenic ascomycete causing the bakanae disease (“foolish seedlings”) in rice plants. This disease is triggered by the best known secondary metabolites produced by the fungus, namely gibberellins. Additionally, F. fujikuroi is able to produce several other well investigated secondary metabolites which we can easily detect and quantify by now (i.e. bikaverin, fusarubin, fusarin C). Besides these known ones, the fungus also possesses the potential to produce a broad spectrum of further, yet unknown, secondary metabolites. A genome-wide bioinformatical screening approach revealed that the F. fujikuroi genome encodes 45 key enzymes for secondary metabolite production, like 18 polyketide synthases (PKSs) and 16 nonribosomal peptide synthetases (NRPSs), all organized in putative gene clusters. However, first microarray analyses showed that the majority of these gene clusters is silent in the wild type under the tested standard conditions (low and high nitrogen concentration, alkaline and acidic pH), which could explain the limited knowledge of produced metabolites. The aim of this project is now to activate silent pathways of F. fujikuroi in order to discover and identify novel secondary metabolites. Therefore, key enzymes (PKSs) and cluster internal transcription factors are overexpressed under control of the constitutive gpd promoter. Comparative expression and product analyses (Northern blot, qPCR, HPLC/LC-MS) of the wild type and the mutant strains are ongoing. Furthermore, previous experiments indicated that fungal-plant interaction triggers the expression of known secondary metabolite clusters. Hence, the expression of the novel cluster genes will be monitored during rice infection and colonization in comparison to the expression on maize plants, which are no natural hosts of F. fujikuroi, in order to gain further insight into the specificity fungal-plant interaction. Keywords: Fusarium fujikuroi, secondary metabolites, PKS, infection

45

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

The type of interaction between type B Trichothecenes on the intestine varies with the dose 1,2,3

1,2

1,2

1,2

I. Alassane-Kpembi , M. Kolf-Clauw , T. Gauthier , R. Abrami , F. A. 3 1, 2* 1,2 Abiola , I. P. Oswald , O. Puel 1

INRA, UMR 1331 Toxalim, Research center in Food Toxicology, F-31027, Toulouse France; Université de Toulouse, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France; 3Institut des Sciences Biomédicales Appliquées, Cotonou, Bénin E-mail: [email protected] 2

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although cytotoxic effects of individual mycotoxins have been widely studied, data on combined toxic effects of mycotoxins are limited. The aim of this study was to assess interactions that occur in situations of co-exposure to type B trichothecenes. Proliferating Caco-2 cells were exposed to increasing doses of type B trichothecenes, alone or in binary or ternary mixture. MTT and Neutral red uptake, respectively linked to mitochondrial and lysosomial integrities, were used for measurement of intestinal epithelial cell viability. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON400/500 pbb) and high (>1000 ppb) toxin concentration from weekly mean temperatures and duration of high humidity around flowering and harvesting were constructed. The observed proportions of cases above 400/500 ppb were 8, 8, 26%, and of above 1000 ppb were 5, 4, 15%, in spring wheat barley and oat (n=183, 252, 432), respectively. Using the prediction as a guide, the chance of finding positives above 400/500 ppb in the population changed to 80, 14, and 37%, above 1000 ppb to 56, 11, and 31% in wheat, barley and oat respectively. In smaller populations (n=130, 174, 339), where also total occurrence of DON producing species Fusarium graminearum and F. culmorum was used as a predictor, the chances of finding positives above 400/500 ppb were 85, 68, and 39%, above 1000 ppb 89, 75%, and 77% in wheat, barley and oat, respectively. Using solely the information on the occurrence of the DON producers, the chances of finding positives above 400/500 ppb were 41, 52, and 66%, 1000 ppb 67, 75%, and 77%. Prediction based of spatially rather coarse gridded weather data points out very effectively high risk regions and fields of spring wheat. Though useful, the prediction is less powerful with barley and oat. Using information on the occurrence of DON producing species improves strongly the power of the prediction. Its usefulness will further be further enhanced with improving spatial resolution. Keywords: Fusarium, mycotoxin, forecasting

78

SESSION 5: DISEASE CONTROL AND FORECASTING MODELS

Forecasting of Fusarium Head Blight and Deoxynivalenol in Wheat with FusaProg to support Growers and Industry 1

1

1

2

H. R. Forrer , T. Musa , A. Hecker , F. Mascher , S. Vogelgsang

1

1

Research Station Agroscope Reckenholz-Tänikon ART, Reckenholzstrasse 191, CH-8046 Zürich; Agroscope Changins-Wädenswil ACW, Route de Duillier 50, CH-1260 Nyon 1, Switzerland E-mail: [email protected] 2

Derived from the determination of fusaria and deoxynivalenol (DON) in 300 wheat samples from 2001-2003 of the Swiss canton Aargau and the analysis of the effect of the corresponding cropping and weather data, we designed in the Fusarium graminearum (FG) and DON forecasting system www.fusaprog.ch (Musa et al. 2007, OEPP/EPPO Bulletin, 37,2,283-289). FusaProg is based upon a division of the cropping system into four groups, namely wheat grown with or without maize as previous crop as well as plough or conservation tillage. Mean values for the incidence of FG and the DON-content of the wheat samples of the four groups were used as starting values to calculate plot specific and regional FG and DON risks. In addition other factors as the wheat cultivar and, most important, the actual weather and growth stage of wheat are considered. FusaProg was validated with another set of Swiss data and optimised with findings of FHB onfarm experiments (Vogelgsang et al. 2011, Mycotoxin Research 27:81-96). The validation of FusaProg with 82 Swiss samples from 2004-2008 showed that 82% of the plot specific forecasts of DON-contaminations above or below a threshold of 0.5 ppm were correct. A corresponding analysis of a Bavarian dataset with 547 samples from 1993 to 2000 resulted in 73% correct appreciations. However, using 1 ppm as threshold, originally used to develop the FusaProg forecast, the ratio of correct estimates increased up to 87%. Regional maps of FusaProg displaying the FG infection risk during anthesis and the DON contamination risk until harvest were used successfully to support the industry from 2007 to 2012 by the transfer of wheat. Actually we try to optimise the reliability of FusaProg using a new FG infection risk model developed with data from FG spore catches from 2008 to 2010. First results are promising and will be presented. Keywords: Fusarium, mycotoxin, forecast, sporetrap

79

SESSION 5: DISEASE CONTROL AND FORECASTING MODELS

Mycotoxins risk assessment in cereals and corn, from monitoring to predictive models A. Froment, A. Nussbaumer, T. Varraillon SYNGENTA 1 avenue des Prés 78286 Guyancourt France E-mail: [email protected]

Several species of Fusarium can produce mycotoxins during the growing period of the crops. At harvest, these toxins may be detected and sometimes at high level in cereals and corn grain depending of the year. EU Food Safety Regulation has fixed thresholds on these mycotoxins concerning all the grain food process. Since 2000, Syngenta has organised a large monitoring of more than 26 000 plots. Agronomic and climatic data and grain samples have been collected for mycotoxins analysis in France. To minimize the risk, Syngenta has developed Good Agriculture Practices recommendation (variety tolerance, crop residue management …). Over the years, the database has been used to assess and define models to predict the mycotoxins risk before harvesting. Predictions are based on different agro-climatic statistical models. Yearly monitoring improve their accuracy. Qualimetre® was the first service in France to forecast the grain mycotoxins level for soft wheat in 2004, durum wheat in 2005 and corn in 2006. After calculation which integrates local agronomic practices and extended wheather information, the grain collectors receive reports with quantification of mycotoxins levels by agronomic practices according to area of collect and for each plot the probability to be under the grain regulation threshold. The forecast is available one month before harvesting for deoxynivalenol in soft and durum wheat and for deoxynivalenol, zearalenone and fumonisins in corn. These models are now widely used by grain collectors to operate with the mycotoxins risk, food safety and regulation. About sixty grain collectors used Qualimetre® in 2012 surveying 4.5 million hectares in France and Italy. Since 2009, a new model to forecast T2-HT2 risk is under development on spring barley. Keywords: mycotoxins, predictive model, food safety, regulation

80

SESSION 5: DISEASE CONTROL AND FORECASTING MODELS

Biological strategy applied to maize preharvest agroecosystem in Argentina to prevent fumonisin contamination 1, 2

M. Etcheverry

, A. Nesci

1, 2

1,3

, P. Pereira , M. Sartori

1,3

1

Laboratorio de Ecología Microbiana. Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas Físico Químicas y Naturales. Universidad Nacional de Río Cuarto, Ruta Nacional N° 36 Km 601, Río Cuarto (5800), Córdoba, Argentina - 2Members of the Research Career, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina - 3Doctoral Fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina E-mail: [email protected]

Biocontrol activity of Bacillusamyloliquefaciens, Microbacteriumoleovorans against F. verticillioides colonization of field-grown maize and fumonisin production in grains at harvest were investigated. Seed treatment during presowing and spraying of maize ears during flowering were evaluated as inoculation methods of selected agents. The impact of inoculation on F.verticillioides count in maize roots and in grains at harvest level as well as on fumonisin content of the grains was evaluated in field trials conducted under natural fungal infection and also alter inoculation with different strains of F. verticillioides. Treatments effect on maize growth and the diversity and activity of bacterial and fungal populatiotions naturally associated with maize roots was also assessed. Additionaly, F verticillioides count and fumonisin B1 content were significantly reduced in grains of plants grown from B. amyloliquefaciens treated seeds. Studies on inoculum production, physiological improvement and formulation process were assessed. Cells of B. amyloliquefaciens grown in liquid media amended with glycerol showed better tolerance at low aw and high survival under heat stress. Furthermore, both biocontrol agents showed accumulation of betaine and ectoine in cells of B. amyloliquefaciens and M. oleovorans can improve the tolerance of both bacteria to water potential modifications and desiccation in the process of freeze drying and production of formulated, which could improve bacterial growth and survival under conditions of low water availability and also increase the potential for biological control of F. verticillioides in field. MSB medium was select for biomass production due to the high growth and survival after freeze-drying process. Formulated were development of fresh cells and freeze-dried of B.amyloliquefaciens and M. oleovorans. The efficacy of four stickers as seed coating was studied, In field trial of maize, the pre-sowing application of freezedried formulated B. amyloliquefaciens and M. oleovorans and paraffinic oil as sticker, caused a decrease of fumonisin B 1 in grain harvest treatments including pre-sowing inoculation of F. verticillioides. Keywords: biocontrol, Bacillus amyloliquefaciens, Microbacterium oleovorans, Fumonisin B1

81

SESSION 5: DISEASE CONTROL AND FORECASTING MODELS

Maize kernel antioxidants and their potential involvement in Gibberella and Fusarium Ear Rot resistance 1

1

1

1

1

V. Atanasova-Penichon , A. Picot , L. Pinson-Gadais , N. Ponts , S. Pons , 1 1 1 1,2 3 G. Marchegay , F. Turtaut , M-N. Verdal-Bonnin , C. Barreau , J. Roucolle , 4 1 P. Carolo , F. Richard-Forget 1

INRA UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS20032, 33882 Villenave d'Ornon cedexFrance; 2CNRS, INRA UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS20032, 33882 Villenave d'Ornon cedex-France; 3Monsanto SAS Peyrehorade, Croix de Pardies, F-40300 Peyrehorade, France; 4Euralis Semences, 117 avenue de Vendôme, F-41000 Blois, FRANCE E-mail: [email protected]

Gibberella and Fusarium Ear Rot (mainly caused by Fusarium graminearum and Fusarium verticillioides, respectively) are the two main diseases affecting European maize crops. The two former fungi pose a serious threat to food safety because of their ability to produce a wide range of mycotoxins, including type B trichothecenes (produced by F. graminearum) and fumonisins (synthesized by F. verticillioides). Since 2007, type B trichothecenes and fumonisins are strictly regulated for the cereals commercialized in Europe. The maize variety is one of the key factors that can significantly influence fungal development and mycotoxin production on kernels. Plants can reduce mycotoxin accumulation by two mechanisms: metabolic transformation of the toxin and inhibition of toxin biosynthesis. This second mechanism involves the occurrence of biochemical compounds that are able to modulate the biosynthesis pathways. Our previous data showed that maize antioxidant secondary metabolites such as phenolic compounds, tocopherols and carotenoids are present in the earliest maize kernel stages, indicating that the mycotoxin-producing fungal species are likely to face them during ear colonization and initiation of mycotoxin biosynthesis. The potential involvement of maize antioxidants in plant resistance to Gibberella and Fusarium Ear Rot and mycotoxin accumulation was the focus of this work. The effect of phenolic compounds, tocopherol, and carotenoids on fungal growth and type B trichothecene and fumonisin accumulation was investigated in vitro. The highest inhibitory activities were obtained for -tocopherol and some of the phenolic compounds including ferulic acid and its dimeric forms, caffeic and chlorogenic acid. Using a set of genotypes with moderate to high susceptibility to Gibberella and Fusarium Ear Rot, we assayed the significantly lowest levels of chlorogenic acid, ferulic acid and its dimeric forms in immature kernels of the very susceptible group. Overall, our data support the fact that these compounds may contribute to resistance to Gibberella and Fusarium Ear Rot and/or mycotoxin accumulation in various maize genotypes. Keywords: Fusarium, mycotoxins, antioxidants, maize

82

SESSION 5: DISEASE CONTROL AND FORECASTING MODELS

Trichoderma gamsii 6085 as a tool for the biological control of FHB on wheat 1

1

2

2

3

S. Sarrocco , F. Matarese , L. Moncini , G. Pachetti , A. Moretti , G. 1 Vannacci 1

Department of Agriculture, Food and Environment, University of Pisa, 56124 - Pisa, Italy - 2Centro Ricerche Strumenti Biotecnici nel Settore Agricolo-forestale (CRISBA), c/o ISIS "Leopoldo II di Lorena" Cittadella dello Studente, 58100 – Grosseto, Italy - 3Institute of Sciences of Food Production, National Research Council, 70126 - Bari, Italy. E-mail: [email protected]

Fusarium head blight (FHB) is a worldwide destructive disease of small cereals, particularly wheat. The disease is caused by a complex of Fusarium spp. with Fusarium graminearum and Fusarium culmorum as the most prevalent. Associated with yield reduction, contamination of grains by mycotoxins represents the most important consequence of FHB. Trichothecenes, as deoxynivalenol (DON), and its acetylated derivates, and nivalenolo (NIV) are the most dominant mycotoxins associated with FHB of wheat. Management of FHB and its mycotoxins is based on strategies such as host resistance, agricultural practices and fungicides, but none of these methods alone is able to significantly reduce the disease. In this scenario many efforts have been initiated to identify FHB antagonists such as beneficial fungi to be used in biocontrol strategies (Sarrocco et al. Phytopathol. Med. 2012). In the last years our attention was directed towards Trichoderma gamsii 6085 as a beneficial isolate for the biocontrol of FHB. This isolate was selected for its ability to grow in presence of DON and the role of some PDR-ABC transporters in mycotoxin resistance was investigated. Under laboratory conditions the antagonistic ability, as mycoparasite and competitor for natural substrates, of T. gamsii 6085 towards F. graminearum and F. culmorum mycotoxigenic isolates was assessed in addition to a reduction of DON production (Matarese et al. Microbiology 2012). During two following growing seasons of wheat (2010/2011 and 2011/2012) T. gamsii 6085 was used as inoculant of soil before sowing and of spikes at anthesis both with interesting results. Particularly, when applied on spikes the isolate was able to colonize spikelets components, demonstrating an endophytic lifestyle and showed a reduction of both disease index and disease severity (Sarrocco et al. J. Plant Pathol 2013). Results here reported are encouraging and since this is the first report of the use of T. gamsii as biocontrol agent of FHB, further researches are scheduled in order to deeply investigate this system. Keywords: Trichoderma gamsii, FHB, biocontrol, mycotoxin

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84

KEYNOTE LECTURE SESSION 6: FUTURE CHALLENGE WORLWIDE

FOR

EUROPE

AND

The risks related to Fusarium mycotoxins at global level: emerging problems and possible solutions A. F. Logrieco Institute of Sciences of Food Production, CNR, Via Amendola 122/O, 70126, Bari, Italy E-mail: [email protected]

Fusarium mycotoxins are still a very hot topic because of the high frequency of their occurrence on a wide range of crops, especially cereals, which are the main staple food, worldwide. Several reasons are responsible of such occurrence, among which some are determined directly by human choices and other are related to natural events such as climatic changes. The need to increase the cultivation profitability for farmers is related to the extension of some crops such as durum wheat in geographical areas inappropriate for its cultivation, since this crop becomes more sensitive to Fusarium pathogenicity. On the other hand, food security to the increasing amount of the world population is causing the extension of some crop cultivation such as maize, in some emerging countries of Africa, where maize is replacing traditional crops (e.g. finger millet and sorghum) and is more exposed to the contamination of toxigenic Fusarium species. As further reason of concern, the climatic change can significantly induce the appearance of emerging problems influencing the distribution of toxigenic Fusarium species and related mycotoxins. Therefore, new mycotoxin/commodity combinations are emerging, providing evidence of a great plasticity and capability of these fungi to continuously select new genotypes provided of higher aggressiveness and mycotoxin production. In order to better control the risks related to Fusarium mycotoxin contamination of food commodities, an approach along the whole food chain, “farm to fork”, is extremely important to identify the critical points along the chain, where the major risks for mycotoxin contamination occur. Global networking, awareness and dissemination activities together with a survey on the main effective pre- and post-harvest solutions, carried out in EU project MycoRed will be provided in the presentation. The MycoRed outcome may represent effective integrated strategies for Fusarium mycotoxin minimization in food and feed chain at the global level. This presentation has been supported by the EU Project MycoRed 222690 FP7KBBE-2007-2A Keywords: Fusarium, mycotoxins, pathogenicity

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SESSION 6: FUTURE CHALLENGE FOR EUROPE AND WORLWIDE

Future challenges of Fusarium and mycotoxins on cereals in Northern Europe P. Parikka, K. Hakala, K. Tiilikkala MTT Agrifood Research Finland, Plant Production Research, FI-31600 Jokioinen, Finland E-mail: [email protected]

Expected changes in climatic conditions in the North are generally beneficial to field crop production and allow growing of more species and cultivars. Longer growing seasons enhance productivity but predicted increase in rainfall can cause risks for crop quality. The Fusarium species causing head blight on cereals are common all over Europe but their importance is different depending on the climatic conditions. The increase in importance of F. graminearum reported earlier in Central Europe has been observed during the past ten years, especially in Norway where high deoxynivalenol contents have been frequently analysed in oats in some areas. Signs of the same development have also been observed in Sweden and Finland, where DON contaminations have previously been lower. Due to environmental and economical reasons, reduced tillage and no-till practices have become more common in cereal production. In Finland, increase of F. langsethiae, the most important producer of T-2 and HT-2- toxins has already been observed on oats and barley under reduced tillage. While DON production is enhanced by high humidity, F. langsethiae can infect and produce toxins in dry conditions. F. poae also benefits of warm and dry conditions and increase risk of nivalenol contamination in grain. Interest to grow maize for silage increases with warmer growing seasons also in the North and can result in higher DON contaminations when tillage is reduced. Crop rotation is recommended to control Fusarium head blight but short rotations may not be effective enough. Insect damages on cereal heads can increase in warming conditions and lead to heavier Fusarium infections and risk of mycotoxins in grain. Chemical control of Fusarium head blight has not necessarily decreased mycotoxin contents in grain. Cultivar resistance to Fusarium infections would be the best and the most sustainable method to control mycotoxin contaminations, especially in oats. Keywords: cereals, climate, tillage, mycotoxins

86

SESSION 6: FUTURE CHALLENGE FOR EUROPE AND WORLWIDE

Climate change impacts on mycotoxins in cereal grain production H. J. van der Fels-Klerx, M. de Nijs RIKILT Wageningen UR, PO Box 230, NL-6700AE Wageningen, The Netherlands E-mail: [email protected]

Emerging mycotoxins in cereal grains include newly detected mycotoxins, as well as known mycotoxins with no or low presence in certain commodities or geographical areas that (suddenly) re-occur at high concentrations. A risk for changes in presence of mycotoxins is the quick shift of origin of raw cereal materials or the (re-)introduction of cereals in production areas. Major diving forces, however, are believed to be (projected) climate changes. Quantitative estimates on impacts of climate change on mycotoxin occurrence in cereals are, however, scarce. The EMTOX project aimed to assess the impact of climate change effects on the presence of mycotoxins in cereal grains in North West Europe in 2040, using a quantitative approach. Climate model data for the period 2031-2050, based on the IPCC A1B emission scenario, were used as the starting point. Empirical models were developed for wheat phenology and for prediction of DON concentrations in wheat (Olesen et al. Food Add. & Cont. 2012, van der Fels-Klerx et al. J. Food Protec. 2012). While climate change data were input for both models, the output of the wheat phenology model was, in addition, used in the DON prediction model. Outcome of the modelling study showed that – in general - climate change will result into increased DON contaminations in wheat in North West Europe in 2040. The predicted increase was higher for spring wheat than for winter wheat. Results showed high variation between regions (van der Fels-Klerx et al. Food Add. & Cont. 2012a,b). Given the study results, industrial and governmental food safety managers should be aware of the risks of mycotoxins in raw materials. Levels of mycotoxins need to be closely monitored, in particular in high risk situations associated with favourable climatic conditions for fungal infections and mycotoxin production. The use of predictive models will be helpful in this respect. Keywords: emerging mycotoxins, climate change, modelling, deoxynivalenol

87

SESSION 6: FUTURE CHALLENGE FOR EUROPE AND WORLWIDE

Uneven and surprising colonization of water pipes of hospitals and non-hospital sites by Fusarium oxysporum and F. dimerum 1

1

1

2

3

C. Steinberg , J. Laurent , V. Edel-Hermann , M. Barbezant , N. Sixt , F. 4,5 6 4,5 2 4,5 Dalle , S. Aho , A. Bonnin , P. Hartemann , M. Sautour 1

INRA, UMR1347 Agroécologie 17 rue Sully, BP 86510, F-21000 Dijon, France; 2Department Environment and Public Health Nancy University, Hospital Hygiene Unit, 9 Avenue de la Forêt de Haye, BP154, 54505 Vandoeuvre-Nancy Cedex, 11 France; 3Environmental Microbiology, Plateau Technique de Biologie du CHU, 2 rue Angélique Ducoudray, BP 37013, 21070 Dijon Cedex, France; 4 Parasitology and Mycology laboratory, Plateau Technique de Biologie du CHU, 2 rue Angélique Ducoudray, BP 37013, 21070 Dijon Cedex, France; 5Université de Bourgogne, UMR1347 Agroécologie 17 rue Sully, BP 86510, F-21000 Dijon, France; 6Hospital Hygiene and Epidemiology unit, Hôpital du Bocage, BP 77908, 21079 Dijon Cedex, France E-mail: [email protected]

Fusarium oxysporum and F. dimerum are ubiquitous soil-borne fungi found in terrestrial ecosystems worldwide, but they were recently detected in the water distribution systems of hospital buildings. A survey that included various hospital buildings at different locations, various non-hospital complexes and a set of private houses was conducted over 2 years in two French cities. The fungi were present only in public hospital buildings and were not detected in the other water distribution systems or in the water inlets, wherever they were. This surprising distribution can be explained by a combination of three mains factors: i) the complexity of the water distribution system of a set of regularly renovated buildings; this complexity includes the diversity of materials used for the pipes and the existence of loops in which water may stagnate; ii) the application of a sanitizing process that creates ecological voids and makes resources of interest available for opportunistic invasive fungi; iii) the real potential of strains of F. oxysporum and F. dimerum to adapt in order to exploit and to tolerate urban aquatic environments. The very low diversity among the various isolates of F. oxysporum and of F. dimerum suggests the existence of a well-adapted population (special form) of each of the two soil-borne species specialized in the colonization and exploitation of the spatial and trophic resources provided by the urbanized water supply system of public hospital buildings. The risk of fusariosis caused by such special invasive and opportunistic forms needs to be taken seriously to prevent any contamination of immunocompromised patients. Keywords: Soil-borne fungi, aquatic niche, adaptation, disease risk assessment

88

SESSION 6: FUTURE CHALLENGE FOR EUROPE AND WORLWIDE

Dermatologic infections by Fusarium species in a tropical clinic 1

1,2

1

3

A. D. van Diepeningen , P. Feng , S. Ahmed , M. Sudhadham , S. 7 1,5,6,7 Bunyaratavej , G. S. de Hoog 1

CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherland; 2The third affiliated hospital of Sun Yat-Sen University, Sun-Yat Sen University, Guangzhou, China; 3Suansunandharajabhat University, Bangkok, Thailand; 4Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; 5 Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, the Netherlands; 6Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China; 7Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. Department of Biology, Faculty of Science and Technology, E-mail: [email protected].nl

In a set of 464 fungal isolates from a dermatological ward in Thailand, forty-four strains (9.5%) proved to be Fusarium spp. The affiliated clinical diagnoses of the Fusarium infections ranged were onychomycosis (61%) or other infections of nail and skin. Multilocus DNA sequence-based genotyping of the infections was done based on partial sequences of Elongation factor 1-apha (EF1-alpha), the internal transcribed spacer (ITS) and RNA dependent polymerase subunit II (RPB2). The analysis revealed that 94% of the isolates belonged to the Fusarium solani species complex (FSSC), only one strain matched with the Fusarium oxysporum (FOSC) complex 33, while six others belonged to the Fusarium incarnatumequiseti species complex (FIESC). No members of the Giberella fujikuroi Species Complex (GFSC) were detected. Especially within the FSSC different sequence types could be recognized; one cluster being similar to Fusarium falciforme (previously known as Acremonium falciforme). We discuss the results of our dermatological study in comparison to previous similar studies in tropical and moderate areas: different species complexes and different ratios between species complexes were observed. Furthermore, in our study we saw approximately equal numbers of male and female patients, while in the other studies infections in women were prevalent. Keywords: Fusarium solani species complex, Fusariosis, Onychomycosis

89

90

POSTER PRESENTATIONS

91

92

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P1 - RNA-Seq analysis reveals new gene models and alternative splicing in the Fusarium graminearum C. Zhao

1,2,3,4

1,2

2,5

3

, C. Waalwijk , P. J. G. M. de Wit , D. Tang , T. van der Lee

1,2

1

Plant Research International, Wageningen, The Netherlands; 2Graduate School Experimental Plant Sciences, Wageningen, The Netherlands; 3State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; 4Graduate University of Chinese Academy of Sciences, Beijing 100049, China; 5Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands E-mail: [email protected]

The genome of Fusarium graminearum has been sequenced and annotated, but correct gene annotation remains a challenge. In addition, posttranscriptional regulations, such as alternative splicing and RNA editing, are poorly understood in F. graminearum. Here we took advantage of RNA-Seq to improve gene annotations and to identify alternative splicing and RNA editing in F. graminearum. In total 25,720,650 reads were generated from RNA-Seq. Using the genome annotation in Broad database, transcripts were detected for 84% of the predicted genes. 74.8% of the reads matched to exonic regions, 10.6% to untranslated regions (UTRs), 12.9% to intergenic regions and only 1.7% to intronic regions. We identified and revised 655 incorrectly predicted gene models (10% of all tested gene models), including revisions of intron predictions, intron splice sites and prediction of novel introns. 231 genes were identified with two or more alternative splice variants, mostly due to intron retention. In-frame analysis showed that the majority of the alternatively spliced transcripts identified in F. graminearum cause premature termination codons (PTCs), of which most are located in intronic regions and these transcripts are potential targets of the nonsense mediated mRNA decay (NMD). Apart from PTC isoforms, some alternatively spliced transcripts encoding proteins with diverse length were identified. The effects of the diversity in length on the biological function of proteins are still unknown, but several functions including binding properties, intracellular localization, enzymatic activity or stability might be affected. Interestingly, the expression ratios between different transcript isoforms appeared to be developmentally regulated. Surprisingly, no RNA editing was identified in F. graminearum. Moreover, 2459 novel transcriptionally active regions (nTARs) were identified and our analysis indicates that some of these could be genes that were missed in the annotation. Finally, we identified the 5’ UTR and/or 3’ UTR sequences of 7666. A number of representative novel gene models and alternatively spliced genes were validated by reverse transcription polymerase chain reaction and sequencing of the generated amplicons. Our results demonstrate that posttranscriptional regulations can be studied efficiently using our developed RNA-Seq analysis pipeline and may be important in adaptation of F. graminearum to changing external environmental conditions that occur during different growth stages. Keywords: transcription, RNA-Seq, regulation, secondary metabolism 93

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P2 - Genome-wide transcriptional response to ambient pH and Pac1 regulatory factor in Fusarium graminearum 1

1

1

J. Merhej , N. Ponts , F. Richard-Forget , C. Barreau

1,2

1

INRA, UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS20032, 33883 Villenave d'Ornon-France; CNRS, UR1264 MycSA, 71 Avenue Edouard Bourlaux, 33883 Villenave d'Ornon-France E-mail: [email protected] 2

The pathogenic fungus Fusarium graminearum produces type B trichothecene mycotoxins during cereal plants infection. Trichothecenes accumulate in cereal grains and represent a threat for health. Expression studies of the Tri genes implicated in trichothecene biosynthesis have demonstrated that acidic extracellular pH is a determinant inducer. Actually, it has been shown that the FgPac1 pH regulatory factor negatively regulates the expression of Tri genes under neutral or basic pHs. Beside their role in regulating secondary metabolites in fungi, Pac transcription factors are known to regulate various classes of genes, especially the genes whose roles involve communication with the environment. In this study, a genome-wide transcriptional analysis conducted in two different pH conditions using a strain deleted for FgPac1 and a strain expressing a constitutively active form of FgPac1was carried out to investigate the global regulation by the pH in F. graminearum. The data obtained identified a set of genes related to various functions whose expression is affected by the change in pH. Our results also point toward a potential role of a regulation by calcium in response to the ambient pH and identified a γ-aminobutyric acid (GABA) shunt activated in response to acidic pH. Finally, a clustering approach followed by cisregulatory motifs search highlights the presence of complex stress-response regulatory circuits impacted by the change in external pH. Keywords: Fusarium, pH, Pac1, microarrays

94

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P3 - Significance of the hydrophobin FgHyd5p in Fusarium graminearum E. Minenko, R. F. Vogel, L. Niessen Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising, Germany. E-mail: [email protected]

Fusarium graminearum and Fusarium culmorum are causative agents of beer gushing. Class II hydrophobins have been demonstrated to induce it. Hydrophobins are a group of small secreted proteins ubiquitously found in filamentous fungi. Some hydrophobins were shown to have functions in fungal development, while others seem to lack known function. Also, cellular functions of FgHyd5p are still unknown. FgHyd5p, the F. graminearum homologue to FcHyd5p (F. culmorum) is a small secreted protein typified by the presence of 8 cysteine residues at conserved positions. FgHyd5p is characterized by formation of low stability aggregates and solubility in organic solvents. We showed that FgHyd5p does not have an effect on morphology of F. graminearum since colony morphology and growth rate, hyphal length and diameter as well as biomass production were shown to stay unaffected in Fg∆hyd5 knockout strains. Knockout of the fghyd5 gene causes a reduction of spore length and an increase in spore numbers. Using an F. graminearum reporter strain expressing sGFP under the control of the Phyd5 promoter we found that FcHyd5p was not expressed in media with different carbon or nitrogen sources, respectively. Moreover we did not observe any pH dependent fghyd5 gene expression. FgHyd5p showed an effect on the hydrophobicity of hyphae resulting in an easy wettable phenotype of transformants. Results of our studies also showed that FgHyd5p has an effect on the virulence of F. graminearum. Wheat and barley plants infected with spores from an Fg∆hyd5 knockout strain at EC 0-9 were significantly longer in EC 12 as compared to plants that were infected with the F. graminearum parent strain. Using the F. graminearum sGFP reporter strain and histological analysis we showed that the gene is expressed during plant infection and may thus play a role as a virulence factor during the infection of cereals. Keywords: Fusarium, hydrophobin, expression, virulence

95

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P4 - Studying gene expression in fungus and planta to understand the interaction Fusarium verticillioidesmaize 1

1

2

I. Lazzaro , P. Battilani , A. Lanubile , A. Marocco

2

1

Institute of Entomology and Plant Pathology; 2Institute of Agronomy, Genetics and Field Crops, University Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy E-mail: [email protected]

Fusarium verticillioides is a plant pathogen able to produce fumonisin in maize kernels. A study on the pathosystem F. verticillioides-maize has been developed speculating on both in vitro and in planta perspectives. The former studied the effects of temperature (T) and water activity (a w) on fumonisin B (FB) production and expression of FUM genes in F. verticillioides strains. The latter monitored which genes were differentially expressed in resistant and susceptible maize lines at several time points after inoculation by a fumonisin-producing strain of F. verticillioides. The in vitro study showed that FUM gene expression was sensibly affected by aw, rather than T, indicating that fungal metabolism is more overturned by low aw than by T decrease. Most of FUM genes were highly expressed at aw=0.990 compared to 0.955, similarly to FB production, underlining that gene expression and secondary metabolite production followed the same trend. At 21 days of incubation, FUM14 and FUM3 -regulating the production of FB1 and FB2 from FB3 and FB4, respectively – were maximally expressed, while FUM21 – coding for a transcription factor for FB biosynthesis – was 10x less expressed; aw=0.900 was inhibitory for culture growth and FB production. The in planta study showed that in kernels at 48 h after inoculation (hai) about 800 genes were differentially regulated and nearly 10% assigned to the defence category. During the very early stages of incubation a small proportion of the host transcripts was induced and none of them was involved in defence processes. Early response genes encoded signalling or regulatory components. The highest number of differentially expressed genes was attained at 48 hai. The late response genes encoded effector proteins. When resistant and susceptible maize genotypes were compared, in the resistant line the expression of defence genes was detected before inoculation, while in the susceptible genotype they were induced only after pathogen inoculation. Keywords: Fusarium, maize, fumonisin, biosynthesis, defence genes

96

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P5 - Comprehensive inventory on coding and noncoding features of the genome of Fusarium fujikuroi. 1

1

2,4

2

3

C. Sieber , M. Münsterkötter , P. Wiemann , B. Tudzynski , M. Hippler , S. V. 3 3 1 Bergner , T. Bald , U. Güldener 1

Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany; 2Institut für Biologie und Biotechnologie der Pflanzen, Molecular Biology and Biotechnology of Fungi, Westfälische WilhelmsUniversität Münster, Schlossplatz 8, 48143 Münster, Germany; 3Institut für Biologie und Biotechnologie der Pflanzen, Plant Biochemistry and Biotechnology, Westfälische WilhelmsUniversität Münster, Schlossplatz 8, 48143 Münster, Germany; 4Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, WI, United States E-mail: [email protected]

The plant pathogen Fusarium fujikuroi synthesizes a vast array of secondary metabolites and causes „bakanae“ disease on rice. Here we present the fully annotated genome sequence of F. fujikuroi assembled in 12 contigs which correspond to 12 chromosomes. Compared to closely related fungi of the Gibberella fujikuroi species complex (GFC) considerable differences regarding the presence of secondary metabolism gene clusters are highlighted, and some of them might be linked to host specificity of the respective species. Utilizing DNA microarray and proteomics experiments we demonstrated a nitrogen and pH specific expression and translation. A deeper analysis of the genome sequence revealed a diversity of previously unknown interspersed repeat families which can be found in high frequency all over the genome. While some of the families occur exclusively in F. fujikuroi, others distribute throughout the GFC. DNA microarray experiments demonstrated that some elements are part of the transcriptome and still seem to propagate further in the genome. Moreover, some elements seem to have a preference to appear between collinear blocks. Main questions are: What influence do interspersed repeats have on genome structure and the speciation process of fungi? Do repeats contribute to hostpathogen interaction and specification? Keywords: Fusarium, fujikuroi, noncoding, secondary-metabolism, regulation

97

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P6 - A retrotransposon based approach for the detection of intraspecific variation among Fusarium oxysporum formae speciales 1

1

2

S. Tonti , M. Dal Prà , P. Nipoti , I. Alberti

1

1

INRAN - Istituto Nazionale per la Ricerca degli Alimenti e la Nutrizione, Via Ca’ Nova Zampieri 37, S. Giovanni Lupatoto (VR), Italy; 2DipSA - Dipartimento di Scienze Agrarie, Alma Mater Studiorum, Università degli Studi di Bologna, Viale Fanin 40, 40127 Bologna, Italy. E-mail: [email protected]

Fusarium oxysporum (Schlechtendahl emend. Snyder and Hansen) is the most diffused and destructive fungal plant pathogen. Its division in to formae speciales, accordingly to the difference in host specificity, is crucial for phytopathological diagnosis and phytosanitary purposes. To date, different approaches have been attempted in order to develop specific primers able to discriminate between the different formae speciales. Retrotransposon based techniques are good tools for the detection of the intraspecific variation among microorganisms. Furthermore polymorphic DNA bands obtained can be cloned and then sequenced in order to design specific primers. We tried a REMAP (Retrotransposon Microsatellite Amplified Polymorphism) analysis on 18 strains belonging to 10 different formae speciales. To this aim four different PCR reactions were carried on combining 4 SSR primers ((AG) 8G, (GA)8T, (GA)8C, (AC)8T) with a primer designed on the LTR region of the retrotransposon skippy. Banding patterns obtained were scored manually. Genetic distance between strains and the resulting UPMGA tree were calculated with Nei and Li/Dice algorithm implemented in the Freetree software. Polymorphic bands were not detected, while DNA banding pattern resulted to be distinctive of some formae speciales. To our knowledge, this is the first attempt to characterize the F. oxysporum species complex with a REMAP analysis. Keywords: Fusarium oxysporum, REMAP, formae speciales

98

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P7 - FCSTUA controls pathogenicity and morphophysiological traits in Fusarium culmorum 1*

2*

1

1

2

F. Spanu , M. Pasquali , B. Scherm , V. Balmas , L. Hoffman , K. Hammond3 2 1 Kosack , M. Beyer , Q. Migheli 1

Dipartimento di Agraria - Plant Pathology and Entomology Unit and Unità di ricerca Istituto Nazionale di Biostrutture e Biosistemi, Università degli Studi di Sassari, Via E. De Nicola 9, I-07100 Sassari, Italy; 2 CRP - Gabriel Lippmann, 41, rue du Brill, L-4422 Belvaux, Luxembourg; 3Wheat Pathogenomics Programme, Plant Pathology and Microbiology Department, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK E-mail: [email protected] *The first two authors have equally contributed to the present work.

Fusarium culmorum is one of the most harmful causal agents of the crown and foot root (CFR) and the Fusarium head blight (FBH) diseases on durum wheat. Here we identified and characterised the main function of FcstuA gene, an APSES protein having 99% homology to the FgstuA protein, in the biological cycle of F. culmorum. Two wild-type strains, FcUK99 (a deoxynivalenol producer) and Fc233B (unable to produce toxin in vitro) were used to FcstuA deletion by homologous recombination. The functional analysis of deletion mutants obtained from both wild-type strains showed that FcStuA mutants displayed lack of monophialides and decreased germination efficiency of conidia, stunted vegetative growth, altered pigmentation on solid substrates, and loss hydrophobicity of the mycelium. Glycolytic process efficiency was strongly impaired and growth was partially restored on glutamic acid. Growth on pectin-like sources ranked in between glucose and glutamic acid with the following order (the lowest to the highest growth): beechwood xylan, sugarbeet arabinan, polygalacturonic acid, citrus pectin, apple pectin, potato azogalactan. DON production in the mutants originating from FcUK99 strain was significantly decreased (- 95%) in vitro. Furthermore, Fcstua was shown to play an important role in pathogenicity, as ΔFcstuaA mutants underwent complete loss of pathogenicity in both CFR and FHB pathosystems on durum wheat and were unable to colonise different plant tissues (apple, potato and tomato). No differences between mutants, ectopic and wild-type strains were observed concerning the level of resistance towards four fungicides belonging to three classes, the demethylase inhibitors epoxiconazole and tebuconzole, the succinate dehydrogenase inhibitor isopyrazam and the cytochrome bc1 inhibitor trifloxystrobin. StuA, given its multiple functions in cell regulation and pathogenicity control, is proposed as a potential target for novel disease management strategies. Keywords: durum wheat, trichothecenes, polygalacturonase, fungicide resistance, APSES proteins

99

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P8 - Interactions between different Fusarium species to uncover multi-toxin synergistic mechanisms K. Bala, B. Blackwell, A. Z. Morales, A. Johnston, C. Brown, D. Schneiderman, S. Gleddie, L. J. Harris Eastern Cereal & Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, K1A 0C6, Canada E-mail: [email protected]

Fusarium head blight disease (FHB) is a major threat to wheat, barley, maize and other economically important crop plants worldwide. FHB is caused by a number of species in the genus Fusarium, including F. graminearum, F. culmorum, F. sporotrichioides and F. avenaceum. Multiple species of Fusarium have been found to infect grain in Canada. Synergistic effects of these species may cause high accumulation of mycotoxins in crops and development of more aggressive strains. We present here deoxynivalenol (DON) profiles of approximately 16 new strains of F. graminearum isolated from various regions of Canada in 2011. Based on translation elongation factor (TEF) and TRI8 gene sequencing, these strains were identified and characterized as 15-A and 3-ADON genotypes. All isolates were analyzed in vitro by HPLC to characterize metabolite profiles and in planta for disease severity. Strains producing maximum 15-ADON and 3-ADON in vitro were identified and selected for co-cultivation studies. One 15-ADON and one 3ADON strain were co-inoculated in a pair-wise fashion with five different strains of F. avenaceum and one F. sporotrichioides strain in liquid cultures. Crude culture filtrates and extracts were sampled at different time points to assess metabolite profiles by HPLC and LC-MS. LC-MS results showed a predominance of F. graminearum metabolites from the 3-ADON strain and F. avenaceum co-cultures. We are currently investigating whether 3-ADON and 15-ADON genotypes of F. graminearum interact differently with F. avenaceum and F. sporotrichioides. We are analyzing the transcriptome of F. graminearum from co-cultures of F. graminearum with F. avenaceum and F. graminearum with F. sporotrichioides to gain insights into the mechanisms of TRI gene regulation during interaction with other Fusarium species. Keywords: Fusarium, co-cultivation, 15-ADON, 3-ADON

100

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P9 - Agrobacterium-mediated insertional mutagenesis of Fusarium oxysporum f. sp. cubense for identification of key genes in the infection cycle of the pathogen 1

2

1

1

1

A. A. Rabie , T. Meyer , L. J. Rose , G. Mostert , I. Beukes , A. C. L. 3 1 Churchill , A. Viljoen 1

University of Stellenbosch, Department of Plant Pathology, Private Bag X1, Matieland 7602, South Africa, 2 University of Pretoria, Forestry and Agricultural Biotechnology Institute, Department of Microbiology and Plant Pathology, Pretoria 0002, South Africa, 3 Cornell University, Department of Plant Pathology, Ithaca, NY, USA. E-mail: [email protected]

The causal agent for Fusarium wilt of bananas, Fusarium oxysporum f. sp. cubense (Foc), is regarded as one of the most destructive pathogens in the world. The most effective way in controlling the disease is by planting resistant banana cultivars. Frequent studies on the infection biology of Foc have been conducted, but the genetic basis for pathogenicity is still poorly understood. Forward and reverse genetics have proven to be a valuable tool for identifying in vivo essential genes involved in the molecular mechanisms of pathogenicity and virulence of a pathogen. In this study, random vector integration was conducted through the implementation of Agrobacterium-mediated insertional mutagenesis (ATMT). Transformation was achieved with four A. tumefaciens strains using a vector conferring Hygromycin B resistance and expression of the green fluorescent protein gene. Vector insertion was confirmed with molecular methods and fluorescent microscopy. Transformants were screened for sporulation potential, alterations in growth rate and pigmentation, the number of T-DNA inserts and in planta alterations in virulence and pathogenicity. Results indicated the best transformation efficiency was obtained with A. tumefaciens strain EHA105/S. The majority of transformants contained one or two insertions. On-going studies suggest a significant reduced growth rate by some transformants, as well as irregularities in pigmentation. TAIL-PCR will be carried out on mutants showing a reduced virulence in order to isolate and identify DNA sequences flanking the TDNA. The identification of pathogenicity genes could lead to an improved understanding of disease development and the development of novel management strategies. Key words: Fusarium oxysporum f. sp. cubense, ATMT, GFP, pathogenicity

101

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P10 - Interspecific Hybrids between Fusarium fujikuroi and Fusarium proliferatum ,

N. M. I. Mohamed Nor¹ ², B. Salleh¹, C. P. Toomajian², J. P. Stack², J. F. Leslie² ¹School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia; ²Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506-5502, USA E-mail: [email protected]

Interspecific hybrids offer unusual opportunities to study speciation and the segregation of traits that differ between species but that are often fixed within a species. We have recovered strains that appear to be hybrids between F. fujikuroi and F. proliferatum from rice fields in Southeast Asia and from a native tallgrass prairie in the United States. These hybrids can cross with standard mating type testers of both species and have DNA sequence profiles that are consistent with their putative hybrid conditions. The existence of these hybrids may indicate that reticulate evolution is occurring or that these two species have yet to completely finish the speciation process. We also have created such hybrids under laboratory conditions by crossing strains of opposite mating type on carrot agar. Based on the segregation of AFLPs, there are some fingerprint patterns that reoccur independently multiple times amongst the progeny, suggesting non-random segregation of at least portions of the genome. The parental strains differ in numerous traits, including secondary metabolite production and pathogenicity towards apples, onions and rice. Evaluation of recombinant progeny is providing the opportunity to identify portions of the genome involved in the speciation process and to identify regulatory and structural genes of importance in pathogenicity and secondary metabolite production. Keywords: speciation, hybridization, secondary metabolites, recombination

102

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P11 - Identifying indicators of soil suppressiveness to fungal diseases 1

1

2

1

2

K. Siegel , S. Aimé , E. Chapelle , V. Edel-Hermann , J. Raaijmaakers , P. 1* 1 Lemanceau , C. Steinberg 1

INRA, UMR1347 Agroécologie, 17 rue Sully, BP 86510, 21065 Dijon, France; 2Wageningen University; Bld 107, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; * Coordinator of the EU project Ecofinders FP7-ENV-2010-264465 E-mail: [email protected]

Soils suppressive to soil-borne diseases are defined by a low disease incidence in spite of the presence of a virulent pathogen and a susceptible plant. In many cases, the inhibition of the disease development relies on the activity of the resident soil microbiome. To identify taxonomic microbial indicators linked to the suppressiveness phenotype of soils, culture independent-based methods have been employed to analyse and compare microbial dynamics in two different soils suppressive to either Rhizoctonia solani damping-off disease of sugar beet or Fusarium wilt disease on flax. Fungal and bacterial taxonomic biodiversity were estimated from ITS and 16S genes by amplicon pyrosequencing. To that end, metagenomic DNA was extracted from the rhizosphere of plants grown in soils with different level of suppressiveness. We obtained 218650 reads in total (125602 for fungi and 93048 for bacteria). At this moment, the analyses of fungal communities are in progress. 114641 reads was kept after filtering by bioinformatic pipeline, distributed into 2303 clusters and 3379 singletons. Although, the bioinformatic and statistical analysis are not finished yet, we have already noticed a difference in the taxonomic diversity composition between suppressive and conducive soils which could explain the suppressive/conducive character of given soil. The next step is to achieve the bacterial communities in Fusarium wilt suppressive/conducive soils and to assess the microbial diversity of other soilborne diseases suppressive soils. Once the analyses of sequencing data are finished and the taxonomic assignments done, the comparison of microbial diversity of all studied soils will be performed in order to find out the similarities or/and differences in these soils which will provide the suppressiveness indicators. Keywords: Châteaurenard, Ecofinders, Fusarium oxysporum, Soil-borne disease

103

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY

P12 - Disentangling mycotoxin regulatory pathways in Fusarium graminearum by quantitative genetics B. Laurent, N. Ponts, V. Atanasova-Penichon, C. Barreau, M. FoulongneOriol INRA UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS20032, 33882 Villenave d'Ornon, France E-mail: [email protected]

Fusarium graminearum is a major causal agent of Fusarium Head Blight, and Maize Ear Rot. During infection, this fungus produces extremely stable trichothecene mycotoxins that accumulate in grains, such as deoxynivalenol, or DON. This toxin represents a threat for human and animal consumers, and maximum levels of contamination of cereals commercialized in Europe are now strictly regulated. The biosynthetic pathway leading to trichothecene accumulation has been well described but the genetic determinism behind the regulation of toxin production remains, however, largely unknown. To begin to answer this question, we have initiated an original approach of quantitative genetic: how many quantitative trait loci (QTL) are involved in the regulation of toxin production, what are their effects, where are they located on the genome, do they interact with each other? So far, there is no published data suitable to perform QTL analyses for toxin production in F. graminearum. We started our search for QTLs influencing DON accumulation using the intraspecific progeny that has been previously generated for creating the reference linkage map (Gale et al. 2005, Genetics), and in which toxin levels segregated as a polygenic trait. Alignment of the map with the genome will allow the identification of candidate genes that may underlie these QTLs. In addition, we have initiated the creation of a new segregating progeny adapted for the quantitative analyses of several traits related to toxin production. Upon completion, this work will provide sound basis to understand the biology and the genetic of trichothecene production in Fusarium, and open new possibilities to elaborate innovative strategies to combat this pathogen. Keywords: Fusarium, mycotoxin, biosynthesis, QTL

104

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

P13 - Masked mycotoxins in durum wheat: a greenhouse experiment 1

1

1

2

2

1

A. Dall’Erta , A. Tonelli , M. Cirlini , P. Lancioni , A. Massi , C. Dall’Asta , G. 1 Galaverna 1

Dept. of Food Science, University of Parma, Parco Area delle Scienze 17/a, 43124 Parma, Italy; Società Produttori Sementi Spa, Via Macero 1, 40050 Argelato (BO), Italy. E-mail: [email protected] 2

The incidence of Fusarium related diseases such as Fusarium Head Blight (FHB), caused by filamentous fungi, is increasing in cereal crops, inducing extensive production losses and severe toxicological problems due to mycotoxin accumulation. Among several strategies to counteract the infection, selection of plant genotypes resistant to Fusarium related disease is one of the most promising approaches. Among cereals, soft wheat and barley have been extensively studied, demonstrating that the in field resistance towards FHB is related to the ability to convert deoxynivalenol (DON) and other Fusarium toxins into masked forms (e.g. conjugates with glucose, glutathione or sulphate), which may be stored within plant vacuoles or vegetal cell membranes. Recently, occurrence of masked mycotoxins (deoxynivalenol-3glucoside) have been proved also in durum wheat, a key crop in Italy, where pasta production is totally based on this cereal (Dall’Asta et al. World Mycotoxin Journal, 6(1), 83-91, 2013). In addition, durum wheat is used also for the production of several high quality bakery products such as IGP/PDO breads. In this study, we present results about the ability of four different durum wheat lines to convert DON to deoxynivalenol-3-glucoside upon fungal inoculation or DON contamination under greenhouse controlled conditions. In particular, three groups have been considered: plants treated with F. graminearum, plants treated with DON and control plants. Both inoculation and contamination were made on the flowering ears and induced typical FHB symptoms in the plant. Plant ears were sampled at different times from the inoculation and analysed for DON and D3G occurrence. Data were statistically analysed and compared to those obtained for control plants. Results demonstrated that the ability to convert DON to its masked forms is genotyperelated and may help breeding program aimed at identifying resistant varieties. Keywords: Fusarium Head Blight, deoxynivalenol-3-glucoside, durum wheat, masked mycotoxins

105

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

P14 - Metabolomics of growth and type B trichothecenes production in Fusarium graminearum 1

1

1

L. Legoahec , V. Atanasova-Penichon , N. Ponts , C. Deborde 3,4 2,3 2,3 1 Maucourt , S. Bernillon , A. Moing , F. Richard-Forget

2,3

, M.

1

INRA, UR1264 MycSA, 71 avenue Edouard Bourlaux, CS20032, F-33882 Villenave d’Ornon cedex, France; 2INRA, UMR1332 Fruit Biology and Pathology, 71 avenue Edouard Bourlaux, CS20032, F33882 Villenave d’Ornon cedex, France; 3Metabolome Facility of Bordeaux Functional Genomics Center, IBVM Centre INRA de Bordeaux, F-33882 Villenave d'Ornon cedex, France; 4Univ. Bordeaux, UMR1332 Fruit Biology and Pathology, Centre INRA de Bordeaux, F-33882 Villenave d'Ornon, France E-mail: [email protected]

The plant fungal pathogen Fusarium graminearum can produce type B trichothecenes, a family of sesquiterpene molecules with toxic properties upon human or animal ingestion. Deoxynivalenol, or DON, and its acetylated forms belong to this family of secondary metabolites and are frequent contaminants of cereals worldwide. The biosynthesis of trichothecenes initiates with the condensation of two molecules of farnesyl pyrophosphate, at the end of the mevalonate pathway in Fusarium, and is under the control of various factors such as the redox parameters of the environment or the carbon source. For example, supplementing liquid submerged cultures of F. graminearum with caffeic acid, a phenolic acid with known antioxidant properties, reduces the accumulation of DON and its acetylated forms in the medium. Such a result, however, gives a partial glimpse of the effect of phenolic acids, from the trichothecene production point of view only. The present study analyzes F. graminearum metabolome in conditions when DON and its acetylated forms are produced. Liquid chromatography coupled with mass spectrometry and proton nuclear magnetic resonance were used to characterize the metabolites produced by the fungus, secreted in the culture medium or not, over the course of 14 days. Fifty-two polar and semi-polar metabolites were identified in the culture medium, i.e., the exo-metabolites, and/or in the mycelium, i.e., the endo-metabolites, comprising amino acids and derivatives, sugars, polyketides, and terpenes including trichothecenes and DON precursors. Sample composition varied over time in terms of primary metabolites as well as secondary metabolites. Data analysis further revealed correlations, positive or negative, between metabolic pathways. In the presence of caffeic acid, metabolomic profiles were modified, counting those resulting from primary metabolism even though fungal biomass production was not affected by the treatment. Several metabolites affected by the treatment were identified for both the exo- and endo-metabolome, in particular DON and its precursors. For the first time, these results expose a unique outlook of a hidden aspect of Fusarium’s response to antioxidant treatment. Keywords: metabolomics, secondary metabolite, Fusarium graminearum

106



SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY

BIOCHEMISTRY,

P15 - Effect of pH and temperature on Fusarium langsethiae growth and on T2 and HT2 toxins production in liquid medium 1

2

2

1

1

E. Rondags R. Fournier , P. Boivin , X. Framboisier , M. Fick 1

Laboratoire Réactions et Génie des procédés, CNRS (UMR 7274), Université de Lorraine, 2, avenue de la Forêt de Haye, TSA 40602, 54518 Vandœuvre-lès-Nancy, France; 2Institut Français des Boissons de la Brasserie-Malterie, 7, rue du Bois de la Champelle, 54500 Vandoeuvre-lès-Nancy, France E-mail: [email protected]

Fusarium langsethiae is a pathogenic fungus affecting cereal cultures and the downstream transformation chains. Contamination by this micro-organism is generally associated with the production of the highly toxic T2 and HT2 mycotoxins. However, in the barley-malt-beer chain, the relationship between Fusarium langsethiae contamination level, growth and toxins production is only partially understood, mainly because contamination occurs on natural complex substrates in polyphasic media. In order to assess this concern, growth and toxins production were monitored in perfectly stirred liquid conditions, as a function of the medium initial pH and cultivation temperature. This study shows that Fusarium langsethiae is able to grow at pH’s ranging from 4.5 to 9 and at temperatures between 10 and 30°C. Optimal growth conditions are close to pH neutrality and 25°C. T2 and HT2 production occurs from pH’s of 4.5 to 9, with an optimum located between pH 6.5 and 7.5. As far as temperature is concerned, toxin production takes place in the 15 to 30°C range, with a strong optimum at 25°C. The effects of temperature and pH on Fusarium growth and toxinogenesis have then been modelized with classical quadratic functions in order to dispose of a prediction tool in liquid media. What is more, no relationship was established between growth and T2 and HT2 production. At last, comparisons between Erlenmeyer’s flasks cultures without immobilized biomass and bioreactor cultures containing mainly adsorbed Fusarium langsethiae indicate that the toxinogenesis is due to immobilized non growing cells. This finding could explain the lack of relationship between Fusarium langsethiae and T2 and HT2 contamination levels in the fields and on the cereal-derived products. This could have strong consequences on the prevention strategies developed either in the field or in the transformation processes. Keywords: Fusarium langsethiae, T2, Barley-Malt-Beer, toxinogenesis

107

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

P16 - Volatile compounds in grain of various wheat cultivars naturally infected and inoculated with Fusarium culmorum 1

1

2

1

M. Buśko , K. Stuper , T. Góral , A. Ostrowska , J. Perkowski

1

1

Poznań University of Life Sciences, Department of Chemistry, Wojska Polskiego str. 75, 60-637 Poznań Poland; 2Department of Plant Pathology, Plant Breeding and Acclimatization Institute NRI, Radzików, 05-870 Blonie, Poland; E-mail: [email protected]

In the present work 30 winter wheat cultivars were investigated for the volatile organic compounds (VOCs). The metabolic profile was established for both healthy and inoculated with Fusarium culmorum grain. VOCs were extracted by mean of HS-SPME method and further analyzed using GC-MS. In samples of grain growing in natural conditions and after inoculation with F. culmorum several dozens of compounds were qualitatively determined. They belong to terpenes, alcohols, aldehydes and ketones, benzene derivatives and hydrocarbons. On the base of obtained chromatograms and mass spectra after deconvolution and estimation of retention indexes 42 compounds were identified. The concentration sum of VOCs for controlled and inoculated samples was similar. However, for particular compounds groups it was differentiated. In controls a higher concentration was stated than in inoculated samples for benzene derivatives, hydrocarbons and terpenes. On the other hand, alcohols, aldehydes and ketones prevailed in inoculated samples. In all of groups, except of terpenes, obtained trends were equal. Above observations for terpenes confirm their key significance in toxic metabolites biosynthesis by Fusarium. These observations also confirm the fact, that lack or low concentrations of particular terpenes in profile of VOCs are related with rich profile of other terpenes. Among determined terpenes there were 6 the most important for trichothecenes biosynthesis: α-pinene, 3-carene, indane, β-chamigrene, thujopsene and trichodiene. For α-pinene, 3-carene, indane a higher concentration was stated in control samples whilst for β-chamigrene, thujopsene and trichodiene in inoculated samples. The most important are results obtained for trichodiene, which is intermediate metabolite in trichothecenes biosynthesis. It cannot be excluded that other terpenes are significant for this metabolomic pathway and could be useful for understanding mechanism of Fusarium toxins biosynthesis. This work was partially supported by the National Science Center in Poland project no. 2704/B/P01/2011/40 Keywords: Fusarium, volatiles, wheat, SPME

108

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

P17 - Semiochemical interactions between toxigenic Fusarium fungi and insects T. Y. Gagkaeva, O. P. Gavrilova, I. V. Shamshev, O. G. Selitskaya, E. I. Savelieva All-Russian Institute of Plant Protection (VIZR), Sh. Podbelskogo 3, 196608, St.-Petersburg – Pushkin, Russia E-mail: [email protected]

The aim of the study was to evaluate chemical nature and functional abilities of semiochemical interactions between toxigenic Fusarium fungi and insects. An analysis of variations of Fusarium fungi (F. graminearum, F. culmorum, F. cerealis, F. poae, F. sporotrichioides, F. langsethiae) in formation of olfactory responses of insects (rice weevil Sitophilus oryzae) was performed. The evaluation of olfactory stimulation with Fusarium fungi on test-insects was carried out after growing of the strains on two substrates: potato sucrose agar and autoclaved wheat grains. In the laboratory experiments F. langsethiae and F. poae strains have shown the clear attractive effect to the tested insects. The pathogenic to plants F. graminearum and F. culmorum strains have demonstrated the repellent effect. Generally beetles of rice weevil responded similar reaction to the cultures of fungal strains growing on both substrates. The method of solid-phase microextraction was used to clarify the spectra of volatile organic compounds above the surface of living fungal cultures. Screeningportraits of metabolomic profiles of Fusarium strains after 7 days of cultivation were obtained. A characteristic profile of sesquiterpenes, that are isomeric carbohydrates with molecular mass 204 and are genetically connected with trichodien, is richest in F. langsethiae in compare with another Fusarium spp. The culture of F. langsethiae produced maximal amount of trichodien also actively emitted volatile compounds. Under equilibrium condition of steam above surface of F. langsethiae, F. sporotrichioides и F. poae cultures were identified ethylacetate, isobutyl, isoamyl and amyl alcohols. Amylacetate and isoamylacetate, ethers of acetic acid and corresponding amyl alcohols were found only in the culture of F. poae. Most likely just these chemicals responsible for a sweet fruit smell that is typical for this fungus. The investigation was supported by the project No. 12-04-00927-а of the Russian Foundation for Basic Research. Keywords: Fusarium, insects, sesquiterpenes, olfactory stimulation

109

SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

P18 - Effect of cry 1Ab toxins on FUM gene cluster expression and on fumonisin production by Fusarium verticillioides 1

1

1

1

L. O. Rocha , G. M. Reis , V. M. Barroso , L. J. Andrade , B. Corrêa

1

1

University of São Paulo, Biomedical Sciences Institute, Avenue Professor Lineu Prestes, 1374, São Paulo-Brazil E-mail: [email protected]

F. verticillioides is most commonly associated with maize worldwide and produces high levels of fumonisins, one of the most important mycotoxins. Previous studies have demonstrated that cry 1Ab protein from Bacillus thuringiensis sbsp. kurstaki could be suitable for Fusarium toxins management due to the control of crop insect injuries. The aim of this study was to assess the impact of cry 1Ab protein expressed in the 30F35YG (YieldGard) corn hybrid on FUM gene expression and on fumonisin production by F. verticillioides ICB 13BA (GU 989110/NCBI accession number). We also evaluated the influence of B. thuringiensis sbsp. kurstaki spores and cry 1Ab crystals on the F. verticillioides growth. In order to eliminate the original microbiota, Bt corn samples and its respective non-Bt isogenic were previously irradiated with 20 kGy. Plates were inoculated with 5 x 8 o 10 F. verticillioides spores/mL and incubated at 25 C and relative humidity of 97.5%. After 10, 20 and 30 days, samples were analysed regarding FB 1 contamination and expression of 14 FUM genes. The statistical analyses were performed using the softwares R 2.9, Gamlss package and SAS 9.1. An initial 8 fungal inoculum of 5 x 10 spores/mL was cultured with 10 μg/mL of bacterial spores and crystals. F. verticillioides biomass was estimated by dry weight of mycelia during 1, 3 and 5 days . The results showed reduced fumonisin B 1 contamination on 30F35 YG in comparison to its isogenic non-Bt corn during the periods of the analyses (p= 0.0095, Weibull distribution). There was no correlation between FUM gene cluster and fumonisin production according to Pearson’s correlation test (except for FUM19, p5500 ppb. 2 The coefficient of determination (R ) between F. graminearum DNA and DON levels was 0.95*** in the 25 oats samples of the year 2011 and 0.73*** in the 38 2 oats samples of the year 2012 obtained from MTT, while the R value from the 20 oats samples from a food company was only 0.22*. In the latter case the DON level was estimated by using the RIDA®QUICK SCAN, while in MTT the mycotoxin levels were measured by using ROMER™ MycoSep-GC-MS and the grain flour was sieved through the 1 mm sieve in order to homogenize it before the analysis. The sieved flour was also used for DNA measurements, while for the samples from the food company the flour was not homogenized by sieving. No correlation was found between F. culmorum DNA and DON levels. F. graminearum DNA levels were in all cases in agreement with DON levels, when DON was measured by GC-MS. When compared to RIDA®QUICK SCAN kit results (DON) the variation in DNA levels was much higher. There was also a significant correlation between the combined T-2 and HT-2 and combined F. langsethiae and F. sporotrichioides * DNA levels (0.33 in 2011 and 0.89*** in 2012). According to our results F. graminearum is clearly the main DON producer in Finnish oats, while F. langsethiae is probably the main T-2/HT-2 producer. Keywords: F. graminearum, F. langsethiae, TaqMan qPCR, DON

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SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY

BIOCHEMISTRY,

P22 - Geographic differences in trichothecene chemotypes of Fusarium graminearum in the Northwest and North of Iran 1

1,2,3

4,5

2

A. D. van Diepeningen , M. Davari , S. H. Wei , A. Babay-Ahari , M. 2 4 4 6 Arzanlou , C. Waalwijk , T. A. J. van der Lee , R. Zare , A. H. G. Gerrits van 1 1,7,8,9 den Ende , G. S. de Hoog 1

CBS-KNAW Fungal Biodiversity Centre, 3584CT Utrecht, the Netherlands; 2Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran; 3Department of Plant Protection, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran; 4Plant Research International, Wageningen University and Research Centre, Wageningen, the Netherlands; 5College of Plant Protection, Shenyang Agricultural University, Shenyang, China; 6Department of Zotany, Iranian Research Institute of Plant Protection, Tehran, Iran; 7Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam; 8Peking University Health Science Centre, Research Center for Medical Mycology, Beijing, China; 9SunYat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China E-mail: [email protected]

The diversity and prevalence of Fusarium species and their chemotypes on wheat in the North-West and North of Iran was determined. Wheat in these areas is severely affected by Fusarium Head Blight (FHB), with Fusarium graminearum as prevalent species causing 96% of the infections in the North-West and 50% in the Northern provinces. Fungal isolates were identified based on morphological characters and sequences of the internal transcribed spacer (ITS) region, and parts of translation elongation factor 1-α (TEF1-α) and RNA polymerase subunit II (RPB2) sequences. Phylogenetic and phylogeographic analyses show little haplotype variation between the F. graminearum strains collected from the different locations, but the isolates differ significantly in their trichothecene chemotypes as determined with a Luminex-Multilocus genotyping assay. Fusarium graminearum strains producing 15-ADON were abundant in Ardabil (NW of Iran), while in Golestan province (N of Iran) at the other side of the Caspian Sea especially nivalenol-producing strains and a variety of other Fusarium species were observed. Strains producing 3-ADON were rarely found in both areas. This is the first detailed study on Fusarium infections in Iranian wheat, showing large differences in prevalent etiological agents and in mycotoxin chemotypes geographically. Keywords: Fusarium graminearum Species Complex (FGSC), Fusarium

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SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY



BIOCHEMISTRY,

P23 - Co-occurrence of mycoflora, aflatoxins and fumonisins in maize and rice seeds from markets of different districts in Cairo, Egypt 1-2

3

A. K. Madbouly , M. I. M. Ibrahim , A. F. Sehab4, M. A. Abdel-Wahhab

3

1

Faculty of Science, Microbiology Department, Ain Shams University, Abbassia, Cairo, Egypt; 2Faculty of Science, Biology Department, Tabuk University, Tabuk, Saudi Arabia; 3Food Toxicology & Contaminants Department, National Research Center, Dokki, Cairo, Egypt; 4Plant Pathology Department, National Research Center, Dokki, Cairo, Egypt E-mail: [email protected]

The mycoflora and mycotoxins contamination of commercial maize and rice grains collected from local markets of the major five zones of the province of Cairo, Egypt, represented by 20 different districts were studied. A total number of about 23 species belonging to 12 different genera of fungi were isolated and identified. About 70% of the samples were infected with Aspergillus flavus and Aspergillus niger, with percentages of 33%, 40% recovered from maize and 46%, 27% recovered from rice, respectively. The percentages of infection of maize ranged from 16% to 142%. The percentages of infection of rice seeds ranged from 6% to 93%. Total aflatoxins and fumonisins detected in maize averaged 9.75 and 33 mg/kg, respectively. Total aflatoxins and fumonisins detected in rice averaged 5.15 and 1014 mg/kg, respectively. Keywords: fumonisins, aflatoxins, Fusarium mycotoxins, mycoflora

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SESSION 2: SECONDARY METABOLITES BIOSYNTHESIS, FEED AND FOOD SAFETY

BIOCHEMISTRY,

P24 - The impact of Fusarium and Microdochium species on the safety and quality of UK malting barley 1

1

2

R. V. Ray , L. K. Nielsen , S. G. Edwards , D. J. Cook

1

1

University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, United Kingdo; 2Harper Adams University, Newport, Shropshire, TF10 8NB, United Kingdom E-mail: [email protected]

Fusarium head blight (FHB) can cause significant reductions of yield, safety and quality of cereals. The SAFEMalt project (Strategies Against Fusarium Effective in MALTing barley) is an UK based 3-year multi-partner research initiative spanning the malting barley supply chain from crop breeder through agronomist and merchant to brewer. The ultimate output of the project is the development of a targeted toolkit for the protection of malting barley quality. The first objective of the project was to identify the predominant Fusarium/Microdochium species in UK barley and to evaluate the impact of their sub-acute infections on the malting and brewing characteristics of commercially grown spring barley. Species specific real-time PCR and mycotoxin quantification assays were carried out on spring malting barleys collected in 2010 and 2011 and on selected samples from previous mycotoxin screening survey between 2007 and 2009. The predominant Fusarium species present in UK malting barley in 2010 (n = 88) and in 2011 (n = 76) were F. avenaceum, F. poae, and F. tricinctum with each species detected in 80-100% of all samples. The predominant Microdochium species was M. nivale. The main mycotoxins detected in 2010 and in 2011 were HT2/T2 and nivalenol correlating positively with quantified DNA of F. langsethiae and F. poae, respectively. Selected malting barley cultivars were further micro malted and subjected to malt and wort analysis of key quality parameters. Preliminary results suggest that the immediate effects of contamination with F. avenaceum and M. nivale were increased water sensitivity of barley and decreased wort filtration volume. Fungal biomass of F. poae and F. langsethiae correlated with increased wort free amino nitrogen. Further analysis of malting and brewing quality parameters on additional barley samples collected in 2011 is in progress. Keywords: Fusarium, Microdochium, Malting barley, quality

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BIOCHEMISTRY,

P25 - Influence of pre-harvest moisture and harvest time on fusarium mycotoxin concentrations in winter wheat L. L. Kharbikar, E. T. Dickin, S. G. Edwards Harper Adams University, Newport, Shropshire, TF10 8NB, UK E-mail: [email protected]

Fusarium graminearum is the most devastating head blight pathogen and contaminates grain with the mycotoxins deoxynivalenol (DON) and zearalenone (ZON). Rainfall at anthesis is known to influence the infection and subsequent production of these mycotoxins in wheat but less is known of the impact of rainfall later in the growing season. In 2008 the UK suffered the highest recorded levels of DON and ZON and this was possibly related to the delayed wet harvest. Anecdotal evidence from end-users suggested that more grain deliveries at intake failed to meet legislative limits when harvested after rather than before the delays. The impact of pre-harvest moisture and harvest time on mycotoxin concentrations in winter wheat was studied in two controlled environments. After inoculation with isolates of F. graminearum at anthesis, plants were bagged for 96 h. At hard dough maturity ears were kept moist by watering for 2 minutes every hour for 8 hours a day using a watering can and bagged overnight or left dry until harvest and harvested when ripe one week later (early harvest) and again a week later (late harvest). Ears were harvested, dried and then milled. Mycotoxin concentrations were determined using R-Biopharm ELISA kits. Low level of ZON in the first experiment suggested the water treatment was not sufficient to stimulate ZON production. Therefore, in a second experiment plants were mist irrigated for 2 hours every day and bagged overnight to retain the moisture after hard dough maturity until harvest. DON levels were very high as a result of the severe infection achieved within these experiments; however the impact of preharvest moisture and harvest date was greater for zearalenone. Results indicate that, late season moisture can significantly increase DON and ZON in wheat and early harvesting, if practical, is strongly advised to reduce the mycotoxin levels in wheat. Keywords: Pre-harvest rainfall, Fusarium graminearum, zearalenone, wheat

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BIOCHEMISTRY,

P26 - Contamination of wheat grain with microscopic fungi and their metabolites in Poland in the years 2006–2009 1

2

K. Stuper , T. Góral , J. Perkowski

1

1

Department of Chemistry, Poznan University of Life Sciences, Wojska Polskiego str. 75, 60–637 Poznan, Poland; 2Department of Plant Pathology, Plant Breeding and Acclimatization Institute NRI, Radzików, 05-870 Blonie, Poland E-mail: [email protected]

Microscopic fungi are microorganisms commonly found in cereal products. Pathogens of cereals colonising kernels are responsible, among other things, for deterioration of the technological value of grain. However, the greatest threat is posed by Fusarium mycotoxins produced by toxin-forming strains of these microorganisms. The aim of the present investigations was to determine the level of contamination with microscopic fungi and mycotoxins from the group of trichothecenes in wheat grain from Poland in a 4-year cycle. In the period from 2006 to 2009 studies were conducted on the content of fungal metabolites (ergosterol [ERG] and type A and B trichothecenes) and the content of microscopic fungi expressed in colony-forming units (CFU) in wheat grain. A total of 129 grain samples were examined. Analysed wheat samples had similar contents of both the investigated fungal metabolites and levels of microscopic fungi. Contents of microscopic fungi were low. Concentration of ERG was on 1 average 2.64 mg/kg, while in colony forming units this value ranged from 10 3 CFU/g to over 10 CFU/g. The total concentration of type A and B trichothecenes was also low and within the four years of the investigations did not exceed 0.062 mg/kg. Concentration of DON did not exceed 1250 µg/kg, established as safe in grain for human consumption, in any of the tested samples. For the results collected in the years 2006–2009 and presented in this paper correlations were calculated between the amount of mycoflora and analysed metabolites in three possible combinations. They were 0.7096 for ERG/total toxin concentration, 0.6086 for ERG/log CFU/g, and 0.4016 for the concentration of total toxins/log CFU/g. Highly significant correlations between the content of trichothecenes and the concentration of ERG indicate that the level of this metabolite is closely related to the content of mycotoxins in grain. Keywords: CFU, ergosterol, trichothecenes, wheat

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BIOCHEMISTRY,

P27 - Fumonisins occurrence in maize cobs infected with opposite mating type strains of F. verticillioides 1

2

2

2

M. Wit , P. Ochodzki , A. Waskiewicz , R. Warzecha , P. Goliński 1 1 Jablonska , W. Wakulinski

3

, E.

1

Department of Plant Pathology, Warsaw University of Life Sciences SGGW, Nowoursynowska 159, 02-766 Warsaw, Poland; 2Plant Breeding and Acclimatization Institute, 05-870 Blonie, Poland; 3 Department of Chemisty, Poznań University of Life Sciences, Wojska Polskiego 75, 60-625 Poznan, Poland E-mail: [email protected]

Fusarium verticillioides is along with F. subglutinans and F. proliferatum the principal causative factors of pink ear rot. Occurrence of the species is variable and strongly related to high temperature and limited precipitation. F. verticillioides is hetherothalic fungus with dimictic mating system governed by two idiomorphs MAT1-1 and MAT1-2. The strains of opposite mating types are widespread in Poland and frequency of their occurrence is statistically equivalent. Diverse phenotypic traits of opposite mating strains of some fungi inclined us to verify fumonisins accumulation in maize cobs inoculated with MAT1-1 and MAT1-2 F. verticillioides strains. Material and methods: Ten genotypes of four maize varieties (Zea mays var. indentata, Zea mays var. indurata, Zea mays var. saccharata and Zea mays var. everta) were used in these studies. Cobs were inoculated 7 days after silk emergence with 2 ml conidial suspension of opposite mating type of the fungus. Cobs Infection degree was evaluated according to 6 degree scale while ergosterol and fumonisins content were determined by HPLC method detailed described elsewhere. Results: During four years of field studies, it was found that neither infection degree of particular Zea mays varieties determined phenotypically and by ergosterol content nor Fumonisins level were not dependent on MAT type. All the mentioned factors were significantly influenced by environmental condition of cropping season, maize variety and kernel composition (especially water, amylose, amylopectin content). Acknowledgment: The work was financed by the Ministry of Science and Higher th Education, project No. N N310 376933 and participation in 12 European Fusarium Seminar is supported through 7FP-Regpot-2011-1-286093 Keywords: fumonisin, mating types

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BIOCHEMISTRY,

P28 - Phylogenetic diversity of and fumonisin gene cluster distribution within Fusarium isolates from wild banana in China 1

2

1

3

4

4

F. Van Hove *, S. De Saeger , I. Lazzaro , Y. Qi , D. Zhang , A. Wu , F. 1 Munaut 1

Université catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology, Mycothèque de l'Université catholique de Louvain (BCCMTM /MUCL), Louvain-la-Neuve, Belgium; 2 Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium; 3 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS), China; 4College of Life Science and Biotechnology, Shanghai Jiao Tong University (SJTU), China. E-mail: [email protected]

Banana fruit is one of the most important crops and is currently the second most important fruit produced all over the world. Several Fusarium species have been identified as important pre- and post-harvest pathogens of cultivated banana. However, there are no studies that have been conducted to identify Fusarium contaminants on wild banana. The first objective of this work was to study the phylogenetic and genetic diversity of Fusarium isolates from the F. fujikuroi species complex contaminating fruits of wild banana plants growing in two southern Chinese Provinces, Hainan and Yunnan. The second objective was to study the distribution and configuration of the fumonisin gene cluster within these Fusarium species. Keywords: Fusarium, fumonisin gene cluster, banana, phylogeny

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BIOCHEMISTRY,

P29 - Different levels of fumonisin production and FUM gene cluster expression on 2B710Hx corn hybrid 1

1

1

1

G. M. Reis , L. O. Rocha , V. M. Barroso , L. J. Andrade , B. Corrêa

1

1

University of São Paulo, Biomedical Sciences Institute, Avenue Professor Lineu Prestes, 1374, São Paulo-Brazil E-mail: [email protected]

Maize is the third most cultivated crop worldwide, and Brazil is currently, the fourth largest producer in the world. Fusarium verticillioides is commonly associated with maize and it is capable to produce high levels of fumonisins. Studies regarding the use of transgenic corn have shown that fumonisin contamination could be reduced due to the damage decrease caused by insects on grains. The aim of this study was to evaluate the effect of cry 1F toxin produced by Bacillus thuringiensis subsp. aizawai, expressed in the 2B710Hx (Herculex) corn hybrid on the fumonisin production and on FUM gene expression by F. verticillioides ICB 13BA (NCBI accession number; GU 989110). 2B710 Hx corn samples and its respective isogenic non-Bt were previously irradiated with 20 kGy in order to eliminate fungi contaminants from field. Plates were inoculated 8 o with 5 x 10 spores/mL and incubated at 25 C and Aw of 0.99. After 10, 20 and 30 days, samples were analysed regarding FB1 and FB2 contamination and FUM1, FUM3, FUM6, FUM7, FUM8, FUM10, FUM11, FUM13, FUM14, FUM15, FUM17, FUM18, FUM19 and FUM21 gene expression. The statistical analyses were performed using the softwares R 2.9, Gamlss package and SAS 9.1. The results showed reduced fumonisin B1 contamination on 2B710 Hx during the 30 days of analyses (p< 0.005). Regarding FB2, there was no statistical difference between the isogenic and transgenic hybrids. There was no correlation between FUM gene cluster and fumonisin production according to Pearson’s correlation test (except for FUM19, p 95%), and 30 ° C (> 98 %) for F. solani compared to control seedlings. In vivo pathogenicity tests were carried out by using two methods, ear infection and soil inoculation (to evaluate disease severity on the collar and the root of wheat seedlings). Pathogenicity test on the collar showed that all Fusarium spp. and M. nivale isolates induced symptoms on the collar and root also those obtained from diseased ear. However F. graminearum and F. culmorum isolates obtained from diseased collars caused disease severity indices higher than the isolates obtained from ears. Also, it was found in this study that isolates belonging to the genus Fusarium isolated from the collar induced typical symptoms of Fusarium head blight on the ear and the disease severity of isolates was independent of isolate origin (collar or ear). Results obtained in this study showed that there is no correlation between the pathogenicity of all Fusarium and M. nivale isolates on the collar and on the ear. Keywords: Fusarium, Microdochium, In vitro growth, pathogenicity

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P47 - Trichothecene production by Fusarium graminearum isolates from Argentina and its relationship with aggressiveness and fungal colonization of the wheat spike 1

1

1

2

I. Malbrán , C. A. Mourelos , P. A. Balatti , Q. Migheli , G. A. Lori

1

1

Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, (1900) La Plata, Buenos Aires, Argentina; 2Dipartimento di Agraria - Plant Pathology and Entomology Unit, Università degli Studi di Sassari, Via E. De Nicola 9, I - 07100 Sassari, Italy E-mail: [email protected]

In Argentina, at least 20 epidemics of Fusarium Head Blight (FHB) of wheat, caused by Fusarium graminearum, have been registered in the last 50 years. The damages induced by the disease are further aggravated by the frequent presence in affected grains of mycotoxins, mostly deoxynivalenol (DON), which may cause health problems to human and animals. In the present work we investigated the correlation between the aggressiveness of F. graminearum isolates from Argentina, its capacity to colonize the spike and the accumulation of DON in wheat grains. Fourteen isolates of F. graminearum were tested for aggressiveness in point inoculated (PI) spikes of field grown wheat in two years of trials. Inoculated spikes were hand threshed and the resulting rachis were superficially disinfected, cut in their component parts and plated on potato dextrose agar medium (PDA) to analyze the colonization of the vascular system. ® Grains were ground and DON was quantified by means of the RIDASCREEN FAST DON ELISA kit (R-Biopharm). Isolates significatively differed in the severity of the symptoms of FHB (F = 7.34; p < 0.01), DON production (F = 5.91; p < 0.01) and percentage of colonized portions of the rachis (F = 7.22; p < 0.01). A close correlation was found between the severity of FHB and DON (r = 0.9073), colonization and severity (r = 0.7106), and colonization and DON (r = 0.8081). The presence of the pathogen in the rachis was verified even in asymptomatic spikes in a percentage that was highly superior to that of symptomatic spikelets for all treatments. Furthermore, even isolates with a limited capacity of induction of visible symptoms of FHB were able to colonize the vascular tissue of wheat spikes and to produce considerable amounts of DON. Keywords: Fusarium head blight, deoxynivalenol, wheat

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P48 - Study of in vitro growth and pathogenicity of some isolates of Fusarium spp. causal agent of Fusarium head scab (FBH) of wheat in Algeria R. Renane Département of botanique Institut national Agronomique (INA), El Harrach, Algiers, Algeria, E-mail: [email protected]

The study of the effect of temperature on in vitro growth of Fusarium spp. isolates obtained from wheat spike exhibiting typical symptoms of head scab (ear blight) showed that the optimum growth was at 25°C for all isolates belonging to the four species of the Fusarium genus. The species were F. avenaceum, F. culmorum, F. moniliforme and F. solani; with a lack of growth of all isolates at 35°C. Among the species studied, the F. culmorum isolates showed the highest rates of growth at all temperatures tested (15, 20, 25 and 30°C). It was also noticed that the growth rate of the four species studied increased between 20 and 25°C, and decreased between 25 and 30°C. Pathogenicity tests of Fusarium spp. isolates were carried out in vitro and in planta. The pathogenicity test in vitro was assessed by examining the coleoptile growth rate of wheat seedlings. The results obtained showed that all Fusarium spp. isolates were pathogenic. These induced retardation in coleoptile growth compared to the control at 20, 25 and 30°C. The most pathogenic of the four species was F. moniliforme isolates which conferred a complete reduction in coleoptile growth (100 %) at 25°C and 30°C. For the other isolates the highest rate of reduction in coleoptile growth was (95.92 %) for F. culmorum and 95.26% for F. avenaceum at 25°C, while the highest rate of reduction 95.28% for F. solani was obtained at 30°C. Pathogenicity carried out by soil inoculation and evaluated by the severity attack at the collar level estimated by a disease scale from 0 to 3 showed that the highest disease index (2.58) was conferred by F. avenaceum isolates, followed by F. moniliforme (1.28), F. solani (1) and least by F. culmorum (0.53). Results obtained in this study showed that there is no correlation between in vitro growth and agressiveness of Fusarium isolates used in this study and also between agressiveness assessed by the reduction in coleoptile growth and the attack at the collar level. Furthermore it was shown that Fusarium isolates which induced head scab of wheat was also aggressive on root and collar of wheat. Keywords: wheat, Fusarium, Trichoderma , biological control

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P49 - Pathogenicity of Fusarium temperatum and Fusarium subglutinans on maize stalk and ear under artificial inoculation under field conditions 1

2

2

1

1

J. Lević , F. Munaut , J. Scauflaire , S. Stanković , D. Ivanović , V. Krnjaja

3

1

Maize Research Institute, Zemun Polje, Belgrade-Zemun, S. Bajića 1, 11185 Belgrade, Republic of Serbia; 2Université catholique de Louvain, Earth and Life Institute, Laboratory of Mycology, Mycothèque de l’Université catholique de Louvain, Université catholique de Louvain, Croix du Sud 2, L7.05.06, 1348 Louvain-la-Neuve, Belgium; 3Institute for Animal Husbandry, Autoput 16, 11080, Belgrade-Zemun, Republic of Serbia E-mail: [email protected]

Pathogenicity of Fusarium temperatum, a new species morphologically similar and phylogenetically closely related to F. subglutinans, was observed on stalk and ear of maize (Zea mays L.). In 2010, this species was established in the culture collection of the Maize Research Institute, stored since 2004. Based on interspecies mating compatibility analyses and confirmed with AFLP fingerprint profiles (done by Munaut), two of 20 tested F. subglutinans isolates belonged to the species F. temperatum. These isolates originated from sorghum (Sorghum bicolour (L.) Moench.) grain. In 2011 and 2012, pathogenicity of 20 (2 F. temperatum and 18 F. subglutinans, respectively) and 4 isolates (2 of each F. temperatum F. and F. subglutinans), respectively, was tested on stalk and ear of two maize hybrids. The artificial inoculation of stalk was done by insertion of a Fusarium-inoculated toothpick into the second internode 7 days after silking. Control plants were inoculated with a sterile toothpick. After 6 weeks, disease intensity was evaluated on the stalk longitudinal section by the 0-6 scale (0 necrosis localised at the inoculation spot; 6 - necrosis spreads to other internodes). Ear inoculation was done with 2-ml spore suspension in silk channels 3-5 days after silking. Control plants were treated with 2 ml sterile water. The disease intensity was assessed on ears immediately prior to harvest, according to the 1-7 scale (1 - no symptoms; 7 - 76-100% infected kernels). There were not statistically significant differences in pathogenicity between isolates of F. temperatum and F. subglutinans, either on stalk or ear. Differences in hybrids responses and symptoms that isolates of fungal species caused on stalks and ears were poorly visible. According to our knowledge, the F. temperaturm occurrence on sorghum seed as well as its pathogenicity on maize ear and stalk are detected for the first time in the world. Keywords: F. temperatum, F. subglutinans, maize, sorghum

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P50 - Effect of timing of inoculation and Fusarium species on the development of Fusarium head blight and deoxynivalenol contamination in oat 1

1

2

A. Xue , Y. Chen , C. Ren , and M. Savard

1

1

Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; 2Baicheng Academy of Agricultural Sciences, 17 Sanhe Road, Baicheng, Jilin 137000, China E-mail: [email protected]

Fusarium head blight (FHB) is a destructive disease of oats in Canada. To supplement the development of FHB-resistant cultivars, we examined the influence of timing of inoculation and pathogenicity of four common Fusarium spp. on development of FHB and deoxynivalenol (DON) content using 12 oat genotypes under controlled environment conditions. Both timing of inoculation and Fusarium spp. significantly affected the development of FHB symptoms and DON contents in the harvested grain. Early inoculations at or before the complete emergence of ears resulted in little or no visible FHB symptoms but DON contaminations ranging from 0.9-3.7 ppm were detected in the harvested grains from these symptomless plants. Severe levels of FHB were observed on all the oat genotypes with infected spikelets (IS) ranging from 40 to 75% and DON content ranging from 6.3 to 10.2 ppm, when inoculated at or after the 50% anthesis stage. The timing of inoculation at the 50% anthesis was considered the most appropriate as it allowed a sufficient time for disease development and assessment prior to the physiological maturity of the plants. Of the four Fusarium spp., F. culmorum and F. graminearum were equally highly pathogenic, having areas under the disease progress curve (AUDPC) of 45.3 and 47.3, and DON of 10.4 and 14.3 ppm, respectively. DON was not detected in the harvested grain from plants inoculated with F. avenaceum or F. sporotrichioides. Fusarium sporotrichioides resulted in the lowest AUDPC (31.2) and were significantly less pathogenic than the two highly pathogenic species. Fusarium avenaceum resulted in AUDPC of 36.7, which was not significantly different from those of neither the highly pathogenic species nor the weakly pathogenic species. The Fusarium spp. ´ oat genotype interactions were not signficant, suggesting that breeding for resistance to F. graminearum may also give enhanced resistance to other Fusarium spp. Keywords: Fusarium head blight, oat, Avena sativa, deoxynivalenol

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P51 - Selective pathogenicity and virulence of Fusarium graminearum species complex members on maize, wheat and sorghum 1

1

1

1, 2

I. Beukes , C. de Klerk , L. J. Rose , G. J. van Coller

3

, B. Flett , A. Viljoen

1

1

University of Stellenbosch, Department of Plant Pathology, Private Bag X1, Matieland 7602, South Africa; 2Directorate Plant Science, Western Cape Department of Agriculture, Private Bag X1, Elsenburg 7607, South Africa; 3Grain Crops Institute, Agricultural Research Council, Private Bag X1251, Potchefstroom 2520, South Africa E-mail: [email protected]

Fusarium head blight (FHB) of wheat, gibberella ear rot (GER) of maize and grain mold (GM) of sorghum is caused by, amongst others, members within the Fusarium graminearum species complex (FGSC). Grains infected with these pathogens are commonly contaminated with trichothecene mycotoxins, which is associated with health complications in humans and animals. It has been suggested that host specificity exists within the FGSC, possibly due to differences in cyclic hydroxamic acids and related benzoxazolinone compounds or other antimicrobial composites in different cereal hosts. In this study, the host preference of five FGSC members, represented by 26 isolates obtained from different South African grains was assessed on wheat, maize and sorghum. Radial growth of these isolates on potato dextrose agar at 12 and 24-h illumination did not differ significantly between the members. Sporulation of cultures under the same light conditions resulted in F. boothii producing the most and F. acaciae-mearnsii the least spores. Wheat heads were inoculated with five isolates each from five FGSC members, previously isolated from South African grown wheat, to establish whether a difference in virulence could be observed between them. Inoculations showed that all FGSC members were pathogenic to wheat, with F. cortaderiae being the least and F. graminearum s.s the most virulent. Macroconidia from different isolates of the same FGSC member were then combined and inoculated onto sorghum and maize plants in the field, and disease development and trichothecene production measured after harvest. Inoculation with F. meridionale resulted in the highest disease incidence and severity on maize kernels. Infection with F. meridionale was supported by the production of nivalenol in both maize and a brown sorghum cultivar. Fusarium graminearum s.s. and F. cortaderiae could not be associated with GER of maize or GM of brown sorghum, and was unable to produce their associated mycotoxins within these grains. Keywords: Fusarium graminearum species complex, host preference

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P52 - Aggressiveness and deoxinivalenol production of Fusarium graminearum isolates from different inoculum sources 1

1

1

2

C. A. Mourelos , I. Malbrán , P. A. Balatti , Q. Migheli , G. A. Lori

1

1

Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, (1900) La Plata, Buenos Aires, Argentina; 2Dipartimento di Agraria - Plant Pathology and Entomology Unit, Università degli Studi di Sassari, Via E. De Nicola 9, I - 07100 Sassari, Italy. E-mail: [email protected]

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe, is one of the most important fungal diseases affecting wheat. In addition to the severe yield and quality losses the disease causes, trichothecene contamination of infected grains poses a serious threat to human and animal health. In Argentina more than three quarters of the total arable land is cultivated under no-tillage. Large quantities of crop residues remain on the soil surface, representing the principal reservoir of F. graminearum. Furthermore, in the southern hemisphere the presence of weeds in the field throughout the entire year has proven to be of epidemiological importance as an inoculum source for FHB development. The aim of this work was to analyze aggressiveness towards wheat of F. graminearum isolates from different sources. Thirty-three isolates obtained from crop residues and symptomless inflorescences of gramineous (GW) and non-gramineous weeds (N-GW) were identified as F. graminearum based on their morphological and cultural characteristics, and confirmed by species-specific PCR. Aggressiveness of the isolates and FHB effect on thousand kernel weight (TKW) were tested by point inoculation (PI) of field grown wheat spikes. Inoculated spikes were hand threshed, grains were ground and DON was quantified by means of the RIDASCREEN® FAST DON ELISA kit (R-Biopharm). All isolates induced FHB symptoms on the spikes and accumulated DON in the grains. The isolates obtained from GW differed significantly in aggressiveness from those from N-GW (F=4.78, p150 wheat fields (n= 663 strains); 2) maize kernels from fields at both central and southern regions (n=104); 3) maize stubble showing perithecia obtained from 20 cultivated and non-cultivated fields (n=271); and 4) rice kernels from fields in the southern growing-region (n=12). Multilocus genotyping (MLGT) was used to determine species identity and trichothecene genotypes for most isolates. For those from maize kernels, partial gene sequences of TEF, tri3 and tri12 were used. For the wheat population, it was found that F. graminearum with a 15-ADON genotype was dominant (83%), followed by F. meridionale with a NIV genotype (12.8%), F. cortaderiae with mostly NIV and a few 3-acetyl deoxynivalenol (3-ADON) (2.6%), F. austroamericanum with mostly 3-ADON and a few NIV (1.2%) and F. asiaticum with the NIV genotype (0.4%). Frequency of F. meridionale in wheat increased with the decrease of latitudes. For the maize kernel population, F. meridionale was dominant (72%), followed by F. graminearum with the 15-ADON genotype (14.5%) and F. cortaderiae with the 3-ADON and NIV genotypes (13.5%). For the maize stubble population, F. meridionale was dominant (50%), followed by F. graminearum with the 15-ADON genotype (30%) and F. cortaderiae with the NIV and 3-ADON genotypes (20%). For both maize populations, higher diversity was found at higher latitude and elevation. Finally, F. asiaticum with the NIV genotype was the sole species found in rice kernels. These results show that several species coexist in the subtropical to tropical agricultural regions of Brazil where host and geographic (climatic) region shape species composition. Keywords: Gibberella zeae, multilocus genotype, trichothecenes

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P58 - Fusarium species associated with Head blight and Foot and Root Rot on durum wheat in Sardinia, Italy: Results from a 12- year survey 1

1

2

1

2

V. Balmas , A. Marcello , G. Goddi , B. Scherm , B. Satta and Q. Migheli

1

1

Dipartimento di Agraria - Plant Pathology and Entomology Unit, Università degli Studi di Sassari, Via E. De Nicola 9, I - 07100 Sassari, Italy; 2Agenzia LAORE- Sardegna, Via Caprera 8, 09123 Cagliari, Italy E-mail: [email protected]

Fusarium foot and root rot (FRR) and Fusarium head-blight (FHB) are the most damaging diseases of durum wheat in Italy. The aetiology of the disease has been monitored in Sardinia (Tyrrhenian Islands) during twelve years (2001-2012) over approximately 100 fields in different agroclimatic areas of Sardinia. For each field/year, between 20 to 50 basal stems and spikes were collected in order to identify the main causal agents and their associated species. A total of 171 kernel samples yielded in the last three years (2010-2012), were analysed for the presence of deoxynivalenol (DON) and its acetylated forms, while 74 grain samples were also analysed for contamination by T2-HT2 mycotoxin. The quantitative analysis of mycotoxins was carried out with a Lateral Flow Immunoassay (Rapid One Step Assay, Charm Sciences Inc. – Foss). FRR was found more frequently than FHB, and it was mainly incited by Fusarium culmorum. Similarly, in most of the sampled fields the prevalent species causing head-blight was F. culmorum, but in some few areas of central and southern Sardinia, Fusarium graminearum was the main pathogen. Fusarium sporotrichioides, Fusarium acuminatum, Fusarium poae and Fusarium crookwellense were also isolated, albeit at a lower frequency. DON was detected in 34% of the analysed samples, while 71% of the samples turned out to be positive to T2-HT2. Comparing the data from three years, the highest percentage of DON-positive samples corresponds to the lowest percentage of T2-HT2positive samples. All tested samples remain behind the compulsory limit established by the European Union (EU) for the presence of DON (1750 ppb), whereas 6% (i.e., 17% of all DON-positive assays) showed a DON contamination over 100 ppb. At present, no legal limits are imposed for T2-HT2 concentrations. Nonetheless, only 3% of the examined samples (i.e., 4% of all T2-HT2-positive assays) showed T2-HT2 concentration values over 50 ppb. Keywords: epidemiology, Immunoassay, food safety

deoxynivalenol,

T2-HT2

toxin,

Lateral

Flow

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P59 - Head blight of wheat in South Africa is associated with numerous Fusarium species and chemotypes 1,2

2

2

3

4

G. J. van Coller , A.-L. Boutigny , L. Rose , T. J. Ward , S. C. Lamprecht , A. 2 Viljoen 1

Directorate of Plant Science, Western Cape Department of Agriculture, Private Bag X1, Elsenburg 7607, South Africa; 2Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa; 3Bacterial Foodborne Pathogens and Mycology Research Unit, Agricultural Research Service, United States Department of Agriculture, Peoria, IL 61604, USA; 4Plant Protection Research Institute, Agricultural Research Council, Private Bag X5017, Stellenbosch 7600, South Africa E-mail: [email protected]

Fusarium head blight (FHB) of wheat is caused by numerous Fusarium species, including trichothecene-producers. In South Africa, FHB is mostly associated with irrigated wheat rotated with maize. Twenty symptomatic wheat heads were collected from four cultivars each in irrigated fields during 2008 and 2009 in the Northern Cape Province (12 sites), in KwaZulu-Natal (KZN) (seven sites), the Free State (six sites), at four sites in the Bushveld (2009), and under dryland conditions in the Western Cape (four sites). A total of 1323 Fusarium isolates were obtained from kernels, identified molecularly and morphologically, and chemotyped. Fifteen Fusarium species were isolated, with the F. graminearum species complex (FGSC) dominant at most sites. Other Fusarium spp. included F. avenaceum, F. brachygibbosum, F. cerealis, F. chlamydosporum, F. culmorum, F. incarnatum-equiseti species complex (FIESC), F. lunulosporum, F. oxysporum, F. poae, F. pseudograminearum, F. solani, F. tricinctum, the Gibberella fujikuroi species-complex and an unknown Fusarium species. Fusarium pseudograminearum was dominant at one location in the Free State and in the Western Cape. Isolates representing the FGSC were identified using a microsphere-based Multilocus Genotyping Assay (MLGT). FGSC members included F. graminearum s.s. (85.2%), F. boothii (8.3%), F. meridionale (3.6%), F. acaciae-mearnsii (1.4%), F. cortaderiae (1.1%), and F. brasilicum (0.4%). The 15ADON chemotype was most common in 2008 and 2009, the 3-ADON chemotype in the Western Cape in 2009 and at one location in the Free State (2008 and 2009), and the NIV chemotype was most common at one site in KZN in 2009. This extensive survey reported F. lunulosporum for the first time on wheat worldwide and identified production areas of concern in South Africa regarding mycotoxin contamination.

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P60 - Fusarium mycotoxin contamination Fusarium species in Polish wheat in 2010-2012

and

P. Ochodzki, T. Góral, I. Grzeszczak Department of Plant Pathology, Plant Breeding and Acclimatization Institute NRI, Radzików, 05-870 Blonie, Poland; E-mail: p.o[email protected]

Monitoring of cereals contamination by mycotoxins and fungi play an important role in food safety control system in Poland. In three consecutive years 20102012 grain of two winter wheat varieties: Bogatka (resistant) and Muszelka (susceptible) and one winter durum wheat cv. Komnata (very susceptible) from the Research Centre for Cultivar Testing experimental stations (26, 32 and 23 respectively) were collected and analyzed. Fusarium damaged kernels (FDK %), Fusarium species profile and content of main mycotoxins in grain were analyzed. Trichothecenes B content in 2010 was relatively low and reached 0,132 ppm (0,099-0,345 ppm) for Bogatka and 0,171 (0,105-651ppm) for Muszelka. Among Fusarium species dominated trichothecenes B and zearalenone-producing F. graminearum. Other species (F. culmorum, F. poae, F. langsethiae, F. avenaceum) occurred in a lower extent. In 2011 trichothecenes B content was higher and reached 0,171 (0,014-0,793) 0,533 (0,036-2,304) and 1,994 ( 0,0654,670) for Bogatka, Muszelka and Komnata, respectively, with similar distribution of Fusarium species. Year 2012 was most favourable for Trich B production, with an average content Trich B 0,264 ppm (0,015-1,370), 0,736 ppm (0,021-2,669) and 3,344 (1,2599,229) ppm in Bogatka, Muszelka and Komnata, respectively. Most prevalent mycotoxin detected in grain was DON, while DON- derivaties and NIV were detected in much lower concentration In some of samples, mainly cv. Komnata in T-2 toxin was detected in appreciable amount in 2011 and 2012. Results clearly showed high variability of all of traits in years, but also strong differences between varieties and localizations. Most resistant variety in terms of mycotoxin contamination and FDK scores was Bogatka, while both Muszelka nad Komnata revealed high susceptibility to Fusarium. Results shows need for further improvement of wheat resistance, especially in a case of durum wheat. Project was financially supported by Ministry of Agriculture and Rural Development Keywords: Fusarium, mycotoxins, trichothecenes, resistance, wheat, durum

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P61 - Fusarium head blight of wheat in Algeria: Preliminary investigations into the relationship with some isolates and cultivars resistance 1

2

S. Hattab–Touati , C. Barreau , Z. Bouznad

3

1

Université Tlijène Laghouat, Algeria; 2INRA UR MycSA, 71 avenue Edouard Bourlaux, 33883 Villenave d’Ornon, France; 3Laboratoire de Phytopathologie et Biologie moléculaire, ENSA El Harrach, Alger E-mail: [email protected]

Fusarium head blight (FHB) is an important fungal disease of wheat, where it may contribute to a significant reduction in crop and are therefore of great economic importance. These Fusarium spp have also a potential to produce mycotoxins that cause a potential health risk when contaminated grain. Also there are numerous reports on how species differentially respond to different isolates and cultivars. In Algeria there are two species which are frequently isolated from infected ear and seeds: F. graminearum and F. culmorum, with a higher frequence for the later species. During two last years, several experiments were carried out with four isolates of F. culmorum inoculated in field on common cultivated varieties, to determine their potential chemotype to produce toxins and to investigate into the relationship with their aggressiveness and cultivars resistance. The results obtained by PCR Taq Man using a specific probe both " chemotype " known for F. culmorum suggest that isolates T52006 and T72007 have the capacity to produce Nivalenol and Fusarenone X toxins (chemotype NIV / FX) and isolate BD2011 and BT2011 produces Deoxynivalenol and deoxynivalenol 3Acetyl (chemotype DON / 3-ADON). The toxinogenic potential was also looked for in vitro on sterile grains of rice; the analyses of the TCTB by HPLC / DAD allowed to confirm the chemotypes of 4 isolates and to determine their toxinogenic potential. It is shown that 2 isolates of chemotype DON/ 3-ADON produce levels of toxin much superior to those NIV / FX isolates. A quantification of Trichothecens B ( TCTB) by HPLC / DAD on 104 samples of grains obtained from ears of 8 varieties inoculated in field, showed well and confirmed that isolates T52006 and T72007 produces NIV and FX toxins, while isolates BD2011 and BT2011 produce DON and 3-ADON toxins. The levels of NIV / FX are clearly lower than the levels of DON / ADON. The 8 varieties of wheat showed a significant variation in the level of accumulation, otherwise these first results show that there is a correlation between the level of invasion of the grain and the quantity of accumulated toxin. Keywords: Wheat, Fusarium head blight, F. culmorum, Trichothecenes B (TCTB)

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P62 - Biodiversity of Fusarium spp. on cereals in different regions of Russia T. Kolomiets, E. Kovalenko, M. Kiseleva, L. Pankratova, N. Zhemchuzhina All-Russian Research Institute of Phytopathology; Moscow region, Odintsovo district, str. Institute, b.5, VNIIF; Moscow Russia. E-mail: [email protected]

Monitoring survey of species causing fusarium head blight, root rot and snow mold of cereals was carried out in 11 regions of Russia. The dominating role of Fusarium spp. was indicated. Twelve species of genus Fusarium were identified by long-time mycological tests. Widely distributed species and limited species were detected by comparative analysis of occurrence of pathogens with fusarium effects on wheat, barley and rye crops in different regions. F. nivale (Monographella nivalis), F. culmorum and F. sporotrichoides were predominated with the highest frequency through the pathogens causing fusarium head blight and snow mold on winter rye. F. culmorum, F. oxysporum were predominated through the pathogens causing root rot. F. sporotrichoides was prevailing within the complex of fungi affecting an ear of winter rye and barley. F. oxysporum was indicated with the highest frequency through the pathogens of wheat root rot. It demonstrated higher adaptability than pathogens of fusarium head blight F. graminearum (Gibberella zeae) and snow mold F. nivale (Monographella nivalis). There was distribution zonality of Fusarium spp in Russia excepting F. oxysporum. F. heterosporum (G. gordonii), F. moniliforme (G.moniliformis), F.graminearum (G.zeae) were dominated in southern regions of European Russia; F. culmorum - in northern regions; F. gibbosum - in Urals. F. heterosporum and F. culmorum were equally presented in central Russia. F. heterosporum was indicated with high frequency in Siberia. Occurrence of F. sporotrichioides was increasing in Easten Siberia. This fungus was predominated in Far East. This distribution zonality was dependent on soil and climate traits of the regions. With low frequency of occurrence in the majority of regions of Russian Federation following species have been identified: F. solani (Nectria haematococca), F. avenaceum (G. avenacea), F. sambucinum (G. pulicaris), F. semitectum (F. incarnatum), F. lateritium (G. baccata). Keywords : Fusarium, species, frequency, zonality

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P63 - Interspecific and intraspecific variability of Fusarium fungi T. Kolomiets, L. Pankratova, E. Kovalenko All-Russian Research Institute of Phytopathology; Moscow region, Odintsovo district, str. Institute, b.5, VNIIF; Moscow Russia E-mail: [email protected]

Pathogen fungi of genus Fusarium causing fusarium head blight, root rot and snow mold of wheat have the high genetic variability. Samples from infected plants consist of several species and special forms which are different by morphology and cultural traits, sporulation ability, pathogenicity and phytotoxicity. There were 2-7 morphotypes of each Fusarium species identified by the long-time observation of morphology and cultural traits. The correlation between sporulation ability and morphology of Fusarium isolates was not shown. The reliable differences were shown for pathogenicity and phytotoxicity between Fusarium spp. F. sporotrichioides and F. sambucinum(Gibberella pulicaris) have the high level of pathogenicity. The maximum of phytotoxicity was demonstrated for F. graminearum (G.zea) and F.nivalis (Monographella nivalis). The moderate level of pathogenicity and phytotoxicity was demonstrated for F. oxysporum and F. culmorum. Pathogenic and toxic fungi were presented during the whole of vegetation of wheat cultivars. Fusarium fungi have the high intraspecific variability. A sample from 19 isolates of F. graminearum was composed from 10 isolates with high phytotoxicity, from 8 without any phytotoxicity, and a single with moderate phytotoxicity. A sample from 13 isolates of F. oxysporum consisted of a single isolate with moderate phytotoxicity, 6 isolates with low phytotoxicity and 6 isolates without any phytotoxicity. Isolates with high toxicity have not always high pathogenicity. There were 3 isolates of F. graminearum without any pathogenicity and 6 with moderate pathogenicity from the total sample of high toxigenic isolates. Two directly contrary isolates of F. сulmorum were selected by pathogenicity and phytotoxicity. They are pathogenic but they have low phytotoxicity up to nothing. No isolates with complex of high pathogenicity and phytotoxicity were identified through Fusarium fungi from wheat. Keywords: Fusarium, variability, pathogenicity, phytotoxicity

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P64 - Plant pathogenic fungal interactions in oats A.-K. Kolseth, P. Persson Swedish University of Agricultural Sciences, Department of Crop Production Ecology, P.O. Box 7043, SE 750 07 Uppsala, Sweden E-mail: [email protected]

In Sweden 20% of all the small grain cereals cultivated is oats, a crop that up until now has been considered to be at good break crop in crop rotations including other small grain cereals. In recent years cereal infections with Fusarium langsethiae and F. graminearum in oats have been severe and problematic for the farmers, mainly lowering the quality of the yield through mycotoxin production. F. langsethiae is found more often in oats than other cereals while F. graminearum has not been considered a problem in oats until the last years. The main objective of the presented study was to learn more of the biology of F. langsethiae. The experiment was designed to study under which environmental conditions F. langsethiae spreads to new plants and within plants during the vegetation period, and which of the plant developmental stages are most sensitive to infection by F. langsethiae. An additional objective was to study how F. langsethiae interacts and perform together with the two other common species found in Sweden, F. graminearum and F. culmorum. Analyses of plants reveal no detectable endophytic infection of F. langsethiae suggesting it has an epiphytic life style. Its pattern of infection also confirms it being a minor pathogen, since it did not cause early plant death, which, in comparison, both F. graminearum and F. culmorum did. In addition, F. langsethiae seem to hamper the infection of F. graminearum, either by direct competition or by triggering the immune response in oats or a combination of the two. Keywords: Gibberella zeae, perithecia, ascospores

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P65 - Molecular and chemical analysis of trichothecene diversity of Gibberella zeae populations from corn, wheat and potatoes in eastern Canada 1

2

2

3

R. R. Burlakoti *, L. Tamburic-Illincic , V. Limay-Rios , R. D. Peters , and P. 2 Burlakoti 1

Weather INnovations Incorporated, Chatham, Ontario; 2University of Guelph, Ridgetown Campus, Ridgetown, Ontario; 3Agriculture and Agri-Food Canada, Charlottetown, Prince Edward Island E-mail: [email protected]

Gibberella zeae, a principal pathogen of Fusarium head blight of wheat and ear rot of corn in Canada, causes significant yield and quality losses as well as contaminates grains with trichothecene mycotoxins. The fungus is also a potato pathogen and is routinely recovered from potato tubers showing symptoms of Fusarium dry rot in Canada. Corn, wheat and potatoes are economically important crops in eastern Canada and these crops are commonly rotated or grown in nearby fields. Therefore, comparative analysis of the trichothecene diversity and population structure of G. zeae isolates from these crops will be useful to develop strategies to manage the diseases in these crops. In addition, multiyear comparisons of fungal populations across diverse regions will identify the impact of weather patterns and other agronomic factors on pathogen populations and disease potential. Gibberella zeae isolates were recovered from corn and wheat grain samples collected from 25 diverse locations in Ontario during 2010 and 2011. Fungal isolates from potato were recovered from samples collected in Quebec, New Brunswick, and Prince Edward Island. Approximately 250 single spore strains of G. zeae (187 from corn, 49 from wheat, and 14 from potatoes) were characterized and trichothecene genotypes were identified using TRI3- and TRI-12 based molecular markers. Molecular analysis revealed that the majority of G. zeae strains from corn and wheat were 15-Acetyl-DON (15ADON) types (97% and 98%, respectively). Interestingly, all the G. zeae strains from potatoes were 3-Acetyl-DON (3ADON) types. The ability of representative isolates to produce 3ADON and 15ADON was verified in rice culture with TLC and quantified using an ESI-LC-MS/MS system. Characterization and trichothecene profiling of G. zeae isolates collected in 2012 is in progress. This study will provide base-line data on 3ADON and 15ADON profiles of G. zeae isolates from wheat, corn and potatoes in eastern Canada. Keywords: Gibberella, Trichothecene, population structure

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P66 - PCR validation and chemotyping of causal Fusarium species of Fusarium head blight on south african wheat 1

1

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N. Baloyi , S. L. Sydenham , C. I. P. De Villiers , M. Gryzenhout

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1

ARC-Small Grain Institute, Bethlehem, Free State Province, South Africa, Private Bag X29, 9701; Department of Plant Sciences, University of the Free State, Bloemfontein, Free State Province, South Africa, P.O. Box 339, 9300 E-Mail: [email protected] 2

Species identification and chemotyping of 203 single spore isolates collected from Fusarium head blight (FHB) symptomatic wheat spikes from four widespread and representative localities (Dundee, Groblersdal, Frankfort and Vaalharts) were validated using molecular assays. Single spore isolates were plated on potato dextrose agar (PDA). DNA was extracted with the modified cetyltrimethylammonium bromide (CTAB) method (Saghai-Maroof et al. 1984). Species-specific primers were used for PCR analysis (Obanor et al. 2012). The presence of mycotoxins, NIV, 3-ADON and 15-ADON, was determined using tri12 and tri13 PCR primers. PCR products were run on 2% agarose gels stained with GR-green and sizes were determined with a 100bp DNA ladder. Three Fusarium species were identified: F. graminearum, F. boothii and F. pseudograminearum. In Frankfort both F. graminearum and F. pseudograminearum where found. F.graminearum and F.boothii were both found in Groblesdal. Dundee and Vaalharts only had F.graminearum present. F. graminearum was the predominant causal species of FHB on wheat in all four localities. Two trichothecene types, 15-ADON and NIV, were detected among the isolates, with the 15-ADON type predominating in all the localities. No 3-ADON producing isolates were found. These results identifying F. graminearum as the predominant casual species of Fusarium head blight on wheat in South Africa correlate well with findings published by Boutigny et al. (2011). This information will help breeders and pathologists safeguard wheat, enabling them to target useful/appropriate resistance against the specific causal Fusarium species. Additionally, it will help with the establishment of a Fusarium isolate collection to be kept at the ARC-SGI for future reference. Keywords: chemotype, F. graminearum, species-specific primers, trichothecenes

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P67 - Genetic and mycotoxigenic diversity of isolates belonging to the Fusarium incarnatum-equiseti species complex, and recovered from maize and banana in China 1

2

2

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F. Munaut , J. Scauflaire , M. Gourgue , C. Bivort , Y. Qi , A. Wu , S. de 5 4 1 Saeger , D. Zhang , F. Van Hove 1

Université catholique de Louvain, Earth and Life Institute (ELI), Applied Microbiology, Mycology, Mycothèque de l’UCL (BCCMTM/MUCL), Louvain-la-Neuve, Belgium; 2Université catholique de Louvain, Earth and Life Institute (ELI), Applied Microbiology, Mycology, Louvain-la-Neuve, Belgium; 3 Academy of Tropical Agricultural Sciences (CATAS), Environment and Plant Protection Institute, Haikou, Hainan Province, China; 4College of Life Science and Biotechnology, Shanghai Jiao Tong University (SJTU), Shangai, China; 5Universiteit Gent (UGent), Faculteit Farmaceutische Wetenschappen, Laboratory of Food Analysis, Ghent, Belgium E-mail: [email protected]

In the frame of a research project aiming to describe the Fusarium spp diversity on maize and banana in four Chinese provinces, 80 out of the 201 isolates obtained were identified as belonging to the Fusarium incarnatum-equiseti species complex. Isolates were identified on basis of the Elongation Factor-1 a sequences (EF), which were then submitted to a phylogenetic analysis together with four isolates representative of four phylogenetic species inside the complex (F. incarnatum s.s., F. lacertarum, F. scirpi and F. equiseti). On maize, five main phylogenetic groups were determined (I, II, III, IV and V), from which three groups (I, II and V) were also recovered from banana. Surprisingly, groups I, II, III and V can be correlated to phylogenetic species described in completely different ecosystems by O’Donnell et al. (2009) (from human beings) and by Funnell-Harris et al. (2010) (from sorghum). Furthermore, one possible new phylogenetic species (group IV) was detected. Genetic diversity of the FIESC isolates was analyzed using the AFLP technique. A total of 200 polymorphic bands were generated for 28 FIESC isolates from maize and were used to construct a phenetic tree. The EF phylogenetic groups were clearly clustered separately by AFLP, with bootstrap values >70%. The groups II and V presented an approximate similarity of only 50% and 40 % from the other cluster, respectively. This raises the taxonomical status of the groups. Within each cluster, an important genetic diversity is observed, although no strict correlation can be made with any parameters of collect such as location, field, etc. Preliminary results on banana isolates showed similar results as well as the occurrence of an important genetic diversity. Molecular studies have demonstrated that the FIESC isolates do not possess the FUM gene cluster. Nevertheless, it has been reported that FIESC isolates are able to produce type A and B trichothecenes. Due to the fact that trichothecenes are also produced by Fusarium graminearum, which was frequently isolated from Chinese maize, the origin of trichothecene contaminations on that crop has to be reconsidered. Furthermore, the occurrence of FIESC isolates on both maize and banana, which are often cultivated close to each other, raises the question of possible cross-contaminations. Keywords: Fusarium, FIESC, maize, banana, China, phylogeny, AFLP, mycotoxins

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P68 - Population analysis of Fusarium graminearum sensu stricto from wheat and maize in the United Kingdom 1,2

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R. Basler , S. Edwards , J. Thomas

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1

National Institute of Agricultural Botany, Huntingdon Road, Cambridge, CB3 0LE, United Kingdom; Harper Adams University, Newport, TF10 8NB, United Kingdom E-mail: [email protected] 2

Fusarium graminearum sensu stricto (s.s) is a diverse toxigenic species that contaminates wheat and maize in variable climates. In the United Kingdom wheat is the major arable land crop, 1,969,000 hectares annually, and maize is an emerging crop with an increase of 20% over six years to 146,000 ha. Maize in the UK is primarily intended for silage and improved cultivars and changes in the climate have contributed to increases in UK maize production. The diversity of Fusarium species and their associated mycotoxins was determined in UK maize with a survey of kernel and stalk samples in 2011 and 2012 from fifteen sites. The predominant species isolated were F. culmorum (45%) and F. graminearum s.s. (44%) in addition 12 other Fusarium species were identified at lower frequencies. To compare the genetic architecture in F. graminearum s.s. in UK maize and wheat eighty-two samples of UK wheat kernels were screened for F. graminearum s.s. in 2012. The species diversity and toxigenicity between maize and wheat are being analysed with mini-, variable number tandem repeats, and micro-satellite markers. Presently the molecular phenotyping of the isolates indicates regional characteristics associated with nivalenol producing isolates of maize F. graminearum s.s. to be in the south and south eastern maize growing regions of the UK. Keywords: Fusarium graminearum, nivalenol

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P69 - The French Fusarium Collection: a living resource for mycotoxin research L. Pinson-Gadais, M. Foulongne-Oriol, N. Ponts, C. Barreau, F. RichardForget INRA UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS20032, 33882 Villenave d'Ornon, France E-mail: [email protected]

Fusaria are responsible for prejudicial diseases on cereal crops worldwide, such as crown rot and Fusarium head blight. Beyond economic losses due to infection symptoms, these pathogens can produce several types of mycotoxins that are harmful to livestock and humans. They are extremely diverse at the intra-specific levels in terms of types as well as quantities of toxins that a strain can produce. Developing appropriate strategies to limit contamination with Fusarium mycotoxins requires a greater knowledge about this variability. We have collected a large number of toxinogenic Fusarium strains. Our assortment now includes about 800 strains, mostly from the species graminearum, culmorum, verticilloides, proliferatum, and temperatum. Species were identified based on morphology and real-time PCR. More than half of our strains were further characterized for toxin production using biochemical and/or real-time PCR-based tools. We isolated about 70 F. graminearum strains from either wheat or maize grains originating from different French cereal production areas. Our results show a high representation of 15-acetyldeoxynivalenol-producing strains in our French samples. Within the same chemotype, we observe a large variability in toxin production levels. The F. graminearum strains were characterized with microsatellite markers and show a large genetic diversity. Two groups were delineated according to their genetic background, roughly corresponding to strains isolated from European one hand and America in the other hand. Our results are also in agreement with the fact that only F.graminearum sensu stricto strains seem to be detected in France so far. The demonstrated genetic and phenotypic diversity provides a sound ground for countless downstream studies such as genetic association and quantitative genetics to understand the determinism of toxin production. Such information should be doubtlessly considered in plant breeding efforts and other disease management strategies aimed at reducing the mycotoxin risk in food and feeds. Our collection is a valuable tool to improve our understanding of toxigenic diversity in Fusarium species. It is managed through a database gathering all information collected on each strain, already available upon request and soon publically available as a web-based interface. Keywords: Fusarium, mycotoxin, database, diversity

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P70 - ToxiFusaDB: the online catalogue of the MycSA Fusarium strains collection 1,2

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V. F. Wong Jun Tai , L. Pinson-Gadais , C. Fernandez , C. Barreau F. 2 Richard-Forget 1

INRA - UMR0927 I2M, Département Génie Civil et Environnemental de l'Institut de Mécanique et d'Ingénierie de Bordeaux, 16 Avenue Pey-Berland 33607 Pessac Cedex; 2INRA – UR1264 Mycologie et Sécurité des Aliments (MycSA), 71 avenue Edouard Bourlaux, CS 20032, F-33882 Villenave d’Ornon Cedex E-mail: [email protected]

More than 100 species belong to the Fusarium genus. Among them, phytopathogenic fungi can infect various crops worldwide causing significant economic losses. In addition, toxigenic Fusaria can produce a wide array of mycotoxins and represent a serious threat for food and feed safety. Tracking and understanding Fusarium diversity is therefore critical for the development of efficient strategies to control fungal attacks and mycotoxin contamination. The MycSA research group collected more than 400 strains of Fusarium spp. [F.graminearum, F.culmorum, F.verticillioides and F.proliferatum] contaminating french wheat and corn harvests. All stored strains are genotyped using speciesand chemotype-specific markers. The F. graminearum strains were further characterized with a set of 20 microsatellites markers (see poster “The French Fusarium Collection: a living resource for mycotoxin research”). The toxigenic potential - amount of toxins a strain can produce when cultivated on kernels in optimal conditions – has been determined for all Fusarium strains included in the MycSA collection. According to these original and highly interesting phenotyping data, the MycSA collection is unique in Europe. To promote communication, education, exchange and dissemination of these Fusarium characterized strains among the national and international research community, an online public access catalog has been developed. Catalogue consultation does not require specific knowledge on the Fusarium strains complexity and diversity. The catalogue can be browsed or searched using multiple criteria (Fusarium species, chemotype, toxigenic potential…), and strains of interest can be ordered online. Keywords: Fusarium, Catalogue, Distribution, Php, MySQL, HTML / CSS / JavaScript

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P71 - Real time PCR for FHB quantification: bias analysis? 1

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2

S. Elbelt , S. Fromentin , V. Laval , C. Barreau , F. Carpentier , C. Ducos , C. 1 1 3 3 2 Lannou , F. Techini , J. Toussaint , C. Vitry , L. Pinson-Gadais 1

INRA, UR1290 Bioger_CPP, bat 13, avenue L. Bretignière, F-78850 Thiverval Grignon, France; INRA, UR1264 MycSA, 71 Avenue Edouard Bourlaux, CS 20032, F-33882 Villenave d’Ornon, France; 3ARVALIS Institut du Végétal, bat 13, avenue L. Bretignière, F-78850 Thiverval Grignon, France E-mail: [email protected] 2

A better understanding of the FHB species complex and particularly of the way species interactions influence epidemic development and toxin production requires accurate, sensitive and specific molecular diagnostic tools, such as realtime PCR. Nine qPCR tests specifically designed for the most represented species of the FHB complex on wheat in Europe: F. graminearum, F. culmorum, F. poae, F. avenaceum, F. langsethiae, F. tricinctum, F. sporotrichioides, M. majus and M. nivale were assessed for their potential for diagnosis. We present here the results of quantification analysis performed in three laboratories which use different mix and real time PCR apparatus. The variation in quantification will be discussed. In one laboratory the limits of the diagnostic was further studied by analysing DNA extraction and real time PCR repeatability. Statistical analysis was performed on results obtained from two extractions of 222 samples and three real time PCR analysis performed per extraction. First results show the importance to carefully test real time PCR parameters when developing real time PCR tools for diagnosis or epidemiological studies. Keywords: Molecular diagnostic tools, real-time PCR, FHB, inter-laboratory validation tests

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P72 - Fusarium spp. on maize in Belgium, from biodiversity to biocontrol 1

1

1

2

2

2

J. Scauflaire , C. Liénard , M. Gourgue , G. Foucart , M. Mary , F. Renard , A. 3 1,4 Callebaut , F. Munaut 1

Université catholique de Louvain (UCL), Earth and Life Institute (ELI), Laboratory of Mycology, Croix du Sud 2, B-1348 Louvain-la-Neuve, Belgium; 2Centre Indépendant de Promotion Fourragère (CIPF), UCL, Croix du Sud 2/11, B-1348 Louvain-la-Neuve, Belgium; 3Centre d'Etude et de Recherches Vétérinaires et Agrochimiques (CODA-CERVA), Leuvensesteenweg 17, B-3080 Tervuren, Belgium; 4 UCL, ELI, Laboratory of Mycology, MUCL, Croix du Sud 2, B-1348 Louvain-la-Neuve, Belgium Email: [email protected]

Numerous Fusarium species are important mycotoxin-producing pathogens affecting maize in Belgium. Crop quality is often reduced by Fusarium rot diseases, and mycotoxin contaminations can pose a serious problem for animal health. As several of these mycotoxins are integrated into European legislation, field monitoring and accurate identification of the Fusarium species remain important tasks, allowing relevant preventive or curative measures. During epidemiological surveys performed in Belgium since 2005 (*), an extensive collection of more than 7000 Fusarium isolates characterized at the species level was established at the MUCL collection. A total of 24 different Fusarium species were identified to occur on maize ears and stalks, of which F. graminearum (43%), F. crookwellense (16%), F. avenaceum (14%), F. culmorum (10%) and F. temperatum (5%) were the most abundant. According to pathogenicity tests (and mycotoxin screenings), F. avenaceum (MON, ENN producer), F. culmorum (3A-DON, NIV, ZEN producer) and F. temperatum (MON, BEAU, ENN producer) showed the ability to cause seedling malformation and emergence reduction in early season; while F. graminearum (DON, 15A-DON, ZEN, NIV producer) and F. crookwellense (NIV, ZEN producer) were always involved in ear or stalk rot diseases at the end of the growing season, alone or simultaneously. Impacts of environmental factors, cultural practices and hybrid selection were analyzed in the frame of an integrated disease management system. Although climatic conditions are the major factor affecting Fusarium rot diseases, adequate crop rotation with a non-cereal culture and the selection of a less susceptible maize hybrid offered a significant reduction of Fusarium infestation and associated mycotoxin contamination at the end of the growing season. Biologically-based inputs can be used to interfere with damping-off diseases caused by Fusarium species. Therefore, fungal rhizopheric isolates were collected from Belgian maize fields and screenings are in progress for the selection of biological control agents (BCA) of F. avenaceum, F. culmorum and F. temperatum. Until now, several isolates of genus Trichoderma, Penicillium and Bionectria demonstrated strong in vitro antagonism against these damping-off pathogens and were selected for further greenhouse and field tests. In the context of an integrated disease management system, such fungal BCA should improve crop health, minimize the economic and environmental costs of controlling plant pathogens and promote sustainable agricultural production. (*) Funded by the Service Public de Wallonie, DGARNE, Direction de la Recherche. Keywords: biocontrol, Fusarium spp., maize, mycotoxins

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P73 - Creation of the State Collection of Fusarium fungal strains E. Kovalenko, N. Zhemchuzhina, T. Kolomiets, M. Kiseleva, L. Pankratova All-Russian Research Institute of Phytopathology; Moscow region, Odintsovo district, str. Institute, b.5, VNIIF; Moscow Russia E-mail: [email protected]

The ARRIP State Collection of Phytopathogenic Microorganisms includes more than 500 strains of genus Fusarium, such as F. culmorum, F. oxysporum, F. sporotrichoides, F. semitectum (F. incarnatum), F. javanicum, F. moniliforme (Gibberella moniliformis), F. heterosporum (G. gordonii), F. gibbosum (G. intricans), F. avenaceum (G. avenacea), F. sambucinum (G. pulicaris), F. graminearum (G. zeae), F. lateritium (G. baccata), F. nivale (Monographella nivalis), F. solani (Nectria haematococca), F. redolens, F. tricinctum, responsible for such plant diseases as Root Rot, Scab (Head Blight), Leaf Blotch (Snow Mold), Fusarium Wilt. The collection was created as the result of long-term monitoring of the specific and interspecific composition of pathogenic mycoflora on the fields of agricultural cultures in different regions of Russia and it is regularly replenished with new specimens. Intraspecific variability of pathogens has been studied concerning their morphological properties, pathogenicity and phytotoxicity. The cataloguing of Fusarium strains with the indication of full characteristics of their main properties has been carried out. Fusarium species are characterized by a high genetic variability and are able to lose the viability and change their physiological properties in the course of long-term storage. Included into the State collection strains are regularly tested and re-cultured to maintain stable morphological and pathogenic properties. Strains of fungi are stored using the following methods: in test tubes on the standard nutritious medium and on grain at 0 ° +4 С, in Eppendorf microtubes filled with 50% glycerin at -80 C and as lyophilized ° material (mycelium and spores) on strips of filter paper in cryotubes at -80 C. As required, pathogenic strains are removed from the storage and used for the research and applied activities. Keywords: Fusarium, collection, cataloguing, storage

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P74 The molecular characterization and determination of genetic variability in Fusarium verticilloides strains isolated from maize in Turkey 1

2

2

B. Kansu , P. Marin , M. T. Gonzalez-Jaen , B. Tunali

1

1

Ondokuz Mayis University, Agriculture Faculty, Department of Plant Protection, 55139, SamsunTurkey; 2Department of Genetics, Faculty of Biology, Complutense University of Madrid, Jose Antonio Novais 12, 28040 Madrid-Spain E-mail: [email protected]

Maize ear rot is one of the most important and common disease in maize fields of Turkey, as well as all over the world. The causal agents of this disease include some of the Fusarium species, but Fusarium verticilloides (Gibberella moniliformis, G. fujikuroi mating population A) is considered the main responsible for decreasing yield in fields. Additionally this species produces several mycotoxins, especially fumonisins, which are dangerous for human and livestock. In order to devise effective strategies to reduce both pathogen growth and fumonisin risk it is necessary to gain knowledge about the genetic variability and the population structure to detect potential populations and lineages that might exist in terms of toxin profile, response to antifungal agents or differential climatic features. These aspects were studied using the partial sequences of the IGS region (Intergenic Spacer of rDNA units) and the translation elongation factor 1 α gene (EF1-α) of a representative sample (66 strains) of F. verticilloides isolated from six different agro-climatic regions of Turkey. Additional strains from diverse geographic origins were also included in the phylogenetic analyses performed using PAUP. The fumonisin-producing ability of the F. verticillioides strains was tested using a specific PCR assay based on FUM1 gene, a key gene of fumonisin biosynthetic pathway. All the strains were positive to the analysis. Additionally, mating type alleles, MAT-1 and MAT-2, were determined in all the strains. Percentages of MAT-1 and MAT-2 idiomorphs were 63.2% and 36.8%, respectively. The separate and combined IGS and EF1-α phylogenies were obtained and discussed in comparison with previous analyses reported and in relation with climatic factors. This research was supported by the Project Office of Ondokuz Mayıs University of Turkey (PYO.ZRT.1904.012.010) and was supported partially by the Ministry of Science and Innovation of Spain (AGL2010-22182-C04-01). Keywords: Fusarium verticilloides, IGS, EF–1, species-specific PCR

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P75 - Monitoring of maize contamination by Fusarium mycotoxins in Poland in 2012 1

P. Ochodzki , E. Czembor

2

1

Department of Plant Pathology, 2Department of Grasses, Legumes and Energetic Plants, Plant Breeding and Acclimatization Institute NRI, Radzików, 05-870 Blonie, Poland E-mail: [email protected]

Evaluation of maize contamination by Fusarium and mycotoxins plays an important role in food safety control systems in the World, including Poland. In 2012 we collected 106 samples grain of 30 maize varieties of different earliness type (9 early-type FAO 230-240, 9 of medium-early FAO 240-260 and 11 of medium-late type FAO 260-290) from 10 experimental stations of Research Centre for Cultivar Testing. Fusarium incidence, species profile and content of most important mycotoxins : deoxynivalenol (DON) and fumonisins (FUM) were analyzed. In 33 samples fumonisins level exceeded level 250 ppb (mean value 1210 ppb, maximum 5990 ppb). In 79 samples DON level exceeded 250 ppb ( mean value 560 ppb, maximum 4770 ppb), and in 3 samples DON content was higher than EU guidance level (1750 ppb). Of 10 experimental stations, most favorable conditions for both FUM and DON production were found in Subcarpathian Province, Southeastern Poland. In this region FUM cumulation in early varieties was lower than in medium-early and medium late ones ( 1060, 1700 and 1580 ppb respectively), whereas DON content ranged from 570 and 660 ppb to 1250 ppb in early, medium-early and medium late types respectively. Very high variability in mycotoxin content between varieties and locations was found. Project was financially supported by Ministry of Agriculture and Rural Development Keywords: Fusarium, mycotoxins, trichothecenes, maize

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P76 - Fusarium verticillioides and F. subglutinans mating types – distribution and molecular structure E. Jabłońska, M. Wit, W. Wakuliński Department of Plant Pathology, Warsaw University of Life Sciences; 159 Nowoursynowska St., 02-776 Warsaw, Poland E-mail: [email protected]

Fusarium genus comprises several toxigenic species associated with maize, including F. verticillioides and F. subglutinans. Both species are the members of Liseola section and causative agents of the ear rot of maize, while F. verticillioides is also considered to be the most prolific producer of fumonisins. The teleomorphs of F. verticillioides and F. subglutinans belong to the Gibberella fujikuroi species complex. Both species are heterothallic fungi with a dimictic mating system typical for numerous Ascomycota and governed by two idiomorphic alleles, MAT1-1 and MAT1-2. High genetic diversity observed in these species and the coexistence of the two mating types in fungal populations at the same geographical scale may suggest the occurrence of the so-called cryptic sex. As the proportion of the two complementary thalli of opposite mating types is an important factor for the occurrence of the sexual process in heterothallic Fusarium species, the aim of the study was to determine the frequency of both idiomorphs (MAT1-1 and MAT1-2) and to describe the MAT locus structure. For this purpose a total of ninety-nine Fusarium isolates collected from infected corn kernels were examined. On the basis of classical mycological methods of morphological identification, 47 isolates were identified as F. verticillioides, and 52 – as F. subglutinans. Subsequently, morphological identification was verified molecularly using SCAR markers. The mating type identification revealed frequency proportions different from an expected result of 1:1 distribution of MAT1-1 and MAT1-2, according to the chisquare test. In the F. subglutinans population the MAT1-1 and MAT1-2 idiomorphs occurred with frequency 35% and 65% respectively, while F. verticillioides mating type proportion was unexpectedly highly segregated with the ratio of 15% MAT1-1 and 85% MAT1-2. The analysis of the MAT locus structure of F. verticillioides revealed the presence of substitutions, most of them being transitions, and deletions occasionally. Keywords: Fusarium, maize, idiomorphs, mating types

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P77 - Geographic distribution and multilocus analysis of Fusarium subglutinans and F. temperatum from maize worldwide A. Susca, A. Villani, G. Mulè, G. Stea, A.F. Logrieco, A. Moretti. Institute of Sciences of Food Production, CNR, Via Amendola 122/O, 70126, Bari, Italy E-mail: [email protected]

Fusarium temperatum is a new described species occurring on maize in Belgium, closely related to F. subglutinans (Scauflaire et al., 2011). Both species are considered morphologically identical and associated to the Fusarium maize ear rot disease complex. Preliminary data indicate that F. temperatum and F. subglutinans correspond to the two cryptic species F. subglutinans group 1 and group 2, respectively. They have a different toxigenic profile. While both species produce fusaproliferin and moniliformin, only F. temperatum produces beauvericin (Moretti et al., 2008). Geographical distribution and the correct identification of these two species represent the baseline to evaluate the plant exposure to mycotoxins and possible toxigenic risk on maize. In this study, 130 strains isolated from maize worldwide and morphologically identified as F. subglutinans were studied by a multilocus sequence analysis of four independent gene regions (β-tubulin, calmodulin, RNA polymerase II and elongation factor 1α). The results obtained showed that 70 strains (54%) were identified as F. temperatum and were isolated from Australia, Germany, Italy, Netherland, Poland, Slovakia, South Africa and Turkey. On the other hand, 60 (46%) strains were identified as F. subglutinans and originated from Argentina, Germany, Italy, Poland, Serbia, South Africa, Turkey, and USA. In addition, the phylogenetic analyses showed for both species different levels of intra-specific variability, in relation to each gene, with higher level of variabillity due to the elongation factor 1α. Overall these data showed that F. temperatum and F. subglutinans are common in maize worldwide although further data are needed to confirm the absence of F. temperatum in American Continent. These results represents reason of further concern worldwide for the toxigenic risk related to consumption of maize kernel contaminated by this pathogen. Keywords: maize, phylogeny, F. subglutinans, F. temperatum

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P78 - Molecular characterization of Fusarium species occurring on olive fruits in Apulia 1

2

2

2

1

1

S. Frisullo , A. Susca , G. Stea , A. Villani , L. Prudente , M. Contursi , P. 1 2 2 Ferrara , A. Logrieco , A. Moretti 1

Università di degli Studi di Foggia, Dipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, Foggia, Italy; 2Institute of Sciences of Food Production, CNR, Bari, Italy E-mail: [email protected]

Olive cultivation is one of the most important crop in Apulia, with 377,526 hectares cultivated and 10,102,300 quintals of olive production. In a survey aimed to evaluate the fungal colonization of olive fruits carried out in the whole Apulia Region, 5 fields for each of the 92 localities selected were investigated. From the survey, together with fungal strains belonging to Botriosphaeriae, Colletotrichum, Diplodia, Neofusicoccum and Penicillium genera, several hundreds of strains belonging to Fusarium genus have been isolated mainly from olive fruits and, at a lesser extent, also from branches. The strains of Fusarium were identified at a morphologically level, resulting species able to produce a wide range of mycotoxins such as cyclohesadepsipeptides, moniliformin and trichothecenes. However, since each Fusarium species can have a specific mycotoxin profile, the toxicological risk related to their occurrence can be highly variable, according with the main species colonizing the olive fruits and must be accurately assessed. Confirmation of strain identification was carried out by using molecular approach. One-hundred and forty-eight representative strains were analyzed by sequencing a portion of calmodulin and β-tubulin genes, which have been proved to be effective for distinguishing species in Fusarium. Data have shown, for the first time, a wide genetic diversity within the population of Fusarium isolated from olives. In particular, F. acuminatum, F. avenaceum, F. longipes, F. merismoides, F. oxysporum, F. proliferatum, F. solani, and F. torulosum were identified. However, many strains could not be assigned to any species and therefore may represent new entities within the genus. The occurrence of some highly toxigenic Fusarium species suggests that a toxicological risk can occur in olive fruits highly contaminated by Fusarium and that such risk must be constantly monitored, also in order to evaluate possible influence of climatic changes on the Fusarium spread on this crop in Apulia. Keywords: olive fruits, Fusarium acuminatum, calmodulin, β-tubulin

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P79 - Evaluation of Fusarium wilt resistance among the accessions of eggplant (Solanum melongena L.) 1

2

3

H. H. Altinok , C. Can , F. Boyaci , V. Topçu

3

1

Department of Plant Protection, Faculty of Agriculture, Erciyes University, Kayseri, Turkey; Department of Biology, Faculty of Science, Gaziantep University, Gaziantep, Turkey; 3West Mediterranean Agricultural Research Institute, Antalya, Turkey E-mail: [email protected] 2

Fusarium wilt (Fusarium oxysporum (Schlechtend: Fr.) f. sp. melongenae; Fomg) is one of the most destructive and widely distributed diseases of eggplant in Turkey. Resistant cultivars are yet to be developed against this pathogen. In this study, eggplant accessions were tested with three highly virulent Fomg isolates (MS-7, UBR-9 and Fomg10) in order to evaluate the presence of field resistance to Fusarium wilt. The plant material for the resistance screening study consisted of 13 accessions of eggplant (Solanum melongena L.), namely, “AGR-703, Amadeo F1, Anatolia F1, Angela F1, Brigitte F1 Corsica F1, Hadrian, Hawk, Kemer, Köksal, Nouma, Sharapova, Yula”. Observations were recorded from leaves at th th th th st th 7 , 11 , 14 , 18 21 , and 25 days using a disease rating scale. The disease severity (%) and the area under disease progress curve (AUDPC) for each of the Fomg isolates were calculated by scale values. Disease incidence ranged from 10% to 95% at eggplant seedlings. According to test results, AGR-703 was found resistant; Köksal and Hawk were moderately resistant, Amadeo F 1, Corsica F1, Anatolia F1, Sharapova, Brigitte F1, Angela F1, Nouma and Yula were moderately susceptible and Hadrian and Kemer were identified as susceptible. Due to its strong resistance against the pathogen, among the all accessions tested, AGR703 is considered as the most appropriate for rootstock in eggplant cultivation where Fusarium wilt is present in the soil. Keywords: Fusarium wilt, cultivar, rootstock, disease severity, eggplant

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SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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FUSARIUM,

P80 - Breeding resistance for Fusarium head blight in supporting higher efficiency of the integrated plant management in wheat Á. Mesterházy, S. Lehoczki-Krsjak, M. Varga, Á. Szabó-Hevér and B. Tóth, P. Nicholson and M. Lemmens Cereal Research Ltd., Alsókikötősor 9, H-6726, Szeged, Hungary E-mail: akos.mesterhazyabonakutato.hu

The experience shows that the breeding of more resistant wheat genotypes is a slow process in spite of the efforts done. The genetic background is complicated; the additive effect of different QTLs is seldom expressed, sometimes different gene combinations might be more susceptible. Most of the QTLs determine FHB reaction, some other only FHB or DON, but only a few influences all. Most of the QTLs are of low or medium effect and many of them cannot be validated. Often they determine traits that influence disease development, but have nothing common with physiological resistances Type I-V. The strong selection for Type II resistance was less effective than thought because fhb1 major QTL determines only 30-40 % os the resistance, the other part is coming from other Type I and Type II QTLs. For this reason a change is now under way to use spraying inoculation more for selection than single floret inoculation. Within the native or local wheat programs many lines and genotypes were found with higher resistance to FHB and their use is more expanded as was before. The high resistance level Asiatic sources resulted in higher resistant plants, but seldom varieties, because the bad agronomy form is hard to correct during the breeding. In the Szeged breeding program several of this type have very high resistance level with acceptable agronomy background. At present the native originated varieties from Szeged give an increasing acreage in Hungary, the leading cultivar is Csillag, but others like Fény, Körös, Berény, and Rozi are also in increasing. The fungicide trials showed that these cultivars in epidemic conditions showed moderate symptoms and combined with fungicide treatment with better technology, also under epidemic conditions the low DON contamination can be secured (below the EU limit 1.25 ppm DON). Acknowledgements: The authors express their thanks for financial support to projects FP7 MycoRed KBBE-2007-2-5-05, contract No. 222690, and GAK (OMFB-00313/2006 ). Keywords: resistance, breeding strategies, DON, QTL, Integrated plant management

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P81 - Breeding of cereal cultivars resistant to Fusarium fungi T. Kolomiets, L. Pankratova, E. Kovalenko All-Russian Research Institute of Phytopathology, Moscow Region, Odintsovo district, str. Institute, b.5, VNIIF, Moscw, Russia E-mail: [email protected]

Breeding of cereal cultivars resistant to Fusarium fungi causing fusarium head blight, root rot and snow mold is the important problem all over the world, including Russia. Genetic diversity of world gene accessions was studied for selection of breeding sources resistant to Fusarium fungi. Ecological and genetic differentiation of species of host plant was taken into account. Total of 5000 wheat cultivars from different genetic groups was screened with identification of different types and rates of disease severity of root rot and snow mold. There was 6,7% resistant lines from winter accession of bread wheat (T. aestivum), 8,8% from spring accessions of bread wheat, 1,4% - from spring accessions of durum wheat (T. durum) selected by screening to resistance. The high resistance was shown for Triticale lines. The samples from Aegilops aucheri, Ae.ovata, Ae.columnaris, Ae.triuncialis, Ae.koteshyi species were resistant to fusarium diseases. Distribution of resistant wheat lines dependent on their geographical origin. Winter wheat cultivars with moderate resistance to fusarium root rot were dominated in Western Europe (Icsu – France, Horpacsi – Hungry, Yasen – Bulgaria etc.) and in North America (Chiefkan, Archamp, Benni, Coker 9227 – USA etc.). Resistant spring bred wheat cultivars were revealed within 8 ecological-geographical groups presented majority of countries. The focus of interest was on accessions introduced from North and South America, India, as well as international and regional nurseries (Laura - Canada, Achut - Nepal, Vectis - USA, BVF-2 - Mexico etc.). Durum wheat cultivars with moderate resistance were dominated in the Mediterranean regions and in the southwesten Asia (Aya de Carvo - Portugal, 80/57 – Italy, Ak-bugda - Dagestan, k.6397 Georgia, k.6397, k. 36269 - Armenia). The origin of wheat cultivars with high resistance to snow mold was West Europe (Glockner, Agent, Aron - Germany, Szemes - Hungary, Turda 81 - Rumania, Sol IV - Sweden). Keywords: Fusarium, wheat, cultivars, breeding

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FUSARIUM,

P82 - Interaction between Quantitative Trait Loci (QTL) for Fusarium head blight (FHB) resistance and Fusarium graminearum 15-ADON and 3-ADON chemotypes in spring wheat L. Tamburic-Ilincic, A. Muckle, A. Schaafsma University of Guelph, Ridgetown Campus, 120 Main St. E. Ridgetown, Ontario, N0P2C0, Canada E-mail: [email protected]

Fusarium graminearum (Schwabe) (FG) is the principal cause of Fusarium head blight (FHB) in North America, one of the most serious diseases of wheat (Triticum aestivum L.). Deoxynivalenol (DON) is the most important mycotoxin produced by FG. 15-acetyl DON (15-ADON) and 3-acetyl DON (3-ADON) chemotypes of FG produce DON and 15-ADON and DON and 3-ADON analogs, respectively. Quantitative trait loci (QTL) associated with FHB resistance from a Wuhan x Nyubai spring wheat population were previously published. In the present study we investigated: 1) which QTL or QTL combination (no QTL, 3B, 5A and 3B+5A) result in the lowest FHB symptoms and DON accumulation and 2) whether FG chemotype and QTL class interacted. We used eight lines (two from each QTL class) and six FG isolates (three from each chemotype group) in field experiments conducted over three years. Wheat lines were spray-inoculated at 50% anthesis with FG isolates and water (control). FHB symptoms were recorded as severity and incidence and their product calculated as FHB index. The harvested grain was analyzed for total DON using ELISA method, while 15-ADON and 3-ADON analogs were confirmed using gas chromatography-mass spectrometry (GC-MS). The lowest level of total DON and FHB index was recorded in wheat lines from the 3B QTL class. In fact lines carrying both QTLs had higher DON content than lines carrying each QTL individually. Lines inoculated with either 15-ADON or 3-ADON FG reacted similarly, with no interaction between chemotype and QTL class. Our results suggest that breeding for FHB resistance in this population using only the 3B QTL is the best strategy for development of wheat lines with lower DON level and FHB index regardless of the FG chemotype used in the inoculations. Keywords: Fusarium, wheat, resistance, mycotoxins

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P83 - Advantage of using native sources of FHB resistance in breeding winter wheat in Ontario, Canada L. Tamburic-Ilincic University of Guelph, Ridgetown Campus, 120 Main St.E. Ridgetown, Ontario, N0P2C0, Canada E-mail: [email protected]

Fusarium head blight (FHB) is one of the most serious diseases of wheat. FHB reduces grain yield and quality, and the fungus produces mycotoxins, such as deoxynivalenol (DON). The most practical way to control FHB is through the development of resistant cultivars. In addition to exotic sources of FHB resistance (such as ‘Sumai 3’ and ‘Frontana’), ‘native’ sources of resistance are commonly used in wheat breeding programs in North America. ‘Native’ winter wheat has better quality and higher yield compared to ‘exotic’ wheat and progenies with good quality, yield and unique FHB resistance could be identified faster. In the current study, we developed a double haploid (DH) population from cross Pioneer 25R47 (susceptible to FHB) and Vienna (moderately resistant to FHB). 110 DH lines were screened for FHB resistance in 2010, 2011 and 2012. Each line was inoculated with a combined suspension of macroconidia of four Fusarium graminearum isolates at 50% anthesis. Plots were misted daily beginning after the first plots were inoculated until three days after the last plots were inoculated. FHB symptoms were recorded as severity (the percent spikelets infected) and incidence (the percent heads infected) and a fusarium head blight index (FHBI) was calculated as the product of severity and incidence divided by 100. The harvested grain was analyzed for DON level using ELISA method (EZ-Quant® www.diagnostix.ca). Average FHBI, DON level and yield for Pioneer 25R47 was 14%, 6.3 ppm and 4.9 t/ha and for Vienna was 4.9%, 4.6 ppm and 5.0 t/ha. Several breeding lines with lower FHB index and DON level than Vienna were identified. The highest yield across the population was 5.4 t/ha. These results indicate that progenies, with better performance than parents, should be identified in relatively short period of time by using DH technology and ‘native’ sources of FHB resistance. Keyword: Fusarium, wheat, resistance

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SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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FUSARIUM,

P84 - Evaluation of German winter wheat cultivars for resistance against Fusarium head blight and mycotoxin reduction B. Rodemann Julius Kühn-Institut, Institute of plant protection in field crops and grassland, Messeweg 11/12, 38104 Braunschweig, Germany, E-mail: [email protected]

Fusarium head blight (FHB) caused by Fusarium graminearum (Schwabe) and Fusarium culmorum (W.G. Smith) belongs to the most damaging disease in cereal crops. Both species were described to produce deoxynivalenol (DON) and DON derivatives and zearalenone depends on the proved isolates. Different studies revealed significant relations between DON content and characteristics of FHB infection. However the relationship between disease symptoms and DON content is not yet well understood in every case. This could be important for minimizing the risk of mycotoxin contamination of grains and foodstuffs. For official cultivar disease ranking, the susceptibility of German cultivars and new breeding lines against FHB was evaluated every year. Under field conditions the FHB nursery was investigated at six different sites in Germany. Furthermore, the FHB and AUDPC were calculated using both incidence and severity. In some cases the DON contamination of the grains was also analyzed. New registered winter wheat cultivars in 2010 and 2011 showed FHB symptoms between 5 to 55% assessed 28 days after inoculation. Investigated grain samples showed DON contents up to 7.4 mg/kg. A strong correlation coefficient (r=0.74**) was observed between visual disease symptoms and mycotoxin concentrations. In accordance to this data a ranking for FHB and DON on a scale from 1-9 (1= low susceptible / low toxin; 9= high susceptible / high toxin) could be made. By cultivation low fusarium susceptible varieties the DON contamination could be reduced by 50-70%. The performance of resistance cultivars in reduction of deoxynivalenol is comparable to the efficacy of fungicides used for fusarium head blight control. Keywords: Fusarium, cultivar, resistance, mycotoxins

177

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P85 - Promising Fusarium head blight resistance in durum wheat N. Prat

1,2,3

1

2

3

, B. Steiner , T. Langin , O. Robert , H. Buerstmayr

1

1

BOKU-University of Natural Resources and Life Sciences Vienna, Department IFA-Tulln, Institute for Biotechnology in Plant Production, Konrad Lorenz Str. 20, 3430 Tulln, Austria; 2INRA-Université Blaise Pascal, UMR 1095, Genetic, Diversity and Ecophysiology of Cereals, 5 chemin de Beaulieu, 63039 Clermont-Ferrand, Cedex 2, France; 3Florimond-Desprez, 3 rue Florimond Desprez, BP 41, 59242 Cappelle-en-Pévèle, France

Cultivated tetraploid wheat, especially durum wheat (Triticum durum), is highly susceptible to the wide-spread disease Fusarium head blight (FHB). While many resistance QTL have been reported in hexaploid wheat (Triticum aestivum) the QTL identified in tetraploid wheat do not provide satisfactory FHB resistance. To overcome T. durum susceptibility attempts have been made to introgress resistance alleles from wild and cultivated relatives. In this study, back-cross lines derived from crosses of T. durum and FHB resistance sources including Triticum dicoccum (cultivated emmer), Triticum dicoccoides (wild emmer) and Triticum aestivum (bread wheat) have been used as resistant parental lines in several biand multi-parental crosses with T.durum. A large population has been developed allowing the evaluation of FHB resistance derived from relatives in an agronomically acceptable durum background. This population was evaluated in 2012 in disease nursery through artificial inoculation at BOKU University in Tulln (Austria). FHB disease symptoms were visually scored, morphological (plant height) and phenological (flowering date) traits were recorded. Based on this first year trial, the population showed a large genetic variation for the different traits evaluated. More interestingly, a large spectrum of response for FHB resistance was observed among the lines ranging from highly resistant to susceptible. These first results are promising and need to be confirmed in 2013 trials. A subset of 500 lines will subsequently be analyzed through both linkage and genome-wide association mapping. The lines will be genotyped in high-density at INRA Clermont-Ferrand (France) using GENTYANE platform and phenotyped at two locations: Florimond-Desprez in Cappelle-en-Pévèle (France) and BOKU University in Tulln (Austria).Through this project, we expect to unveil QTL linked with resistance and/or increased susceptibility and to evaluate the importance of epistatic interactions for FHB resistance in durum wheat. We acknowledge funding of this project by CIFRE (Conventions Industrielles de Formation par la Recherche). Keywords: FHB, disease resistance, tetraploid wheat, durum

178

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P86 - Selection of aggressive Fusarium isolates for breeding 1

M. Ittu , I. Ciocazanu

2

1

National Agricultural Research Development Institute (NARDI) Fundulea, 1 N. Titulescu street, 915200 Romania; 2Pioneer Hi-Breed Seed Agro SRL, com. Ganeasa, sat Sindrilita DN 2, km 19,7, jud. Ilfov, 077010, Romania E-mail: [email protected]

Fusarium species from Gibberella fujikuroi complex (F.verticillioides, F.proliferatum and/or F. subglutinans) are important fungal pathogens that cause pink ear rot (PER), a disease of maize with devastating impact on yield losses and food safety. Use of reliable Fusarium isolates for assessment of host resistance and understanding of relation between phenotypic scoring and Fumonisin (FUM) accumulation are crucial for developing strategies to minimize the associated risks to disease. The objectives of this study were to evaluate: i) selection of aggressive isolates for large scale inoculums production in several Fusarium populations, sampled from South Romania and ii) relation between disease scores and FUM content under artificial field inoculation. New pathogenic isolates are obtained each two years, by specific procedures. Aggressiveness of the new isolates was evaluated under artificial inoculation in seedling stage, data being expressed as reduction of coleoptyle length in inoculated seedlings vs. noninoculated ones (as % of control). Isolates causing greatest reduction in coleoptiles growth were considered as most aggressive and were selected for large scale production of inoculum for breeding purposes. Multienvironment assessment of resistance under artificial field inoculations performed with highly aggressive F. verticillioides isolates, obtained in this way, revealed a very close negative correlation between pink ear rot rating (1=very sensitive, 9=very resistant) and FUM content (ppm) across years, according to correlation index that ranged from r=-0.729, N=61 (2008) to r=-0,815, N=15 (2010). Based on these findings it could be assumed that selection for reduced symptoms (pink ear rot score) should fairly allow identification of genotypes with reduced FUM content. Keywords: aggressiveness, Fumonisin (FUM), resistance, maize

179

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P87 - Developing test method to oats and barley for resistance to Fusarium langsethiae P. Parikka, M. Jalli MTT Agrifood Research Finland, Plant Production Research, FI-31600 Jokioinen, Finland E-mail: [email protected]

Importance of T-2/HT-2 toxins produced by F. langsethiae has increased recently in many countries, especially in Northern Europe and presence of toxins particularly in oats, raises questions of resistance in cultivars. Testing resistance in field conditions is difficult because of competing infections. F. langsethiae is the earliest Fusarium species infecting flowers and it has been detected in oats and barley at heading. Normally, Fusarium infections are dependent on humidity and infections in dry conditions are rare. F. langsethiae, however, can establish infections in field conditions without long-lasting high relative humidity. Development of greenhouse test to F. langsethiae was started at MTT Agrifood Research Finland in 2009 first on oats and later also some barley cultivars were included in the test calibration. Development and transmission of infection was studied by inoculating seedlings and developing plants at different growth stages: starting at young seedling stage and ending at panicle emergence and flowering. Inoculations with F. langsethiae spore suspension were made with a hand sprayer. Temperature and humidity were regulated according to plant development and inoculation periods. High humidity was maintained in greenhouse shortly before and after inoculation. Observations of infection were made one and two weeks after inoculation and of harvested grain by plating kernels on peptone-PCNB agar plates. F. langsethiae sprayed on young oat seedlings did not continue development further in plants and did not infect panicles. Inoculation at shoot development was also unable to proceed up to panicles, and when sprayed at flag leaf stage the fungus could only occasionally infect panicles. Successful infections were obtained when plants were inoculated at head/panicle emergence or one week after that. In oats, the later inoculations resulted in higher infections. Differences between cultivars could be obtained in observations both in oats and barley. Keywords: cultivars, resistance, infection

180

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P88 - Variation for Fusarium head blight resistance and Fusarium toxins accumulation in winter triticale lines inoculated with Fusarium culmorum 1

2

1

1

1

T. Góral , H. Wiśniewska , P. Ochodzki , D. Walentyn-Góral , I. Grzeszczak , 2 3 3 3 3 J. Beletr , Z. Banaszak , M. Pojmaj , D. Kurleto , M. Konieczny , G. 4 4 4 4 Budzianowski , A. Cicha , K. Paizert , H. Woś 1

Department of Plant Pathology, Plant Breeding and Acclimatization Institute NRI, Radzików, 05-870 Blonie, Poland; 2Institute of Plant Genetics, Polish Academy of Sciences, 34 Strzeszyńska str., 60-479 Poznan, Poland; 3Danko Plant Breeders Ltd., Choryń 27,64-000 Kościan, Poland; 4Plant Breeding Strzelce Ltd., 20 Główna str., 99-307 Strzelce, Poland E-mail: [email protected]

Resistance to Fusarium head blight of 29 winter triticale lines, and three cultivars ‘Borwo’, ‘Fredro’ and ‘Mikado’ was evaluated. Triticale was sown in the field experiments in Cerekwica near Poznań and in Radzików near Warsaw. At flowering, triticale heads were inoculated with three Fusarium culmorum isolates. Fusarium head blight index was scored and after the harvest percentage of Fusarium damaged kernels was assessed. Triticale grain was analyzed for the contents of trichothecenes B (deoxynivalenol and derivatives, nivalenol) and zearalenone. The average FHB indexes were similar in both locations and amounted 19.8% in Radzikow, and 19.9%. in Cerekwica. Percentage of Fusarium damaged kernels was higher in Cerkwica (53.7%) than in Radzików (26.8%). An average content of DON in Radzików amounted to 8,690 ppm and was lower than in the second location – 19.543 ppm. In Cerekwica there were also large quantities of NIV in grain. The average content was 10.048 ppm, while in Radzików it was very low – 0.324 ppm. Considerable amounts of DON derivatives in grain in both locations were detected (1,815 ppm of 3AcDON and 1,913 ppm of 15AcDON). The content of the ZON in the grain from Cerekwica was very high and amounted to 1123 ppb, while in Radzików was 6 times lower - 200 ppb. Concentrations of trichotecenes B in both locations as well as zearalenone in both locations correlated significantly. Relationships between FHB index and mycotoxin contents were statistically insignificant in both locations. In contrast, FDK percentages correlated significantly with mycotoxin contents. In both locations the parallel experiments with 36 winter wheat were carried out. Triticale proved to be less infected than wheat (ears, kernels). However, content of mycotoxins (trichothecenes), was higher in triticale grain than in wheat grain. These results showed that there is a threat of contamination of triticale grain with mycotoxins despite weaker FHB symptoms. Keywords: Fusarium, mycotoxins, resistance, triticale

181

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P89 - Identification of physiological traits in wheat conferring passive resistance to Fusarium head blight S. Jones, J. Foulkes, D. Sparkes, R. Ray University of Nottingham, School of Biosciences, Division of Plant and Crop Sciences, Sutton Bonington Campus, LE12 5RD, UK E-mail: [email protected]

Fusarium head blight (FHB), caused by a complex of Fusarium and Microdochium species, is a devastating fungal disease of cereals worldwide that leads to reductions of grain yield, quality and safety. Developing cultivars with improved FHB resistance is considered an essential step towards reducing the impact of this disease. This study aimed to identify and quantify plant and ear traits in wheat conferring passive resistance to FHB through disease escape. Two field experiments were carried out in 2010 and 2011 using 5 UK winter wheat cultivars and 10 doubledhaploid lines derived from a cross between a spring wheat line of large-ear phenotype from CIMMYT, Mexico, and the UK winter wheat cultivar, Rialto. All 15 wheat genotypes were ground inoculated at GS 30 using oat grains infected with a mixture of Fusarium graminearum, F. culmorum, F. avenaceum, F. langsethiae, F. poae, Microdochium majus and M. nivale. Plant and ear characteristics of the wheat genotypes were assessed at GS 65. Visual disease symptoms were scored at regular intervals from mid-anthesis onwards and used to calculate the area under the disease progress curve (AUDPC) for each wheat genotype. Multiple linear regression with groups was used to identify significant physiological traits related to AUDPC for the two seasons. Stem length, flag leaf length and ear length were the predominant plant characteristics which accounted for a significant (P25-68.4 ppm/kg), across the isolates of each Fusarium chemotype. With a few exceptions the resistance scores were better in most resistant genotypes, irrespective of Fusarium chemotype. These findings suggest the importance to develop germplasm with higher tolerance to DON contamination, if increase of divergent Fg populations become problematic and different mechanisms for resistance to pathogen and associated mycotoxins could be found. Keywords: resistance, aggressiveness, DON chemotype, wheat

183

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P91 - Mapping QTLs for Fusarium head blight response in a durum wheat elite population 1

1

1

1

2

2

M. Maccaferri , S. Corneti , A. Ricci , S. Stefanelli , E. Braida , P. Lancioni , 3 1 1 4 4 2 K. Ammar , A. Prodi , A. Pisi , M. Pascale , V. Lippolis , A. Massi , R. 1 Tuberosa . 1

Department of Agricultural Science - University of Bologna, Viale Fanin 44, 40127 Bologna-Italy; Società Produttori Sementi Bologna, Via Macero 1, 40050 Argelato- Italy; 3CIMMYT, Carretera Mexico-Veracruz KM. 45, 56130 Texcoco-Mexico; 4Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), via G. Amendola 122/O, 70126 Bari-Italy E-mail: [email protected] 2

Durum wheat is strongly affected by Fusarium head blight (FHB). Thus, understanding the genetic basis of resistance is a major objective of breeding. In this study, a population of 249 F7:8 recombinant inbred lines (RILs) obtained from the cross Kofa (desertDurum®) x Svevo (Italia cultivar) has been evaluated for FHB response in five artificially inoculated field trials (mix suspension of F. graminearum and F. culmorum conidia) carried out in 2010 and 2011 in Italy and Mexico. Fusarium incidence and severity, grain yield, number of grains per spike, percentage of fusarium-damaged kernels, thousand kernel weight, deoxynivalenol (DON) content were recorded. Heritability values of FHB symptoms were medium to high across trials (from 35 to 62%). Both Kofa and Svevo showed intermediate responses, while the RILs showed extensive transgressive segregation, allowing us to map 14 QTL clusters for FHB responses and DON content. Only two of these QTL clusters, on chromosomes 2A and 7B, were coincident with QTLs for heading date. Two major QTLs consistently proved to affect FHB response across trials were located on chromosome arms 2BL and 3BS, in coincidence with major QTLs for plant height, peduncle length, senescence and grain yield previously identified in this population (Maccaferri et al. Genetics 178: 489-511). In both cases, the alleles responsible for constitutive reduced height and peduncle length, increased senescence rate and decreased grain yield were also associated to increased fusarium susceptibility. The role of these two QTLs is being further investigated with near isogenic lines, particularly to elucidate the pleiotropic vs. tight linkage relationships, in view also of the FHB escape associated to plant eight. Four QTLs for DON content were also found, two of which were DONspecific and did not influence other disease symptoms. The contribution of "AGER - Agroalimentare e Ricerca", project “From Seed to Pasta” is acknowledged. Keywords: FHB, Triticum durum, molecular markers, marker assisted selection

184

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P92 - Forthcoming development of diverse FHB and DON resistant wheat in South Africa S. Sydenham, C. De Villiers ARC-Small Grain Institute, Bethlehem, Free State Province, South Africa, Private Bag X29, 9701 E-mail: [email protected]

In South Africa (SA) Fusarium head blight (FHB) has the potential to become an epidemic on spring irrigation wheat under favourable conditions. Furthermore, there are no moderate to highly tolerable FHB wheat varieties commercially available in SA yet. Additionally, no fungicide is officially registered for the control of FHB on wheat. Of major concern is the fact that deoxynivalenol (DON) mycotoxin levels in wheat grain are not officially monitored as there is no legislation or guidance level in place. To avoid continual dependence on Sumai-3 based resistance, FHB nurseries and novel resistant wheat sources are imported annually, diversifying the available FHB resistance gene pool. Imported resistant donors/nurseries are screened by the point inoculation method in the glasshouse and are further characterised with targeted FHB resistance linked SSR markers. Only novel verified FHB resistant donor sources are selected to be used further in variety development. Targeted directional crosses for two focused programmes (one backcross focused and other gene stacking focused) are carried out in the glasshouse based on phenotypic and molecular data. Field trials of parental resistant donors and developed material will be planted in a honey-comb design and artificially inoculated (grain and liquid inoculum) under irrigation. Superior performing FHB resistant entries will be selected based on glasshouse data, field trial performance and molecular marker data. Objective of this study is to develop diverse Fusarium head blight and DON resistant adapted wheat germplasm by using a combined approach of phenotypic screening and marker-assisted selection. The ultimate goal is to successfully pyramid or stack a number of FHB QTL/genes from diverse donor sources into top performing wheat cultivars that are well adapted to South African irrigation areas, with increased FHB resistance, reduced DON accumulation, reduced kernel damage and reduced yield loss. Intended outcomes include both the successful commercial release of FHB resistant cultivars as well as a series of FHB resistant germplasm registrations. Keywords: Fusarium Head Blight, gene pyramiding, SSR markers, resistance QTL/gene

185

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P93 - Identification and characterization of wheat genes contributing in plant resistance to the mycotoxin deoxynivalenol A. Perochon, C. Arunachalam, K. Heinrich, A. Kahla, S. Walter, G. Erard, F. Doohan Molecular Plant Microbe Interactions Group, School of Biology and environmental Science, College of Life Sciences, University College Dublin, Belfield, Dublin 4, Ireland E-mail: [email protected]

We focus on identifying biochemical pathways involved in the wheat response to the Fusarium virulence factor deoxynivalenol (DON). Using functional genomics techniques, DON-responsive transcripts were identified: these included transcripts encoding a basic leucine zipper transcription factor, a multidrug resistance protein ABC transporter, cytochrome P450s and novel proteins. Based on the results, candidate genes were silenced in wheat heads using virusinduced gene silencing (VIGS). We found that heads with reduced transcript levels developed more DON-induced bleaching as compared to control treatment. These studies have also highlighted a novel, evolutionary divergent protein involved in the wheat response to DON. Transient expression and microscopy studies showed this protein fused to a fluorescent tag localised within punctate areas of the nucleus of wheat cells. Yeast two hybrid experiments suggest that this protein interacts with SnRK1 (SNF1-Related Kinase 1) and NAC transcription factors. Thus, it is likely that this novel protein is involved in genes expression regulation. We are currently characterizing these interactions and the role of this protein in plant stress responses. Keywords: mycotoxin, wheat, VIGS, protein-protein interaction

186

SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P94 - Resistance of winter wheat breeding lines to Fusarium head blight and accumulation of Fusarium toxins in grain 1

2

2

2

1

H. Wiśniewska , T. Góral , P. Ochodzki , D. Walentyn-Góral , J. Belter , M. 1 3 4 3 5 6 Kwiatek , J. Bogacki , T. Drzazga , B. Ługowska , P. Matysik , E. Witkowski , 7 7 K. Rubrycki , U. Woźna–Pawlak 1

Institute of Plant Genetics, Polish Academy of Sciences, 34 Strzeszyńska str., 60-479 Poznan, Poland; 2Department of Plant Pathology, Plant Breeding and Acclimatization Institute NRI, Radzików, 05-870 Blonie, Poland; 3Danko Plant Breeders Ltd., Choryń 27,64-000 Kościan, Poland; 4Małopolska Plant Growing Company – HBP LLC, 4 Zbożowa str., 30-002 Kraków, Poland; 5Plant Breeding Strzelce Ltd., 20 Główna str., 99-307 Strzelce, Poland; 6Plant Breeding Smolice Ltd., Smolice 41, 63740 Kobylin, Poland; 7Poznań Plant Breders Ltd., 5 Kasztanowa str., 03-004 Tulce, Poland E-mail: [email protected]

Fusarium head blight (FHB) is an important cereal disease caused by Fusarium species. Disease is mainly damaging for bread wheat. The aim of this work was (1) to evaluate FHB resistance variability of wheat lines and (2) to analyze concentration of Fusarium toxins in grain. This research was conducted on 71 winter wheat lines differing in genetic background. Cultivars ‘Arina’ and ‘Tonacja’ were used as controls. Lines were sown in two field experiments located in Cerekwica, Western Poland and in Radzików, Central Poland. Field experiments were established as a randomized complete block design. Wheat heads were inoculated at flowering stage with conidial suspension of Fusarium culmorum isolates producing deoxynivalenol (DON), nivalenol (NIV) and zearalenon (ZEA), at a rate of 100 000 spores per mL. The disease was rated 14 and 16 days after inoculation. Fusarium head blight index (FHBi) was calculated. FHBi ranged from 8.3 to 49.9%. Only 3 lines showed infection below 10%. Percentage of Fusarium damaged kernels (FDK) was high, ranging from 50.2% to 98.0%. The FDKs of ‘Tonacja’ and ‘Arina’ cultivars were also high (65.2 and 69.7%). However, the FDK of line ‘DED’ (susceptible check) was 82.2%. Fusarium damaged kernels of only 5 genotypes did not exceed 60.0%. The correlation coefficient of FHBi versus FDK was significant (r=0.575). Grain from 35 lines from two locations, with low FHBi and FDK, was analyzed for the presence of Fusarium toxins. DON amount was the highest, and ranged from 7.041 ppm to 22.651ppm. The total amount of NIV was also high, ranging from 3.331 ppm to 19.285 ppm. The lowest concentration of ZEA was found in ‘Arina’ – 128 ppb, the highest ZEA level was 8432 ppb. Research was supported by the Ministry of Agriculture and Rural Development projects: HOR hn 801 - 13/11 and HOR hn 078-801-9/11. Keywords: Fusarium, mycotoxin, resistance, wheat

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SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE VARIETY DEVELOPMENT

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P95 - Evaluation of Fusarium Head Bblight resistance in a panel of durum wheat (Triticum turgidum L.) 1

2

1

1

1

1

A. Brunazzi , S. Greggio , P. Lancioni , E. Braida , P. Mantovani , R. Bovina , 1 3 4 1 G. Gasparini , S. Tonti , A. Prodi , G. Ferrazzano 1

Società Produttori Sementi S.p.A. (PSB), Via Macero 1, 40050 Argelato (BO), Italy; 2Università degli Studi di Ferrara, Via Paradiso 12, 44121 Ferrara, Italy; 3Ente Nazionale delle Sementi Elette (ENSE) Via Ca’ Nova Zampieri 37, 37057 San Giovanni Lupatoto (VR), Italy; 4Dipartimento di Scienze e Tecnologie Agroambientali (DiSTA), Viale Fanin 44, 40127 Bologna, Italy E-mail: [email protected]

Fusarium Head Blight (FHB) caused by Fusarium spp. is one of the most destructive fungal disease of wheat; it causes dramatic yield and quality losses and contamination of cereal products with mycotoxins, in particular deoxynivalenol (DON). The release of new varieties resistant to FHB and less affected by mycotoxin contamination, is the most efficient way to contrast this disease, with no additional costs for the farmers and with advantages for the consumers health. In this study, we set up a two year field experiment under artificial inoculation with a mixture of virulent and toxigenic strains of Fusarium culmorum and F. graminearum in Argelato (Bologna), in order to evaluate a panel of durum wheat genotypes (100) for their reaction to FHB; three common wheat varieties know to be resistant to FHB (Sumai 3, Gondo and Heilo) were also included as resistant control. At anthesis, ten spikes per genotype were tagged and harvested separately. FHB incidence and severity were rated on the selected spikes in two different times. We also evaluated: Fusarium Damaged Kernels (FDK), Area Under the Disease Progress Curve (AUDPC), Damage Index (DI) and DON content. The disease incidence and severity data matrixes were analyzed by ANOVA and the analyses revealed the presence of significant difference among the investigated genotypes. Highly significant correlation were detected between DI and AUDPC (r=0.96, p