ORIGINAL RESEARCH
published: 06 Ap il 2016
doi: 10.3389/ micb.2016.00406
F on ie s in Mic obiology | www. on ie sin.o g 1Ap il 2016 | Volume 7 | A icle 406
Edi ed by:
Alex And ianopoulos,
Uni e si y o Melbou ne, Aus alia
Re iewed by:
Vijai Kuma Gup a,
NUI Galway, I eland
S e an G. R. Wi sel,
Ma in-Lu he -Uni e si ä
Halle-Wi enbe g, Ge many
*Co espondence:
Ma ias Pasquali
[email p o ec ed];
Susanne Vogelgsang
susanne. ogelgsang@
ag oscope.admin.ch
Special y sec ion:
This a icle was submi ed o
Fungi and Thei In e ac ions,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 18 Decembe 2015
Accep ed: 14 Ma ch 2016
Published: 06 Ap il 2016
Ci a ion:
Pasquali M, Beye M, Log ieco A,
Audenae K, Balmas V, Basle R,
Bou igny A-L, Ch po á J, Czembo E,
Gagkae a T, González-Jaén MT,
Ho gaa d IS, Köycü ND, Ho mann L,
Le i´
c J, Ma in P, Miedane T,
Migheli Q, Mo e i A, Mülle MEH,
Munau F, Pa ikka P, Pallez-Ba hel M,
Piec J, Scau lai e J, Sche m B,
S anko i´
c S, Th ane U, Uhlig S,
Vanheule A, Yli-Ma ila T and
Vogelgsang S (2016) A Eu opean
Da abase o Fusa ium g aminea um
and F. culmo um T icho hecene
Geno ypes F on . Mic obiol. 7:406.
doi: 10.3389/ micb.2016.00406
A Eu opean Da abase o Fusa ium
g aminea um and F. culmo um
T icho hecene Geno ypes
Ma ias Pasquali1*, Ma co Beye 1, An onio Log ieco2, K is Audenae 3, Vi gilio Balmas4,
Ryan Basle 5, Anne-Lau e Bou igny 6, Jana Ch po á 7, El˙
zbie a Czembo 8,
Ta iana Gagkae a9, Ma ía T. González-Jaén10, Inge d S. Ho gaa d11, Nagehan D. Köycü12,
Lucien Ho mann1, Jelena Le i ´
c13, Pa icia Ma in10, Thomas Miedane 14, Qui ico Migheli 4,
An onio Mo e i2, Ma ina E. H. Mülle 15, F ançoise Munau 16, Päi i Pa ikka 17,
Ma ine Pallez-Ba hel1, Jona han Piec1, Jona han Scau lai e16, Ba ba a Sche m 4,
Sla ica S anko i ´
c13, Ul Th ane18, Sil io Uhlig19, Ad iaan Vanheule3, Tapani Yli-Ma ila20
and Susanne Vogelgsang21*
1Depa men o En i onmen al Resea ch and Inno a ion, Luxembou g Ins i u e o Science and Technology, Bel aux,
Luxembou g, 2Ins i u e o Sciences o Food P oduc ion, Na ional Resea ch Council, Ba i, I aly, 3Depa men o Applied
Biosciences, Facul y o Bioscience Enginee ing, Ghen Uni e si y, Ghen , Belgium, 4Depa men o Ag icul u e, Uni e si y o
Sassa i, Sassa i, I aly, 5BIOGER UMR, INRA, Thi e al-G ignon, F ance, 6ANSES, Plan Heal h Labo a o y, Ange s, F ance,
7Di ision o C op Gene ics and B eeding, C op Resea ch Ins i u e, P ague, Czech Republic, 8Depa men o G asses,
Legumes and Ene gy Plan s, Plan B eeding and Acclima iza ion Ins i u e-Na ional Resea ch Ins i u e, Radzikow, Poland,
9Labo a o y o Mycology and Phy opa hology, All-Russian Ins i u e o Plan P o ec ion, S . Pe e sbu g, Russia, 10 Depa men
o Gene ics, Facul y o Biology, Complu ense Uni e si y o Mad id, Mad id, Spain, 11 No wegian Ins i u e o Bioeconomy
Resea ch, Ås, No way, 12 Depa men o Plan P o ec ion, Ag icul u e Facul y, Namık Kemal Uni e si y, Teki dag, Tu key,
13 Labo a o y o Phy opa hology and En omology, Maize Resea ch Ins i u e Zemun Polje, Belg ade, Se bia, 14 Plan B eeding
Ins i u e, Uni e si y o Hohenheim, S u ga , Ge many, 15 Leibniz Cen e o Ag icul u al Landscape Resea ch, Ins i u e o
Landscape Biogeochemis y, Münchebe g, Ge many, 16 Applied Mic obiology, Ea h and Li e Ins i u e, Uni e si é Ca holique
de Lou ain, Lou ain-la-Neu e, Belgium, 17 Depa men Na u al Resou ces and Biop oduc ion, Na u al Resou ces Ins i u e
Finland (Luke), Jokioinen, Finland, 18 Sec ion o Euka yo ic Bio echnology, DTU Sys ems Biology, Technical Uni e si y o
Denma k, Kongens Lyngby, Denma k, 19 Sec ion o Chemis y and Toxicology, No wegian Ve e ina y Ins i u e, Oslo, No way,
20 Molecula Plan Biology, Depa men o Biochemis y, Uni e si y o Tu ku, Tu ku, Finland, 21 Resea ch Di ision G assland
Sciences and Ag o-Ecosys ems, Ins i u e o Sus ainabili y Sciences, Ag oscope, Zü ich, Swi ze land
Fusa ium species, pa icula ly Fusa ium g aminea um and F. culmo um, a e he main
cause o icho hecene ype B con amina ion in ce eals. Da a on he dis ibu ion o
Fusa ium icho hecene geno ypes in ce eals in Eu ope a e sca e ed in ime and space.
Fu he mo e, a common co e se o ela ed a iables (sampling me hod, hos cul i a ,
p e ious c op, e c.) ha would allow mo e e ec i e analysis o ac o s in luencing
he spa ial and empo al popula ion dis ibu ion, is lacking. Consequen ly, based on
he a ailable da a, i is di icul o iden i y ac o s in luencing chemo ype dis ibu ion
and sp ead a he Eu opean le el. He e we desc ibe he esul s o a collabo a i e
in eg a ed wo k which aims (1) o cha ac e ize he icho hecene geno ypes o s ains
om h ee Fusa ium species, collec ed o e he pe iod 2000–2013 and (2) o enhance
he s anda diza ion o epidemiological da a collec ion. In o ma ion on hos plan , coun y
o o igin, sampling loca ion, yea o sampling and p e ious c op o 1147 F. g aminea um,
479 F. culmo um, and 3 F. co ade iae s ains ob ained om 17 Eu opean coun ies
was compiled and a map o icho hecene ype B geno ype dis ibu ion was plo ed
o each species. All in o ma ion on he s ains was collec ed in a eely accessible and
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
upda able da abase (www.ca alogueeu.luxmcc.lu), which will se e as a s a ing poin
o epidemiological analysis o po en ial spa ial and empo al icho hecene geno ype
shi s in Eu ope. The analysis o he cu en ly a ailable Eu opean da ase showed ha
in F. g aminea um, he p edominan geno ype was 15-ace yldeoxyni alenol (15-ADON)
(82.9%), ollowed by 3-ace yldeoxyni alenol (3-ADON) (13.6%), and ni alenol (NIV)
(3.5%). In F. culmo um, he p e alen geno ype was 3-ADON (59.9%), while he NIV
geno ype accoun ed o he emaining 40.1%. Bo h, geog aphical and empo al pa e ns
o icho hecene geno ypes dis ibu ion we e iden i ied.
Keywo ds: ace yldeoxyni alenol, chemo ype, da abase, Fusa ium, geno ype, myco oxin, ni alenol, icho hecene
INTRODUCTION
Fusa ium head bligh (FHB) is one o he mos impo an ce eal
diseases wo ldwide. Se e e ou b eaks o FHB may esul in
signi ican yield losses o up o 50%, depending on he small
g ain ce eal c op (Pa y e al., 1995). McMullen e al. (2012)
sugges ed ha FHB in he Uni ed S a es migh lead o economic
losses in excess o one billion USD pe yea . Mo e impo an ly is
he p oduc ion o seconda y me aboli es, speci ically myco oxins,
con amina ing he ha es ed p oduc s and hus jeopa dizing
ood and eed sa e y (e.g., Snijde s, 1990).
In ce eals, FHB is usually caused by a se o di e en Fusa ium
species, wi h di e en li e s yles and di e en ypes o myco oxins
p oduced. Wi hin he Fusa ium g aminea um species complex
(FGSC; O’Donnell e al., 2000), which p esen ly includes 16
species (Aoki e al., 2012), F. culmo um and F. ce ealis a e among
he mos dominan pa hogens causing head bligh on whea
and o he ce eals wo ldwide (Moss and Th ane, 2004; Osbo ne
and S ein, 2007). O he equen ly de ec ed species a e F. poae,
F. a enaceum,F. langse hiae,F. icinc um,F. spo o ichioides
(Ioos e al., 2004; Xu e al., 2005; Xu and Nicholson, 2009; Somma
e al., 2014), and he non- oxigenic species Mic odochium ni ale
and M. majus (Glynn e al., 2005).
One o he main Fusa ium myco oxin classes a e he
icho hecenes, sesqui e pene epoxides ha inhibi euka yo ic
p o ein syn hesis, which may cause se e e oxicosis in
humans and animals (Ueno, 1983; Ma esca, 2013). Fusa ium
icho hecenes a e g ouped in o wo classes based on he
p esence ( ype B) s. absence ( ype A) o a ke o g oup a he C-8
posi ion (K is ensen e al., 2005). Depending on di e ences in
he co e icho hecene clus e (TRI clus e ), which includes wo
egula o y genes (TRI6 and TRI10) and mos o he biosyn he ic
enzymes equi ed o he p oduc ion o icho hecenes (Kimu a
e al., 2007; Alexande e al., 2011), Fusa ium species as well as
indi idual s ains may p oduce di e en ypes o icho hecenes.
Among he ype B icho hecenes, he ollowing a e
conside ed o ha e a signi ican impac on ood and eed
sa e y: deoxyni alenol (DON), ni alenol (NIV), and hei
ace yla ed de i a i es 3-ace yldeoxyni alenol (3-ADON),
15-ace yldeoxyni alenol (15-ADON), and 4-ace ylni alenol
(4-ANIV, syn. usa enone-X; E iksen e al., 2004; Desja dins,
2006).
Di e en Fusa ium species chemo ypes ha e been desc ibed:
chemo ype I, p oduces DON and/o i s ace yla ed de i a i es
while chemo ype II, p oduces NIV and/o 4-ANIV (Sydenham
e al., 1991). The DON chemo ype can be u he spli
in o chemo ype IA (p oducing 3-ADON) and IB (p oducing
15-ADON; Mille e al., 1991). S uc u al di e ences among
oxins om di e en chemo ypes may ha e consequences on
s ain i ness, since he speci ic pa e n o oxygena ion and
ace yla ion can modi y he bioac i i y and hence he (phy o)
oxici y o hese compounds (e.g. Wa d e al., 2002; Alexande
e al., 2011). As i has been shown in a la ge su ey on Canadian
g ains, DON and NIV, being he wo mos abundan oxins
de ec ed, now ep esen he wo majo conce ns o sa e y o
whea and ba ley p oduc s (Ti lemie e al., 2013).
En i onmen al ac o s may esul in a geog aphical
pa i ioning o subpopula ions ha may coincide wi h
chemo ypes. The success o a gi en chemo ype, which is o
impo ance wi h espec o FHB con ol, is o en ela ed o
local ac o s ( an de Lee e al., 2015). Based on chemo ype
cha ac e iza ion o I alian Fusa ium species, Co a elli e al.
(2015) sugges ed ha clima ic condi ions ha e a s ong impac
on he occu ence o 3-ADON and 15-ADON whe eas NIV
con amina ion occu ed i espec i e o clima ic condi ions.
Yli-Ma ila e al. (2013) p oposed ha he p e alence o a speci ic
chemo ype may also be in luenced by a ce ain hos . Fo example,
NIV-p oducing s ains we e ound o be mo e agg essi e owa ds
maize compa ed wi h DON-p oduce s (Ca e e al., 2002) and
we e associa ed, in F. asia icum, p e e en ially o maize in China
(Ndoye e al., 2012). Maie e al. (2006) pos ula ed NIV o be
a i ulence ac o in maize, which is in line wi h indings ha
associa e an inc ease in NIV popula ions in a eas whe e he
p eceding c op was maize (Audenae e al., 2009; Pasquali e al.,
2010; Sampie o e al., 2011).
Two main easons o de e mine he chemo ype o a s ain
ha e been p oposed (Pasquali and Migheli, 2014): (1) o ob ain
epidemiological in o ma ion on he popula ion colonizing a c op
in a gi en a ea, using chemo ype as a p oxy in he ield; (2)
o in o m on he oxigenic isk o con amina ed ood o eed
de e mined by he p esence o a ce ain chemo ype, wi h he long
e m pe spec i e o de eloping p e en i e models and s a egies
o dec ease he isk.
A p esen , da a on chemo ype dis ibu ion o FGSC a e
a ailable om all con inen s ( e iewed in Pasquali and Migheli,
2014), being F. g aminea um sensu s ic o (s.s.) he mos s udied
species. Less wo k has been de o ed o chemo ype de e mina ion
in F. culmo um (Sche m e al., 2013). Shi s in species popula ion
F on ie s in Mic obiology | www. on ie sin.o g 2Ap il 2016 | Volume 7 | A icle 406
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
ha e been epo ed in many su eys (Xu e al., 2005; Nielsen e al.,
2011; F edlund e al., 2013), bu epo s on chemo ype shi s in
ce ain a eas a e mo e ecen (e.g. Nielsen e al., 2012; Beye e al.,
2014).
Despi e he ac ha in o ma ion om all con inen s is now
a ailable, mos epo s do no include comple e in o ma ion on
he s ains analyzed, such as geog aphic o igin, hos om which
i was isola ed, me hod used o species iden i ica ion, e c. In
addi ion, p ecise cha ac e iza ion o he species is equen ly
lacking, being based only on mo phological obse a ions, hence,
making i un easible o use he da ase o u he s udies.
The main goal o his join s udy was o gene a e an accessible
map o icho hecene geno ypes om h ee FHB causing species
wi h de ailed in o ma ion on how he da a we e ob ained.
This will allow, in he long e m, he acquisi ion o consis en
and homogenous da ase s p o iding a alid compa ison o he
dis ibu ion o chemo ypes du ing yea s and among coun ies.
To each his aim, esea ch ins i u ions om 17 Eu opean
coun ies we e inqui ed o p o ide da a on how he sampling
was pe o med as well as de ailed in o ma ion on c opping
his o y and loca ion. A mo e coo dina ed e o , leading o
common p o ocols o sampling, chemo ype de e mining and
da a epo ing in a mo e accessible way would inc ease he
s anda diza ion o epidemiological da a. Fu he mo e, i could
acili a e he e o o unde s anding which ac o s do a o
es ablishmen and pe sis ence o a speci ic chemo ype. This
collec i e e o is now assembled in a ully accessible and
upg adable da ase o chemo ype di e si y wi hin FGSG and
F. culmo um on ce eals in Eu ope.
MATERIALS AND METHODS
Da a Collec ion
An Excel empla e ile was sen o esea ch pa ne s ag eeing
o pa icipa e in he ini ia i e (Supplemen a y File 1). The
in o ma ion o be submi ed (i applicable) we e as ollows:
chemo ype, yea o isola ion, whe he he s ain was ob ained
by a single spo e o a single hyphae, he loca ion including he
geog aphic coo dina es, he c op hos and cul i a om which
i was isola ed, p e ious c op, me hod o isola ion, me hod used
o species a ibu ion, p ime s and/o gene in case o PCR and
sequencing, name o cul u e collec ion in case i was deposi ed,
s ain ID, and ci a ion o he s ain/s in a publica ion. Whene e
gene ic chemo ype o species was unknown, s ains we e shipped
o he Luxembou g Ins i u e o Science and Technology (LIST)
labo a o y o gene ic chemo yping (Pasquali e al., 2011) and
species iden i ica ion by sequencing EF1alpha (Geise e al.,
2004). The o e all da ase (www.ca alogueeu.luxmcc.lu; a ailable
as o mid-Ap il 2016) was buil h ough in eg a ing da a
communica ed by esea ch pa ne s and by labo a o y esul s
ob ained wi h he p ocedu es desc ibed below.
DNA Ex ac ion and Chemo ype
De e mina ion
Fungal colonies we e g own on PDA as desc ibed in Pallez e al.
(2014) in o de o di ec ly ex ac DNA using a apid p ocedu e.
B ie ly, a 2–5 mm piece o mi aclo h issue (Millipo e, USA)
co e ed by a 5 days old ungal cul u e, was collec ed and added
o 100 µL TE (10 mM T is-Cl, 0.05 mM pH 9 EDTA solu ion,
Sigma-Ald ich, USA). A e 5 min o mic owa e ea men a
900 W and a 30 s cen i uga ion a 13,000 g, 5 µL we e hen used
o PCR eac ions. When iden i ica ion o he s ain was epo ed
o be pu a i e by pa ne s, EF1alpha ampli ica ion was ca ied
ou , ollowed by sequencing as desc ibed in Dubos e al. (2011).
I he species was p e iously de ined, i12 mul iplex PCR o
gene ic chemo yping (Wa d e al., 2002) was ca ied ou . All
PCR eac ions we e pe o med in a 50 µL olume o a oid isk
o PCR inhibi ion due o he quick ex ac ion me hod using 2X
Phusion mas e mix (The mo, USA), 300 nM o each p ime
and wa e . The mal cycle s used we e Biome a T-p o essional
and Ve i i PCR The mal cycle s (Li e Technologies, USA) using
he p og amme as desc ibed in Pasquali e al. (2011). All
eac ions included posi i e con ols o he h ee chemo ypes
and a nega i e con ol o moni o ing po en ial con amina ions.
Reac ions we e isualized on a Bio ad aga ose eady o use gel a
3%, using a UV spec a analyze (Ingenius, Syngene, UK). When
esul s we e ambiguous he eac ion was epea ed a leas once.
Da a assembled om o he labo a o ies we e collec ed by he
Excel empla e ile and uploaded o he da abase page. When
di e se me hods o gene ic chemo yping we e used (Waalwijk
e al., 2003; Jennings e al., 2004; Qua a e al., 2006; S a key e al.,
2007; Yli-Ma ila and Gagkae a, 2010) by he o iginal isola ing
labo a o y, his ac is speci ied di ec ly in he da abase.
S a is ical Analysis and Visualiza ion Tools
Desc ip i e g aphs on species and chemo ype dis ibu ion we e
ob ained using SigmaPlo e sion 12.5 (Sys a So wa e Inc,
USA) and SPSS e sion 19 (IBM, USA). The Eu opean maps
gene a ed o his s udy we e p epa ed using he A cMap
pla o m (ESRI Inc., USA). A Mul iple Co espondence Analysis
ool (B oeksema e al., 2013) was used o s udying he
o e all da ase and i s homogenei y in ela ion o species and
chemo ype dis ibu ion. Logis ic eg essions we e pe o med
using SigmaPlo 12.5.
Da abase Cons uc ion
The Eu opean da abase was cons uc ed by assembling he
o e all da ase on he da abase a chi ec u e de eloped by Piec
e al. (2016). A il e ing op ion o coun y and labo a o y, he
op ion o upload new da ase s, wi h adminis a o alida ion,
was added o he exis ing a chi ec u e. Func ions o he da abase
include he possibili y o ha e a ull o il e ed download o he
o e all da ase .
RESULTS
The cu en wo k ep esen s he i s collec i e a emp o
compile in o ma ion on chemo ype di e si y occu ing in
Eu opean coun ies. Mo eo e , he a ailabili y o a ull open
access da abase p o ides o he i s ime a cen alized sou ce o
in o ma ion o Fusa ium disease eco ds on ce eals, which is o
high alue o esea che s wo king in he myco oxin/Fusa ium
biodi e si y domain.
F on ie s in Mic obiology | www. on ie sin.o g 3Ap il 2016 | Volume 7 | A icle 406
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
The Da abase
The o e all da ase including all in o ma ion collec ed o
his wo k has been assembled in a da abase. Based on he
p e ious a chi ec u e cons uc ed o he LIST cul u e collec ion
(Piec e al., 2016), a da abase wi h imp o ed unc ionali ies
was buil . The o e all map wi h o e lapping F. g aminea um
and F. culmo um species is shown on he i s page o
he da abase (www.ca alogueeu.luxmcc.lu; Figu e 1). Fu he
uploading o da a can be pe o med acco ding o he ins uc ions
in Supplemen a y File 2. Resea che s wo king on Fusa ium
oxigenic di e si y a e in i ed o con ibu e o he da abase o o
download he da ase o u he analysis.
Da a Desc ip ion
In o ma ion o a o al o 1147 F. g aminea um and 479
F. culmo um s ains was included in he da ase collec ed om
he pe iod 2000–2013 and plo ed on he espec i e maps
(Figu es 2A,B). Yea s o isola ion we e close o homogenei y
(Figu e 3A). Luxembou g was he coun y whe e mos s ains
we e ob ained, ollowed by Belgium and Russia (Figu e 3B).
A p esen , chemo ype in o ma ion om some coun ies is
missing in he cu en da ase , he e o e, u he uploading o
in o ma ion will be impo an o ob ain a mo e p ecise pic u e
o chemo ype dis ibu ions in Eu ope.
The majo c op om whe e s ains we e isola ed was whea
(66.7%) ollowed by maize (22.5%), ba ley (5.4%), and o he
c ops (combined 5.3%; Figu e 3C). As can be obse ed by he
map o c op dis ibu ion, whea was sampled in 16 ou o he 17
coun ies, whe eas o he c ops we e sampled in a limi ed numbe
o coun ies (maize n=6; ba ley n=7; oa s =3; Figu e 4).
Oa s we e sampled only in No he n Eu ope, including No way,
Finland and Russia, whe e oa s a e an impo an c op, while no
FIGURE 1 | Spa ial dis ibu ion o chemo ypes and Fusa ium species in Eu ope. 3-ADON, 3-ace yldeoxyni alenol; 15-ADON, 15-ace yldeoxyni alenol; NIV,
ni alenol. F. co ade iae we e isola ed in I aly bu canno be isualized as hey a e o e lapped by o he s ains.
FIGURE 2 | (A) Spa ial dis ibu ion o Fusa ium culmo um chemo ypes in Eu ope. Red squa es, gene ic 3-ADON chemo ype. Yellow squa es, gene ic NIV
chemo ype. (B) Spa ial dis ibu ion o Fusa ium g aminea um sensu s ic o chemo ypes in Eu ope. G een ci cles, gene ic 15-ADON chemo ype. Red ci cles,
gene ic 3-ADON chemo ype. Yellow ci cles, gene ic NIV chemo ype.
F on ie s in Mic obiology | www. on ie sin.o g 4Ap il 2016 | Volume 7 | A icle 406
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
FIGURE 3 | F equency dis ibu ions o (A) he yea s, when he ungal
s ains o he cu en da abase we e isola ed, (B) he coun ies o
o igin o he s ains, (C) he hos plan s om which s ains we e
isola ed, (D) he p e ious c ops (“O he ” include mix u es o legumes
and ce eals, luce ne, lupines, pe ennial o ages, spinach, and suga
bee ), and (E) he species/chemo ype combina ions ound. Only 3
Fusa ium co ade iae s ains we e included and a e hus no isible in his
igu e. In he hos plan igu e, “o he c ops” include his les, soya and
po a oes. “O he poaceae” include o age g asses, einko n whea , i icale,
wild ype ba ley.
maize was sampled in No he n Eu ope, whe e he clima e is no
ye sui able o maize p oduc ion.
P e ious c op in o ma ion was a ailable only o a mino i y
o samples (<50%). The e o e, he analysis o hese da a was
pos poned un il enough da a a e uploaded o allow meaning ul
conclusions (Figu e 3D).
Wi h espec o he o e all dis ibu ion o chemo ypes pe
species o F. g aminea um, he p edominan geno ype was
15-ADON (82.9%, 951 s ains), ollowed by 3-ADON (13.6%, 156
s ains), and NIV (3.5%, 40 s ains). The 15-ADON geno ype
was mos common in isola es om whea and maize, while he
3-ADON geno ype was mos common in no he n Eu ope and
oa s. Fo F. culmo um, he p e alen geno ype was 3-ADON
(59.9%, 287 s ains), while he NIV geno ype accoun ed o he
emaining 40.1% (192 s ains). Th ee F. co ade iae wi h he
NIV chemo ype we e also included (Figu e 3E). The chemo ype
dis ibu ion wi hin each coun y can be accessed h ough he
il e ing op ions a ailable online (see Supplemen a y File 3 o
g aphical ep esen a ion). In e es ingly, only 3-ADON isola es o
F. g aminea um and F. culmo um we e ound in he collec ed
Eu opean isola es om oa s.
Da a Analysis
Mul iple co espondence analyses including yea , coun y, and
hos plan showed no e idence o a p e e en ial associa ion o he
species (F. g aminea um o F. culmo um) wi h speci ic coun ies
(bo h species we e p esen in 16 ou o 17 coun ies), se s o
coun ies, yea s o c ops (Figu e 5A). On he con a y, when he
co esponding analysis was pe o med on he chemo ype da ase ,
i was e iden ha chemo ypes we e no andomly dis ibu ed
o e coun ies, yea s, and c ops (Figu e 5B).
Fo u he analyses o he chemo ype dis ibu ion, we
ocused on he mos abundan popula ion ob ained om he
same hos . A o al o 784 F. g aminea um s.s. s ains we e
isola ed om whea . The 15-ADON chemo ype was a ely
obse ed in No he n Eu ope (Figu e 2B). The la i ude ha
ma ked he No he n limi o he 15-ADON chemo ype
dis ibu ion in F. g aminea um s ains isola ed om whea
in Eu ope was es ima ed by logis ic eg ession: Ha dly any
15-ADON chemo ype s ains o F. g aminea um we e ound
abo e 54.4 ±10.8◦No he n la i ude while he p obabili y
o a 15-ADON chemo ype s ain in a F. g aminea um
popula ion mo e sou hwa ds con e ged o 95.5 ±0.85%
(Figu e 6A).
Simila ly, in F. culmo um, he pe cen age o NIV chemo ype
s ains d as ically d opped o Eas o 7.5 ±0.6◦longi ude
(Figu es 2A,6C). Wes o 7.5 ±0.6◦longi ude, he p obabili y
o obse ing a NIV chemo ype s ain in a F. culmo um
popula ion isola ed om whea con e ged o 62.7 ±12.6%. In
F. g aminea um, he p obabili y o inding a NIV chemo ype
s ain was 15.9 ±2.5% Wes o 5.0 ±0.8◦longi ude Eas , bu
d opped quickly u he eas wa ds Figu es 2B,6B.
The a ailabili y o empo al se ies o s ains allowed also
e i ying he possible shi o species o chemo ypes in egions
wi h high da a densi y. By selec ing he denses sampled
a ea om he a ailable da ase (Luxembou g, yea 2007–
2012), i was possible o obse e how he NIV chemo ype in
F on ie s in Mic obiology | www. on ie sin.o g 5Ap il 2016 | Volume 7 | A icle 406

Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
FIGURE 4 | Ma e ials om which he ungal s ains we e isola ed.
F. g aminea um disappea ed a he end o he sampling pe iod
(Supplemen a y Video 1).
DISCUSSION
By assembling he da ase desc ibed he e, we could es ablish
a comp ehensi e collec ion o Eu opean da a on Fusa ium
di e si y on ce eals. The analysis o he dis ibu ion o gene ically
de e mined chemo ypes con i ms he dominance o he 15-
ADON chemo ype in Wes e n, Sou he n and Cen al Eu ope
in F. g aminea um (Pasquali and Migheli, 2014) bu a he
same ime, we iden i ied some cu en geog aphic limi s o
i s dis ibu ion in whea . Whe he his depends on c opping
and/o clima ic ac o s me i s u he in es iga ion gi en he ac
ha he 15-ADON chemo ype is cu en ly he majo cause o
DON accumula ion in Eu opean whea . Ou da a a e gene ally
in ag eemen wi h ea lie epo s on he e ogeneous chemo ype
dis ibu ion in Eu ope (Yli-Ma ila e al., 2009; Aamo e al.,
2015; an de Lee e al., 2015). Howe e , ea lie epo s did
no es ima e whe e he geog aphical limi s o he chemo ype
spa ial dis ibu ion a e. Ou s udy indica es a limi ed sp ead
o 15-ADON icho hecene geno ypes owa d he No he n
la i udes. This is con i med by he esul s om a ecen s udy
in No way whe e 3-ADON was he domina ing icho hecene
geno ype (Aamo e al., 2015). Howe e , he 15-ADON ype was
ecen ly in oduced in o No way, p obably om o he pa s o
Eu ope (Aamo e al., 2015), which exempli ies he need o
a common da abase o moni o geno ype shi s in Eu ope. A
hypo hesis wo h es ing would be o e i y i pa ame e s such
as empe a u e, ligh i adia ion, c opping p ac ices, and/o hos
plan s ypical o ce ain la i udes may ha e an impac on he
sp ead o he 15-ADON popula ion by combining pheno ypic
es s wi h s udies on he homogenei y o he ungal popula ion.
The dominance o he 3-ADON chemo ype in No he n
Eu ope, which is in acco dance wi h p e ious esul s o
Yli-Ma ila e al. (2009),Yli-Ma ila and Gagkae a (2010),
and Yli-Ma ila e al. (2013), was also con i med. The ole
o oa s as he po en ial p e e en ial hos o he 3-ADON
popula ion o F. g aminea um seems o be con i med in ou
da ase .
We also obse ed ha he NIV chemo ype was p e e en ially
ound in Wes e n Eu ope. The ac ha NIV chemo ypes in
F. culmo um we e a ely ound in Eas e n loca ions, sugges s
ha possibly di e en popula ions associa ed o chemo ype
di e si y migh be adap ed o dis inc c opping p ac ices o o
dis inc clima ic condi ions, hence, u he di e si y s udies on
F. culmo um popula ions a e needed.
Fu he mo e, we could iden i y empo al pa e ns o
chemo ype dis ibu ion ha we e pa ially associa ed o empo al
changes in clima ic condi ions as obse ed in Luxembou g
(Beye e al., 2014). Enla ging he da ase would allow simila
compa isons ac oss Eu opean coun ies as has been done o
example in No h Ame ica and wi hin China. In ac , analyses
o he icho hecene chemo ype dis ibu ions ac oss Canada
(Wa d e al., 2008) e ealed a d ama ic longi udinal cline in
which 3-ADON p oduce s om whea we e signi ican ly mo e
common in Eas e n Canada han in Wes e n p o inces (Wa d
e al., 2008), amoun ing o a 14- old inc ease be ween 1998 and
2004. The au ho s sugges ed ha he apid inc ease in 3-ADON
equency in Wes e n Canada migh indica e ha 3-ADON
popula ions ha e a selec i e ad an age o e isola es om he
esiden 15-ADON popula ion. The eason o he obse ed
shi is unclea bu hey hypo hesized ha i could be due o
changes in ag icul u al p ac ices o en i onmen al condi ions.
Guo e al. (2008) hypo hesized ha he shi occu ing in
Mani oba migh ha e been p oduced by seed shipmen and
long dis ance anspo a ion o spo es. In he s udy by Zhang
F on ie s in Mic obiology | www. on ie sin.o g 6Ap il 2016 | Volume 7 | A icle 406
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
FIGURE 5 | Mul iple co espondence analysis (MCA) wi h yea , coun y,
c op. The wo majo componen s a e plo ed. Dimension 1 accoun ed o
6.66% o he a iance in he da a and dimension 2 accoun ed o 5.55% o he
a iance. Bo h dimensions e lec le els o associa ion be ween he ca ego ies
o he ac o s a he han he ac o s hemsel es. G oups o objec s being
close o each o he sha e many p ope ies, while objec s (in ou cases
Fusa ium s ains) being dis an , do no sha e many p ope ies. I dis inc
g oups occu in he plo , he da a se con ains enough in o ma ion o sepa a e
he g oups based on he ca ego ical da a ha en e ed he MCA. I all g oups
a e in e mixed, he a iables ha en e ed he MCA a e no sui able o
dis inguish he g oups. (A) Colo s indica e di e en Fusa ium species (black,
Fusa ium g aminea um; blue, Fusa ium culmo um). (B) Colo s indica e
di e en chemo ypes (black, 3-ADON; blue, NIV; iole , 15-ADON).
e al. (2010), whe e mo e han 400 isola es o F. asia icum
(pa o he F. g aminea um species complex) om ba ley a
18 si es (10–2000 km apa ) in h ee alleys o Sou he n China
we e analyzed, a signi ican cline o 3-ADON p oduce s was
obse ed in he middle alley, bu no co ela ions wi h clima e
o ag onomy ac o s we e iden i ied.
Ce ainly, he da ase p esen ed he e includes some gaps ha
should be illed in o de o pe o m inc easingly eliable analyses
on he po en ial causes o change in species o chemo ype
dis ibu ion. I is expec ed ha u he upda es which could
include also sowing da e, illage egime, ungicide ea men s,
and o he cul u al p ac ices will s eng hen he da ase and allow
a be e unde s anding o he e ec s o c opping measu es and
en i onmen al ac o s on s ain dis ibu ion.
Ne e heless, i is e iden ha molecula geno yping is a
powe ul ool o suppo o e u e epidemiologically gene a ed
hypo heses (Li in se a e al., 2015). Nume ous da abases ela ed
o ungal di e si y and biop oduc s a e a ailable (Wacke , 2015),
as well as ungal eposi o ies o specimens (Abd-Elsalam e al.,
2010), bu hey a ely in eg a e di e en sou ces o in o ma ion
on s ain di e si y. Ou da abase includes molecula da ase s
associa ed o geog aphic and c opping p ac ices and wi h he
a ailabili y o GPS da a, ou da abase can be indi ec ly en iched
wi h me eo ological da ase s ela ed o he loca ion whe e he
s ains we e collec ed.
I has been sugges ed ha “la ge da ase s con aining
epidemiological da a associa ed o gene ic in o ma ion can help
unde s anding, ecognizing and e en ually, managing ungal
FIGURE 6 | P esence (=1) and absence (=0) o di e en chemo ype
s ains wi hin he Eu opean Fusa ium g aminea um sensu s ic o
and F. culmo um isola e collec ion in ela ion o he la i ude whe e
hey we e ound. (A) 15-ADON chemo ype s ains in F. g aminea um.
(B) NIV chemo ype s ains in F. g aminea um.
(C) NIV chemo ype s ains in F. culmo um. The logis ic eg ession line gi es an
es ima e abou he p obabili y o inding a pa icula isola e wi hin he F.
g aminea um/F. culmo um popula ion.
ou b eaks” (Li in se a e al., 2015). Hence, gi en he impo ance
o sha ed da a o ungal epidemiological s udies and conside ing
he in e ac ions be ween en i onmen al, c opping, and gene ic
ac o s, ou up-scalable and ully open access da abase can
possibly help add essing u u e isks o sp ead o oxigenic
Fusa ium popula ions on ce eals.
F on ie s in Mic obiology | www. on ie sin.o g 7Ap il 2016 | Volume 7 | A icle 406
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
This wo k also ep esen s an example o a Eu opean
pa icipa i e and coope a i e app oach ha can se e as an
example o he es ablishmen o o he epidemiological s udies
p o i ing om he a ailabili y o la ge and well main ained
da ase s. Gi en he complexi y o FGSC, u u e e o s will no
only aim a inclusion o da a om o he coun ies and new
ungal s ains bu will also ha e o in eg a e mul i locus sequence
geno yping in o ma ion (MLST) o be e cha ac e ize di e si y
and possibly e en new species o he le el o popula ion di e si y
(Wa d e al., 2002; Talas e al., 2011; Aamo e al., 2015; Talas and
McDonald, 2015).
A he same ime, his en i ely accessible da ase is essen ial
o allowing u he a ge ed s udies in o de o ully di e en ia e
all included s ains a bo h species and subpopula ion le els,
assuming ha F. g aminea um as well as F. culmo um a e
cons i u ed by di e en subpopula ions (Liang e al., 2014;
Balmas e al., 2015; an de Lee e al., 2015).
In he cu en e sion o he da ase , gene ic de e mina ion o
he chemo ype was ca ied ou o di e en ia e he h ee majo
known chemo ypes in Eu ope. I is o eseeable o u he analyze
he s ains by in es iga ing he p esence o he NX-2 chemo ype
ha has been cu en ly ound only in he USA (F uhmann e al.,
2014; Liang e al., 2014; Va ga e al., 2015).
Employing a lexible, up-scalable and upg adable da abase,
ou wo k ep esen s he i s a emp o build a global da abase in
which s ains, p o ided wi h in o ma ion o GPS da a and hos ,
a e also analyzed using mul i-locus geno yping in combina ion
wi h VNTR (Va iable Numbe Tandem Repea ) sc eening and
po en ially whole genome sequencing ( an de Lee e al., 2015).
The success o such an e o will depend on he u u e
con ibu ions: he a ailabili y o a well-main ained and expanded
da abase is a solid con ibu ion o a sha ed app oach philosophy
o conduc ing esea ch ha will help o speed up scien i ic
p og ess in ungal biology and ag icul u e (Abbà e al., 2015).
AUTHOR CONTRIBUTIONS
MP concei ed and pe o med he expe imen s, coo dina ed he
assembly, analyzed he da a, and w o e he manusc ip . SV
concei ed he expe imen s, coo dina ed he assembly, assembled
and analyzed he da a, and w o e he manusc ip . MB pe o med
he expe imen s, analyzed he da a and w o e he manusc ip . JP
assembled and managed he da abase. AL, KA, VB, RB, AB, JC,
EC, TG, MG, IH, NK, LH, JL, PG, TM, QM, AM, MM, FM, PP,
MP-B, JS, BS, SS, UT, SU, AV, and TY pe o med expe imen s and
helped in w i ing he manusc ip .
FUNDING
The Luxembou g Ins i u e o Science and Technology, LU,
acknowledges he Minis è e de l’Ag icul u e, de la Vi icul u e
e de la P o ec ion des Consomma eu s-Adminis a ion des
Se ices Techniques de l’Ag icul u e o inancially suppo ing
he Sen inelle p ojec . The wo k on I alian s ains has been
inancially suppo ed h ough he M.I.U.R. P ojec AGROGEN
(Labo a o y o GENomics o ai s o AGROnomic impo ance
in du um whea : Iden i ica ion o use ul genes, unc ional
analysis and assis ed selec ion by biological ma ke s o he
de elopmen o he na ional seed chain) (D. D. 14.03.2005
n. 602/Ric). Funding o he esea ch o Ryan Basle was
p o ided by Felix Tho nley Cobbold T us and he John Oldac e
Founda ion.
The wo k o JC was suppo ed by he Minis y o Ag icul u e
o he Czech Republic, P ojec No. RO0415. The esea ch o
MG and PG was suppo ed by he Spanish Minis y MINECO
(AGL2014-53928-C2-2-R). The Minis y o Ag icul u e and
Food, No way unded he wo k o IH. The esea ch o TM
was unded by he Fede al Minis y o Educa ion and Resea ch
(BMBF) (GABI-KANADA #FKZ 0313711A), Bonn and by he
Ge man Academic Exchange Se ice (DAAD), Bonn (code
no.: A/06/92183). PP acknowledges he Finnish Minis y o
Ag icul u e and Fo es y o unding he p ojec FinMyco on
Fusa ium and myco oxins in Finland. The esea ch o JS was
unded by he Di ec ion Géné ale de l’Ag icul u e, Di ec ion
de la Reche che ( e . D31-3159, D31-1162, D31-7055), in he
amewo k o a p ojec en i led “Ca ac é iza ion e dynamique
des usa ioses su maïs en Région Wallonne.” BS acknowledges
suppo by P.O.R. SARDEGNA F.S.E. 2007–2013—Obie i o
compe i i i à egionale e occupazione, Asse IV Capi ale
umano, Linea di A i i à l.3.1 ( esea ch p ojec “Iden i ica ion
o na u al and na u al-like molecules inhibi ing myco oxin
biosyn hesis by Fusa ia pa hogenic on ce eals”). UT hanks he
Danish Di ec o a e o Food, Fishe ies and Ag i Business g an
FFS05-3 o inancial suppo . The wo k o TY was inancially
suppo ed by he Academy o Finland (no. 126917, 131957,
250904, 252162, 267188, and 266984), Ol i Founda ion, Tu ku
Uni e si y Founda ion, a CIMO a el g an o Taha Hussien,
and he No dic ne wo k p ojec New Eme ging Myco oxins and
Seconda y Me aboli es in Toxigenic Fungi o No he n Eu ope
(p ojec 090014), which was unded by he No dic Resea ch
Boa d.
ACKNOWLEDGMENTS
We would like o acknowledge he MycoRed p ojec o
pa onizing he ac i i y in he amewo k o a scien i ic
alliance. The MycoKey p ojec is also acknowledged. F iede ike
Pogoda, Céline B ocho , Se ane Con al, Bo is Un e eine ,
F édé ic Gi aud, Alyssa Cowles, and Tiphaine Dubos om
he Luxembou g Ins i u e o Science and Technology (LU)
and I ene Bänzige and E eline Jenny om Ag oscope (CH)
a e acknowledged o hei excellen echnical suppo in
ungal collec ion, isola ion and managemen . Be jan B oeksema
(LU) is acknowledged o suppo ing he da a analyses. Jana
Palico á and Ta ’ána Sumíko á om he C op Resea ch Ins i u e
(CZ) a e acknowledged o hei excellen echnical suppo
in ungal collec ion, isola ion and managemen . The Finnish
Ce eal commi ee is acknowledged o he esea ch ma e ial.
Veli Hie aniemi is acknowledged o p ojec managemen and
Ma jaana Vi anen o he excellen echnical suppo in ungal
isola ion and managemen [Na u al Resou ces Ins i u e Finland
(Luke)], FI. The Kimen seed heal h labo a o y is acknowledged
o collec ing No wegian F. g aminea um isola es o IH, NIBIO,
NO.
F on ie s in Mic obiology | www. on ie sin.o g 8Ap il 2016 | Volume 7 | A icle 406
Pasquali e al. Fusa ium T icho hecene Geno ypes Eu opean Da abase
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ micb.
2016.00406
Supplemen a y File 1 | Templa e o pa icipan s o he o iginal ini ia i e.
Supplemen a y File 2 | Ins uc ion o upload new da ase on he da abase
and an ou pu example o a gi en s ain.
Supplemen a y File 3 | (A) Fusa ium g aminea um and (B) Fusa ium
culmo um chemo ype dis ibu ion wi hin each pa icipa ing coun y
in % o all samples analyzed (n). 3-ADON, 3-ace yldeoxynia lenol;
15-ADON, 15-ace yldeoxynia lenol; NIV, ni alenol.
Supplemen a y Video 1 | Fusa ium g aminea um sampled du ing 6 yea s
in Luxembou g. E e y yea , a leas 15 ields we e sampled in di e en
geog aphic loca ions in he coun y. Depending on he yea , he species was mo e
o less p esen . The NIV chemo ype disappea ed in he las 4 yea s o sampling in
his species.
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F on ie s in Mic obiology | www. on ie sin.o g 9Ap il 2016 | Volume 7 | A icle 406