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A European Database of Fusarium graminearum and F-culmorum Trichothecene
Genotypes
Pasquali, Matias; Beyer, Marco; Logrieco, Antonio; Audenaert, Kris; Balmas, Virgilio; Basler, Ryan;
Boutigny, Anne-Laure; Chrpova, Jana; Czembor, Elzbieta; Gagkaeva, Tatiana
Total number of authors:
32
Published in:
Frontiers in Microbiology
Link to article, DOI:
10.3389/fmicb.2016.00406
Publication date:
2016
Document Version
Publisher's PDF, also known as Version of record
Link back to DTU Orbit
Citation (APA):
Pasquali, M., Beyer, M., Logrieco, A., Audenaert, K., Balmas, V., Basler, R., Boutigny, A-L., Chrpova, J.,
Czembor, E., Gagkaeva, T., Gonzalez-Jaen, M., Hofgaard, I. S., Koycu, N. D., Hoffmann, L., Levic, J., Marin, P.,
Miedaner, T., Migheli, Q., Moretti, A., ... Vogelgsang, S. (2016). A European Database of Fusarium graminearum
and F-culmorum Trichothecene Genotypes. Frontiers in Microbiology, 7, 1-11. [406].
https://doi.org/10.3389/fmicb.2016.00406
ORIGINAL RESEARCH
published: 06 April 2016
doi: 10.3389/fmicb.2016.00406
Frontiers in Microbiology | www.frontiersin.org 1 April 2016 | Volume 7 | Article 406
Edited by:
Alex Andrianopoulos,
University of Melbourne, Australia
Reviewed by:
Vijai Kumar Gupta,
NUI Galway, Ireland
Stefan G. R. Wirsel,
Martin-Luther-Universität
Halle-Wittenberg, Germany
*Correspondence:
Matias Pasquali
matias.pasquali@list.lu;
Susanne Vogelgsang
susanne.vogelgsang@
agroscope.admin.ch
Specialty section:
This article was submitted to
Fungi and Their Interactions,
a section of the journal
Frontiers in Microbiology
Received: 18 December 2015
Accepted: 14 March 2016
Published: 06 April 2016
Citation:
Pasquali M, Beyer M, Logrieco A,
Audenaert K, Balmas V, Basler R,
Boutigny A-L, Chrpová J, Czembor E,
Gagkaeva T, González-Jaén MT,
Hofgaard IS, Köycü ND, Hoffmann L,
Levi
´
c J, Marin P, Miedaner T,
Migheli Q, Moretti A, Müller MEH,
Munaut F, Parikka P, Pallez-Barthel M,
Piec J, Scauflaire J, Scherm B,
Stankovi
´
c S, Thrane U, Uhlig S,
Vanheule A, Yli-Mattila T and
Vogelgsang S (2016) A European
Database of Fusarium graminearum
and F. culmorum Trichothecene
Genotypes Front. Microbiol. 7:406.
doi: 10.3389/fmicb.2016.00406
A European Database of Fusarium
graminearum and F. culmorum
Trichothecene Genotypes
Matias Pasquali
1
*
, Marco Beyer
1
, Antonio Logrieco
2
, Kris Audenaert
3
, Virgilio Balmas
4
,
Ryan Basler
5
, Anne-Laure Boutigny
6
, Jana Chrpová
7
, El
˙
zbieta Czembor
8
,
Tatiana Gagkaeva
9
, María T. González-Jaén
10
, Ingerd S. Hofgaard
11
, Nagehan D. Köycü
12
,
Lucien Hoffmann
1
, Jelena Levi
´
c
13
, Patricia Marin
10
, Thomas Miedaner
14
, Quirico Migheli
4
,
Antonio Moretti
2
, Marina E. H. Müller
15
, Françoise Munaut
16
, Päivi Parikka
17
,
Marine Pallez-Barthel
1
, Jonathan Piec
1
, Jonathan Scauflaire
16
, Barbara Scherm
4
,
Slavica Stankovi
´
c
13
, Ulf Thrane
18
, Silvio Uhlig
19
, Adriaan Vanheule
3
, Tapani Yli-Mattila
20
and Susanne Vogelgsang
21
*
1
Department of Environmental Research and Innovation, Luxembourg Institute of Science and Technology, Belvaux,
Luxembourg,
2
Institute of Sciences of Food Production, National Research Council, Bari, Italy,
3
Department of Applied
Biosciences, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium,
4
Department of Agriculture, University of
Sassari, Sassari, Italy,
5
BIOGER UMR, INRA, Thiverval-Grignon, France,
6
ANSES, Plant Health Laboratory, Angers, France,
7
Division of Crop Genetics and Breeding, Crop Research Institute, Prague, Czech Republic,
8
Department of Grasses,
Legumes and Energy Plants, Plant Breeding and Acclimatization Institute-National Research Institute, Radzikow, Poland,
9
Laboratory of Mycology and Phytopathology, All-Russian Institute of Plant Protection, St. Petersburg, Russia,
10
Department
of Genetics, Faculty of Biology, Complutense University of Madrid, Madrid, Spain,
11
Norwegian Institute of Bioeconomy
Research, Ås, Norway,
12
Department of Plant Protection, Agriculture Faculty, Namık Kemal University, Tekirdag, Turkey,
13
Laboratory of Phytopathology and Entomology, Maize Research Institute Zemun Polje, Belgrade, Serbia,
14
Plant Breeding
Institute, University of Hohenheim, Stuttgart, Germany,
15
Leibniz Centre for Agricultural Landscape Research, Institute for
Landscape Biogeochemistry, Müncheberg, Germany,
16
Applied Microbiology, Earth and Life Institute, Université Catholique
de Louvain, Louvain-la-Neuve, Belgium,
17
Department Natural Resources and Bioproduction, Natural Resources Institute
Finland (Luke), Jokioinen, Finland,
18
Section for Eukaryotic Biotechnology, DTU Systems Biology, Technical University of
Denmark, Kongens Lyngby, Denmark,
19
Section for Chemistry and Toxicology, Norwegian Veterinary Institute, Oslo, Norway,
20
Molecular Plant Biology, Department of Biochemistry, University of Turku, Turku, Finland,
21
Research Division Grassland
Sciences and Agro-Ecosystems, Institute for Sustainability Sciences, Agroscope, Zürich, Switzerland
Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main
cause of trichothecene type B contamination in cereals. Data on the distribution of
Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space.
Furthermore, a common core set of related variables (sampling method, host cultivar,
previous crop, etc.) that would allow more effective analysis of factors influencing
the spatial and temporal population distribution, is lacking. Consequently, based on
the available data, it is difficult to identify factors influencing chemotype distribution
and spread at the European level. Here we describe the results of a collaborative
integrated work which aims (1) to characterize the trichothecene genotypes of strains
from three Fusarium species, collected over the period 2000–2013 and (2) to enhance
the standardization of epidemiological data collection. Information on host plant, country
of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum,
479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries
was compiled and a map of trichothecene type B genotype distribution was plotted
for each species. All information on the strains was collected in a freely accessible and
Pasquali et al. Fusarium Trichothecene Genotypes European Database
updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point
for epidemiological analysis of potential spatial and temporal trichothecene genotype
shifts in Europe. The analysis of the currently available European dataset showed that
in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON)
(82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV)
(3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV
genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns
of trichothecene genotypes distribution were identified.
Keywords: acetyldeoxynivalenol, chemotype, database, Fusarium, genotype, mycotoxin, nivalenol, trichothecene
INTRODUCTION
Fusarium head blight (FHB) is one of the most important cereal
diseases worldwide. Severe outbreaks of FHB may result in
significant yield losses of up to 50%, depending on the small
grain cereal crop (
Parry et al., 1995). McMullen et al. (2012)
suggested that FHB in the United States might lead to economic
losses in excess of one billion USD per year. More importantly is
the production of secondary metabolites, specifically mycotoxins,
contaminating the harvested products and thus jeopardizing
food and feed safety (e.g., Snijders, 1990).
In cereals, FHB is usually caused by a set of different Fusarium
species, with different life styles and different types of mycotoxins
produced. Within the Fusarium graminearum species complex
(FGSC; O’Donnell et al., 2000), which presently includes 16
species (Aoki et al., 2012), F. culmorum and F. cerealis are among
the most dominant pathogens causing head blight on wheat
and other cereals worldwide (
Moss and Thrane, 2004; Osborne
and Stein, 2007). Other frequently detected species are F. poae,
F. avenaceum, F. langsethiae, F. tricinctum, F. sporotrichioides
(Ioos et al., 2004; Xu et al., 2005; Xu and Nicholson, 2009; Somma
et al., 2014), and t he non-toxigenic species Microdochium nivale
and M. majus (Glynn et al., 2005).
One of the main Fusarium mycotoxin classes are the
trichothecenes, sesquiterpene epoxides that inhibit eukaryotic
protein synthesis, which may cause severe toxicosis in
humans and animals (Ueno, 1983; Maresca, 2013). Fusar ium
trichothecenes are grouped into two classes based on the
presence (type B) vs. absence (type A) of a keto group at the C-8
position (Kristensen et al., 2005). Depending on differences in
the core trichothecene cluster (TRI cluster), which includes two
regulatory genes (TRI6 and TRI10) and most of the biosynthetic
enzymes required for the production of trichothecenes (
Kimura
et al., 2007; Alexander et al., 2011), Fusarium species as well as
individual strains may produce different types of trichothecenes.
Among the type B trichothecenes, the following are
considered to have a significant impact on food and feed
safety: deoxynivalenol (DON), nivalenol (NIV), and their
acetylated derivatives 3-acetyldeoxynivalenol (3-ADON),
15-acetyldeoxynivalenol (15-ADON), and 4-acetylnivalenol
(4-ANIV, syn. fusarenone-X;
Eriksen et al., 2004; Desjardins,
2006
).
Different Fusarium species chemotypes have been described:
chemotype I, produces DON and/or its acetylated derivatives
while chemotype II, produces NIV and/or 4-ANIV (
Sydenham
et al., 1991). The DON chemotype can be further split
into chemotype IA (producing 3-ADON) and IB (producing
15-ADON; Miller et al., 1991). Structural differences among
toxins from different chemotypes may have consequences on
strain fitness, since the specific pattern of oxygenation and
acetylation can modify the bioactivity and hence the (phyto)
toxicity of these compounds (e.g. Ward et al., 2002; Alexander
et al., 2011). As it has been shown in a large survey on Canadian
grains, DON and NIV, being the two most abundant toxins
detected, now represent the two major concerns for safety of
wheat and barley products (Tittlemier et al., 2013).
Environmental factors may result in a geographical
partitioning of subpopulations that may coincide with
chemotypes. The success of a given chemotype, which is of
importance with respect to FHB control, is often related to
local factors (van der Lee et al., 2015). Based on chemotype
characterization of Italian Fusarium species, Covarelli et al.
(2015) suggested that climatic conditions have a strong impact
on the occurrence of 3-ADON and 15-ADON whereas NIV
contamination occurred irrespective of climatic conditions.
Yli-Mattila et al. (2013) proposed that the prevalence of a specific
chemotype may also be influenced by a certain host. For example,
NIV-producing strains were found to be more aggressive towards
maize compared with DON-producers (Carter et al., 2002) and
were associated, in F. asiaticum, preferentially to maize in China
(Ndoye et al., 2012). Maier et al. (2006) postulated NIV to be
a virulence factor in maize, which is in line with findings that
associate an increase in NIV populations in areas where the
preceding crop was maize (
Audenaert et al., 2009; Pasquali et al.,
2010; Sampietro et al., 2011).
Two main reasons to determine the chemotype of a strain
have been proposed (Pasquali and Migheli, 2014): (1) to obtain
epidemiological information on the population colonizing a crop
in a given area, using chemotype as a proxy in the field; (2)
to inform on the toxigenic risk of contaminated food or feed
determined by the presence of a certain chemotype, with the long
term perspective of developing preventive models and strategies
to decrease the risk.
At present, data on chemotype distribution of FGSC are
available from all continents (re viewed in
Pasquali and Migheli,
2014
), being F. graminearum sensu stricto (s.s.) the most studied
species. Less work has been devoted to chemotype determination
in F. culmorum (
Scherm et al., 2013). Shifts in species population
Frontiers in Microbiology | www.frontiersin.org 2 April 2016 | Volume 7 | Article 406
Pasquali et al. Fusarium Trichothecene Genotypes European Database
have been reported in many surveys (Xu et al., 2005; Nielsen et al.,
2011; Fredlund et al., 2013), but reports on chemotype shifts in
certain areas are more recent (e.g.
Nielsen et al., 2012; Beyer et al.,
2014
).
Despite the fact that information from all continents is now
available, most reports do not include complete information on
the strains analyzed, such as geographic origin, host from which
it was isolated, meth od used for species identification, etc. In
addition, precise characterization of the species is frequently
lacking, being b ased only on morphological observations, hence,
making it unfeasible to use the dataset for further studies.
The main goal of this joint study was to generate an accessible
map of trichothecene genotypes from three FHB causing species
with detailed information on how the data were obtained.
This will allow, in the long term, the acquisition of consistent
and homogenous datasets providing a valid comparison of the
distribution of chemotypes during years and among countries.
To reach this aim, research institutions from 17 European
countries were inquired to provide data on how the sampling
was performed as well as detailed information on cropping
history and location. A more coordinated effort, leading to
common protocols for sampling, chemotype determining and
data reporting in a more accessible way would increase the
standardization of epidemiological data. Furthermore, it could
facilitate the effort of understanding which factors do favor
establishment and persistence of a specific chemotype. This
collective effort is now assembled in a fully accessible and
upgradable dataset of chemotype diversity within FGSG and
F. culmorum on cereals in Europe.
MATERIALS AND METHODS
Data Collection
An Excel template file was sent to research partners agreeing
to participate in the initiative (Supplementary File 1). The
information to be submitted (if applicable) were as follows:
chemotype, year of isolation, whether the strain was obtained
by a single spore or a single hyphae, the location including the
geographic coordinates, the crop host and cultivar from which
it was isolated, previous crop, method of isolation, method used
for species attribution, primers a nd/or gene in case of PCR and
sequencing, name of culture collection in case it was deposited,
strain ID, and citation of the strain/s in a publication. Whenever
genetic chemotype or species was unknown, strains were shipped
to the Luxembourg Institute of Science a nd Technology (LIST)
laboratory for genetic chemotyping (
Pasquali et al., 2011) and
species identification by sequencing EF1alpha (Geise r et al.,
2004
). The overall dataset (www.catalogueeu.luxmcc.lu; available
as of mid-April 2016) was built through integrating data
communicated by research partners and by laboratory results
obtained with the procedures des cribed below.
DNA Extraction and Chemotype
Determination
Fungal colonies were grown on PDA as des cribed in
Pallez et al.
(2014) in order to directly extract DNA using a rapid procedure.
Briefly, a 2–5 mm piece of miracloth tissue (Millipore, USA)
covered by a 5 days old fungal culture, was collected and added
to 100 µL TE (10 mM Tris-Cl, 0.05 mM pH 9 EDTA solution,
Sigma-Aldrich, USA). After 5 min of microwave treatment at
900 W and a 30 s centrifugation at 13,0 00 g, 5 µL were then used
for PCR reactions. When identification of the strain was reported
to be putative by partners, EF1alpha amplification was carried
out, followed by sequencing as described in
Dubos et al. (2011).
If the species was previously defined, tri12 multiplex PCR for
genetic chemotyping (Ward et al., 2002) was carried out. All
PCR reactions were performed in a 50 µL volume to avoid risk
of PCR inhibition due to the quick extraction method using 2X
Phusion master mix (Thermo, USA), 300 nM of each primer
and water. Thermal cyclers used were Biometra T-professional
and Veriti PCR Thermal cyclers (Life Technologies, USA) using
the programme as described in Pasquali et al. (2011). All
reactions included positive controls for the three chemotypes
and a negative control for monitoring potential contaminations.
Reactions were visualized on a Biorad agarose ready to use gel at
3%, using a UV spectra analyzer (Ingenius, Syngene, UK). When
results were ambiguous the reaction was repeated at least once.
Data assembled from oth er laboratories were collected by the
Excel template file and uploaded to the database page. When
diverse methods for genetic chemotyping were used (
Waalwijk
et al., 2003; Jennings e t al., 2004; Quarta et al., 2006; Stark ey et al.,
2007; Yli-Mattila and Gagkaeva, 2010) by the original isolating
laboratory, this fact is specified directly in the database.
Statistical Analysis and Visualization Tools
Descriptive graphs on species and chemotype distribution were
obtained using SigmaPlot version 12.5 (Systat Software Inc,
USA) and SPSS version 19 (IBM, USA). The European maps
generated for this study were prepared using the ArcMap
platform (ESRI Inc., USA). A Multiple Correspondence Analysis
tool (
Broeksema et al., 2013) was used for studying the
overall dataset and its homogeneity in relation to species and
chemotype distribution. Logistic regressions were performed
using SigmaPlot 12.5.
Database Construction
The European database was constructed by assembling the
overall dataset on the database architecture developed by
Piec
et al. (2016). A filtering option for country and laboratory, the
option to upload new datasets, with administrator validation,
was added to the existing architecture. Functions of the database
include the possibility to have a full or filtered download of the
overall dataset.
RESULTS
The current work represents the first collective attempt to
compile information on chemotype diversity occurring in
European countries. Moreover, the availability of a full open
access database provides for the first time a centralized source of
information for Fusarium disease records on cereals, which is of
high value for researchers working in the mycotoxin/Fusarium
biodiversity domain.
Frontiers in Microbiology | www.frontiersin.org 3 April 2016 | Volume 7 | Article 406
Pasquali et al. Fusarium Trichothecene Genotypes European Database
The Database
The overall dataset including all information collected for
this work has been assembled in a database. Based on the
previous architecture constructed for the LIST culture collection
(
Piec et al., 2 016), a database with improved functionalities
was built. The overall map with overlapping F. graminearum
and F. culmorum species is shown on the first page of
the database (www.catalogueeu.luxmcc.lu; Figure 1). Further
uploading of data can be performed according to the instructions
in Supplementary File 2. Rese archers working on Fusarium
toxigenic diversity are invited to contribute to the database or to
download th e dataset for further analysis.
Data Description
Information of a total of 1147 F. graminearum and 479
F. culmorum strains was included in the dataset collected from
the period 2000–2013 and plotted on the respective maps
(Figures 2A,B). Years of isolation were close to homogeneity
(Figure 3A). Luxembourg was the country where most strains
were obtained, followed by Belgium and Russia (Figure 3B).
At present, chemotype information from some countries is
missing in the current dataset, therefore, further uploading of
information will be important to obtain a more precise picture
of chemotype distributions in Europe.
The major crop from where strains were isolated was wheat
(66.7%) followed by maize (22.5%), barley (5.4%), and other
crops (combined 5.3%; Figure 3C). As can be observed by the
map of crop distribution, wheat was sampled in 16 out of the 17
countries, whereas other crops were s ampled in a limited number
of countries (maize n = 6; barley n = 7; oats = 3; Figure 4).
Oats were sampled only in Northern Europe, including Norway,
Finland and Russia, where oats are an important crop, while no
FIGURE 1 | Spatial distribution of chemotypes and Fusarium species in Europe. 3-ADON, 3-acetyldeoxynivalenol; 15-ADON, 15-acetyldeoxynivalenol; NIV,
nivalenol. F. cortaderiae were isolated in Italy but cannot be visualized as they are overlapped by other strains.
FIGURE 2 | (A) Spatial distribution of Fusarium culmorum chemotypes in Europe. Red squares, genetic 3-ADON chemotype. Yellow squares, genetic NIV
chemotype. (B) Spatial distribution of Fusarium graminearum sensu stricto chemotypes in Europe. Green circles, genetic 15-ADON chemotype. Red circles,
genetic 3-ADON chemotype. Yellow circles, genetic NIV chemotype.
Frontiers in Microbiology | www.frontiersin.org 4 April 2016 | Volume 7 | Article 406