Mycotoxin Research

https://doi.org/10.1007/s12550-019-00341-3

ORIGINAL ARTICLE

First report of Fusarium foetens as a mycotoxin producer

Jesús M. González-Jartín 1 & Amparo Alfonso 1 & María J. Sainz 2 & Mercedes R. Vieytes 3 & Olga Aguín 4 &
Vanesa Ferreiroa 4 & Luis M. Botana 1

Received: 8 August 2018 / Revised: 28 December 2018 / Accepted: 2 January 2019

# Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Fusarium foetens, a pathogen of Begonia plants, has been recently described as a new fungal species. This Fusarium species
causes a destructive vascular wilt disease which leads to the death of the plant. Moreover, Fusarium species are known to produce
a huge variety of secondary metabolites such as mycotoxins and phytotoxins. Here, we studied the toxicogenic profile of one
F. foetens strain, isolated from maize, employing two methods based on the use of ultra-performance liquid chromatography
coupled to mass spectrometry-ion trap-time of flight detection. The mycotoxins beauvericin and fusaric acid were detected in a
pure culture of F. foetens. In addition, four fusaric acid analogs (10,11-dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric
acid, and a hydroxylated unsaturated fusaric acid analog) were tentatively identified on the basis of their accurate mass and
fragmentation patterns. Therefore, these preliminary data indicate that F. foetens isolated from maize is able to produce Fusarium
mycotoxins including beauvericin and fusaric acid.

Keywords Fusarium foetens . Fusaric acid . Mycotoxin . Begonia

Introduction Mycotoxin contamination of agricultural commodities can

Fusarium is a genus of filamentous fungi in the Ascomycota
that colonizes a wide range of host plants and crops all around
the world. It includes around 70 species, some of which cause
several plant diseases and/or produce mycotoxins in many
economically important crops, resulting in high losses of yield
and quality (Munkvold 2017).
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12550-019-00341-3) contains supplementary
material, which is available to authorized users.
* Amparo Alfonso
amparo.alfonso@usc.es
* Luis M. Botana
luis.botana@usc.es
1
Departamento de Farmacología, Facultad de Veterinaria,
Universidade de Santiago de Compostela, 27002 Lugo, Spain
2
Departamento de Producción Vegetal y Proyectos de Ingeniería,
Facultad de Veterinaria, Universidade de Santiago de Compostela,
27002 Lugo, Spain
3
Departamento de Fisiología, Facultad de Veterinaria, Universidade
de Santiago de Compostela, 27002 Lugo, Spain
4
Estación Fitopatolóxica Areeiro, Deputación de Pontevedra, Subida a
la Robleda s/n, 36153 Pontevedra, Spain
occur in on the field (e.g., Fusarium sp.) and/or during storage
(e.g., Aspergillus sp.). Consequently, most research have fo-
cused on the mycotoxigenic profile of Fusarium species path-
o g e n i c t o c e r e a l s , s u c h a s F. g r a m i n e a r u m a n d
F. verticillioides, as cereals are staple crops susceptible to my-
cotoxin contamination (Sainz et al. 2018). There are three
major groups of Fusarium toxins, namely, trichothecenes,
fumonisins, and zearalenone, frequently found in agricultural
products (Sainz et al. 2015; Smith et al. 1994). However, there
are many other toxins produced by Fusarium such as
moniliformin (MON), beauvericin (BEA), fusaproliferin,
enniatins, or fusaric acid (FA) receiving increasing attention
because they are often found in food and feed (D’Mello et al.
1999; Nazari et al. 2015). One additional problem is the co-
occurrence of two or more mycotoxins in one product since
synergistic interactions among some toxins have had been
observed. For instance, toxicological synergism occurs be-
tween deoxynivalenol and FA, and deoxynivalenol and
fumonisin B1 (Harvey et al. 1996; Smith et al. 1997). The
presence of several toxins in one product is frequent since a
commodity can be infected, at the same time, with more than
one fungus and some fungal strains produce several myco-
toxins (Streit et al. 2012). For instance, F. sporotrichioides
concurrently produces ten type-A trichothecenes such as T-2
toxin, HT-2 toxin, or neosolaniol (Gonzalez-Jartin et al. 2018).

In 2004, Schroers et al. (2004) reported an outbreak of a
new disease in ornamental crops, specifically in Begonia
plants (Begonia x hiemalis) in the Netherlands. The causative
agent was identified as a Fusarium species that differed mor-
phologically from F. begoniae but resembled F. oxysporum; it
was described as a new species, named F. foetens, based main-
ly on phylogenetic analyses. The infection of F. foetens in
Begonia plants causes basal rot, vein yellowing, and wilting
symptoms leading to the dead of the host plant (Schroers et al.
2004). The origin of this species remains unknown. However,
it has been hypothesized that F. foetens proceeded from an
exotic origin since it has an optimal growth at 25–28 °C,
and up to now, this species has been detected in Europe,
Canada, and the USA (Huvenne et al. 2011). Fusarium
foetens can survive in the soil and infect certain plants show-
ing no evidence of disease, and thus, it may spread and infect
other crops (EPPO 2013). After the initial outbreak, the Plant
Protection Service of Netherlands has held F. foetens under
official control in order to avoid propagation, and two pest risk
assessments were published (van der Gaag and Raak 2010).
Several analytical techniques have been developed to study
the metabolite profile of fungi. In the case of mycotoxins, the
most important ones are based on the analysis of fungal ex-
tracts by liquid chromatography-mass spectrometry (LC-MS)
and searching for the monoisotopic mass of the relevant de-
tected ions in databases (Klitgaard et al. 2014). Therefore,
accurate mass measurement is necessary to perform a correct
identification. In addition, some instruments allow
fragmenting the detected ions and thus generate data about
their elemental composition. These data can be subsequently
employed to predict the molecular formulae of the studied
compounds (Ferrer and Thurman 2003). At the moment, the
potential of F. foetens to produce mycotoxins has not been
thoroughly studied. Therefore, we used ultra-performance liq-
uid chromatography coupled to mass spectrometry-ion trap-
time of flight (UHPLC-MS-IT-TOF) to establish the toxigenic
profile of one isolate of F. foetens. First, a multi-toxin ap-
proach was used, and then a specific study and characteriza-
tion of FA was done.
Material and methods
Chemicals
Water was purified in a Millipore Milli-Q Plus system
(Millipore, Bedford, MA). Methanol, acetonitrile, and
acetic acid (glacial, 100%) were supplied by Panreac
Quimica S.A. (Barcelona, Spain), and formic acid was
from Merck (Madrid, Spain). All solvents employed in
this work were HPLC or analytical grade. Ammonium
f o r m a t e w a s f r o m F l u k a (B u c h s , S w i t z e r l a n d ) .
Ultrafree-MC, Durapore membrane centrifugal filters
Mycotoxin Res
(0.45 μm pore size) were purchased from Millipore
(Billerica, MA). FA standard was acquired from Sigma
(Madrid, Spain). Solid standards provided by Sigma
(Madrid, Spain) were as follows: FA, deoxynivalenol,
zearalenone, fumonisin B 1 , enniatin A, enniatin A 1 ,
enniatin B, enniatin B1. Analytical standards of T-2 tox-
in, HT-2 toxin, neosolaniol, fumonisin B2, and MON
were from Romer Labs (Tulln, Austria), and BEA was
from Enzo (Barcelona, Spain).
Fungal isolation and identification
In a study of Fusarium diversity on forage maize in
Galicia (NW Spain), a monosporic isolate of Fusarium
foetens was obtained from an asymptomatic kernel using
the Fusarium-selective Komada medium (Komada 1975),
and then it was subcultured and maintained on potato
dextrose agar (PDA) (Sharlau, Barcelona, Spain). The
species was tentatively identified as F. foetens by observ-
ing macroscopic characteristics of mycelium and micro-
scopic features of fungal structures (Leslie and Summerell
2006). Species identification was confirmed by amplifica-
tion, sequencing, and phylogenetic analysis of a portion
of the sequence of the translation elongation factor 1-
alpha gene (EF-1 α). DNA was extracted from the myce-
lium using the E.Z.N.A® Fungal DNA Mini Kit (Omega
Bio-tek, Inc.). Then the portion of the gene EF-1 α was
amplified with primers EF1 and EF2 as described by
O'Donnell et al. (2000). Products of PCR were purified
by using High Pure PCR Product Purification Kit (Roche
Applied Science) and sequenced in an ABIPrism™ 3130
Genetic Analyzer (Applied Biosystems). The EF sequence
generated was deposited in the NCBI GenBank database
under the accession number MH536333. The sequence
was compared with related EF sequences available at the
NCBI’s GenBank database using the BLASTn program
and aligned with the ClustalW program. Phylogenetic
analyses of nucleotide sequences of the EF-1 α region
were conducted using the neighbor-joining (NJ) approach,
which was performed using the MEGA6 software
(Tamura et al. 2013). The resulting NJ tree showed that
the isolate from this study clustered together with
GenBank sequences corresponding to three isolates of
F. foetens with a strong bootstrap support (100%), and
was phylogenetically distinct from F. oxysporum (Fig. 1).
The ITS region of rDNA was similarly amplified, se-
quenced, and analyzed, but it did not allow the identification
of the Fusarium species.
Mycotoxin analysis
For mycotoxin analysis, the isolate of F. foetens was
subcultured in PDA, PDA amended with streptomycin
Mycotoxin Res

Fig. 1 Neighbor-joining tree

showing the relationship of 35
sequences of Fusarium species
inferred from the sequence of the
elongation factor 1 α gene. The
bootstrap probability values
(1000 replicates) are shown on the
nodes. The EF sequence of
Ilyonectria liriondendri
(GenBank Accession: JF35705)
was used as outgroup. The
sequence of the isolate from this
study is indicated by accession
number in bold. Branch lengths
are proportional to the estimated
number of nucleotide
substitutions. Scale bar is 0.05
nucleotide substitutions per site

(PDAs), PDA amended with chloramphenicol and strep- Chromatography

tomycin (PDAcs), malt extract agar (MEA), Komada me-
dium, Spezieller Nährstoffarmer agar (SNA), yeast extract
sucrose agar (YES), and dichloran Rose-Bengal chloram-
phenicol agar (DRBC) at 25 °C in the dark. Three repli-
cates were prepared for each cultivation medium. After
1 week of incubation, three agar plugs (6 mm diameter)
were cut from each resultant monosporic culture. Agar
plugs were subsequently extracted with 0.5 mL of an ace-
tonitrile/water/acetic acid mixture [79:20:1 (v/v/v)] by
shaking for 3 min using a vortex mixer. Finally, the ex-
tract was filtered through a 0.45-μm centrifugal filter.
The analysis of these samples was performed by two different
methods. In both cases, the UHPLC system was from
Shimadzu (Kyoto, Japan) interfaced to an IT-TOF instrument.
The UHPLC system consisted of two pumps (LC-30AD), an
autoinjector (SIL-10AC) with a refrigerated rack, a degasser
(DGU-20A), a column oven (CTO-10AS), and a system con-
troller (SCL-10Avp).
Chromatographic method 1: the column was a 100 mm ×
2.1 mm (inside diameter), 1.8 μm, Waters ACQUITY
HSS T3 column (Waters, Milford, MA). The temperature

was kept at 40 °C. Mobile phases were as follows: (a)
water containing 0.1% formic acid and 5 mM ammonium
formate and (b) methanol. The elution gradient (18 min)
was as follows. Initially, the level of eluent B was hold at
0% for 1 min, and then it was linearly increased to 50% B
within 3 min. After 4 min at 50% B, the gradient was
increased to 100% B within 4 min and kept for 3 min.
Finally, the gradient was switched to 0% B over 0.5 min,
and the column was re-equilibrated for 2.5 min. The flow
rate of the mobile phase was maintained at 0.3 mL/min,
and the injection volume was set at 5 μL.
Chromatographic method 2: the column was a 100 mm ×
2.1 mm (inside diameter), 1.7 μm, Waters BEH C18 col-
umn (Waters, Milford, MA). The temperature was main-
tained at 40 °C. Mobile phases were as follows: (a) water
containing 0.1% formic acid and (b) acetonitrile, and the
elution gradient (13 min) was as follows. Initially, the
level of eluent B was hold at 0% for 1 min, then it was
linearly increased to 30% B within 2.5 min. After that,
30% B was kept for 2 min. Thereafter, the gradient was
increased to 100% B over 1.5 min and maintained for
3 min. Finally, the gradient was switched to 0% B over
0.5 min, and the column was re-equilibrated for 2.5 min.
The flow rate of the mobile phase was kept at 0.35 mL/
min, and the injection volume was set at 5 μL.
Mass detection
The mass spectrometer was an IT-TOF instrument from
Shimadzu (Kyoto, Japan). It was equipped with an
electrospray ionization (ESI) interface and the operating
conditions were as follows: curved desolvation line and
heat block temperature, 200 °C; nebulizing gas flow,
1.5 L/min; detector voltage, 1.65 kV; drying gas pres-
sure, 105 kPa; ion trap pressure, 1.8 × 10−2 Pa; pressure
of TOF region, 1.4 × 10−4 Pa. With the chromatographic
method 1, the MS method was performed in full scan
MS mode with a mass range of 150–900 in positive
and negative mode. The ion accumulation time was set
to 20 ms, with an event time of 300 ms and three repe-
titions. With the chromatographic method 2, the MS
method was performed in full positive scan MS mode
with a mass range of 150–300. The ion accumulation
time was set to 20 ms, with an event time of 300 ms
with three repetitions. Relevant ions were isolated in
MS1 scan for collision-induced dissociation (CID) MS1–
2
experiments within a tolerance range of 1 Dalton (Da).
The mass range of the method in MS2 was 50–300 and
the ion accumulation time was set to 30 ms. The colli-
sion energy parameter was set at 100%, argon was used
as the collision gas at 100%, and the frequency parame-
ter was set to 45 kHz.
Mycotoxin Res
Accurate mass measurement and Formula
assignments
A standard sample (reference: 641225-06613-08) from
Shimadzu (Kyoto, Japan) was employed as an external refer-
ence to calibrate the mass range prior to data acquisition. MS1–
2
data, acquired with mass accuracy, was employed to predict
elemental composition of selected ions. With these MS1–2
data, the Formula Predictor software (Shimadzu, Kyoto,
Japan) generates a list of possible candidates for each ion
allowing errors lower than 10 mDa.
Results and discussion
Fusarium foetens has been recently described as a new phy-
topathogenic agent, but its toxicogenic properties have so far
not been studied (Schroers et al. 2004). This species is related
to F. oxysporum (Fig. 1), which has been reported to produce
BEA, enniatins, FA, and MON (Gruber-Dorninger et al. 2017;
Rabie et al. 1982). The production of metabolites is
conditionated by culture medium, and filamentous fungi usu-
ally yield a high variety of compounds in PDA (Frisvad et al.
2008). Hence, to elucidate if F. foetens is able to produce
mycotoxins, one isolate was incubated in PDA, during a week
at 25 °C in the dark.
A simple and quick method to study metabolite profiles of
fungi is to analyze agar plugs from pure Petri dish cultures
(Smedsgaard 1997). Thus, three agar plugs were obtained
from each culture of F. foetens and then extracted with a sol-
vent widely employed for the analysis of mycotoxins (Sulyok
et al. 2006). Thereafter, the F. foetens extract was analyzed by
UHPLC-MS-IT-TOF in scan mode using the chromatographic
method 1, which was previously optimized to detect Fusarium
mycotoxins like trichothecenes (deoxynivalenol, neosolaniol,
T-2 toxin, HT-2 toxin), fumonisin B1, zearalenone, MON,
BEA, or enniatins (Fig.-S1), and other mycotoxins.
When the presence of BEA was checked in the extract, by
selecting the exact mass of the [M+NH 4 ] + ion at m/z
801.4433, a high intense peak was detected at the retention
time (RT) 14.35 min (Fig. 2a). As shown in Fig. 2b, the accu-
rate mass spectrum of the peak revealed the ion at m/z
801.4438; hence, there is a low mass difference (0.4 mDa)
between the measured mass and the theoretical mass of BEA
(m/z 801.4433). In addition, an ion at m/z 784.4188 was de-
tected that can be attributed to [M+H]+ of BEA (theoretical m/
z 784.4168, error 2.0 mDa). A commercial standard of BEA
was analyzed employing the same conditions. As before, the
extract chromatogram of the [M+NH4]+ ion of BEA yielded a
peak at 14.35 min (Fig. 2c); the accurate mass spectrum
showed the [M+H]+ and [M+NH4]+ ions at m/z 784.4201 at
m/z 801.4456, respectively (Fig. 2d). Therefore, the coinci-
dence between the RT and the exact mass of the [M+H]+
Mycotoxin Res
Fig. 2 a UHPLC-MS-IT-TOF chromatogram of a F. foetens extract. b Accurate MS spectrum of the compound eluting at 14.35 min. c UHPLC-MS-IT-
TOF chromatogram of a standard solution of BEA (300 ng/mL). d Accurate MS spectrum of BEA
and [M+NH4]+ ions allow to unequivocally identify this com-
pound as BEA. The production of this mycotoxin was also
detected when the fungus was grown on wheat (Gonzalez
et al. 2016).
Next, acquisition data was reanalyzed, and a high intense peak
was detected at 7.1 min (Fig. 3a). The extract ion chromatogram
of the peak showed the ion at m/z 180.1013 (Fig. 3b). The accu-
rate exact mass of the ion may correspond to FA (m/z 180.1019);
therefore, to identify the peak, an analytical standard of FA was
used. However, chromatographic conditions were not suitable to
analyze this mycotoxin since the peak showed a tail up to 2 min
and, due to this, the RT was not reproducible.
To perform an accurate identification, analytical column, mo-
bile phases, and elution gradient were optimized. As shown in
Fig. 4a, when the analytical standard was analyzed with the
chromatographic method 2, the tail of the peak was eliminated,
and thus RT was reproducible. One additional advantage of the
new method was that the analysis time was shortened to 13 min.
Hence, the fungus extract was reanalyzed employing the new
conditions. The accurate theoretical mass of the [M+H]+ ion of
FA at m/z 180.1019 was searched in the standard sample (Fig. 4a)
and in the fungal extract (Fig. 4d). As a result, in both cases, a
high intense peak was obtained at the RT 5.75 min. The accurate
Fig. 3 a UHPLC-MS-IT-TOF
chromatogram of a F. foetens ex-
tract obtained with the chromato-
graphic method 1. b Accurate
MS1 spectrum of the compound
eluting at 7.1 min
mass spectrum of the standard shows the ion at m/z 180.1031
(Fig. 4b) while the peak of the fungal extract proceeds from the
ion at m/z 180.1033 (Fig. 4e). Therefore, in both cases, there were
a low mass difference between the accurate mass measured and
the theoretical mass of FA (m/z 180.1019), being this difference
of 1.2 mDa and 1.4 mDa, respectively. On the other hand, the ion
at m/z 162.0930 was observed in the MS1 scan of the standard
sample (Fig. 4b). The accurate mass difference of this ion regard-
ing the [M+H]+ ion of FA (m/z 180.1031) is − 18.0101 Da, that is
a loss of water. The same loss (− 18.0106 Da) was observed in
the spectrum of the fungal extract (Fig. 4e). In order to perform a
more complete identification, the fragmentation process of [M+
H]+ ions was studied. MS-IT-TOF technology allows the predic-
tion of molecular formulae on the basis of the recorded data,
acquired with mass accuracy, and the fragmentation pathway
employing and ad hoc predictor software (Gonzalez-Jartin
et al. 2017). Thus, [M+H]+ ions were fragmented employing
the IT-TOF in MSn mode applying CID. Thereafter, elemental
composition of parent and product ions were predicted with the
predictor software. The fragmentation of the [M+H]+ ion of the
standard at m/z 180.1031([C10H13NO2+H]+) leads to three main
fragments in MS2 (Fig. 4b, c). The main fragment ion at m/z
162.0927 (− 18.0104 Da) was formed due to the loss of a water

Fig. 4 a UHPLC-MS-IT-TOF chromatogram of a standard solution of FA
(250 ng/mL) obtained with the chromatographic method 2. b Accurate
MS1 spectrum of FA. c Accurate MS2 spectrum of the ion at m/z
180.1031. d UHPLC-MS-IT-TOF chromatogram of an extract of
molecule [M+H-H2O]+. The second most intense fragment was
the [M+H-CO]+ ion at m/z 152.1087 formed by the loss of a
carbonyl moiety (− 27.9944 Da). Finally, the ion at m/z
134.0981 (− 46.0050 Da) was obtained by the loss of CO and
H2O [M+H-CH2O2]+. The detected fragments match with those
previously found in the literature (Crutcher et al. 2017; Niehaus
et al. 2014). Detailed data about accurately measured m/z, theo-
retical m/z, elemental composition, and mass errors expressed as
mDa of precursors and production ions are shown in Table 1.
Next, the [M+H]+ ion at m/z 180.1033 found in the fungal extract
was submitted to the same fragmentation process, and the same
fragments were obtained in MS2 spectrum (Fig. 4e, f). In this
way, the coincidence between retention times, exact masses, and
fragmentation pathways allow us to unequivocally identify as FA
the peak detected in the F. foetens extract at 5.75 min.
Consequently, it was demonstrated for the first time the ability
of F. foetens to produce mycotoxins.
In order to elucidate if F. foetens produces some FA analog,
ions showing neutral losses of water were searched in MS1 scan
since, as it was mentioned before, FA shows losses of these
molecules (Fig. 4b, e). Figure 5 shows five peaks with this pat-
tern: peak 1 corresponding to the ion at m/z 212.0924 (1.2 min),
peak 2 at m/z 194.0810 (4.3 min), peak 3 at m/z 196.0962
(4.4 min), peak 4 at m/z 178.0877 (5.25 min), and FA
Table 1 Data for MSn
compounds: mass stage, Ion MS Elemental composition
elemental composition,
accurately predicted m/z, [M+H]+ 1 C10H13NO2
accurately measured m/z, the [M+H-H2O]+ 2 • C10H11NO
mass errors expressed as [M+H-CO]+ 2 • C9H13NO
milli-Daltons (mDa)
[M+H-CH2O2]+ 2 • C9H11N
Mycotoxin Res
F. foetens obtained with the chromatographic method 2. e Accurate
MS1 spectrum of the compound eluting at 5.75 min. f Accurate MS2
spectrum of the ion at m/z 180.1033
(5.75 min). Since the fragmentation pathway of FA has been
previously set, the new ions were fragmented to establish if they
corresponded to some modified form of FA. As it was explained,
the fragmentation of the [M+H]+ ion of FAyielded losses of H2O
(exact mass 18.0105 Da), CO (exact mass 27.9949 Da), and
CH2O2 (exact mass 46.0054 Da). As Fig. 6 shows, the fragmen-
tation of new [M+H]+ ions led to the same mass losses detected
in FA. Therefore, the match of fragmentation patterns points out
that the detected ions are analogs of FA. Again, the formula
predictor software was applied to elucidate the element compo-
sition of parent and product ions, and detailed data about exact
mass, predicted formulae, and mass errors are reported in
Table 2. First, the peak 1 was studied; the ion at m/z 212.0924
(Fig. 6a) has a probable formula of C10H13NO4. This molecular
formula matched with 10,11-dihidroxyfusaric acid, and this FA
analog was isolated from a F. verticillioides (= F. moniliforme)
culture (Burmeister et al. 1985). Moreover, a compound with the
same molecular formula was tentatively identified in a Fusarium
oxysporum extract (Zhu et al. 2016). The ion at m/z 194.0810
detected in peak 2 (Fig. 6b) has a probable formula of
C10H11NO3. In the literature, there were found no references
about a natural compound with the same elemental composition.
On the other hand, the m/z of the fusaric acid methyl ester (m/z
194.1176) is similar to the m/z of the compound; however, the
Predicted m/z (Da) Measured m/z (Da) Error (mDa)
180.1019 180.1031 1.2
162.0913 162.0927 1.4
152.1070 152.1087 1.7
134.0964 134.0981 1.7
Mycotoxin Res

Peak 1 Peak 3
RT 1.2 min RT 4.4 min
m/z 212.0924 m/z 196.0962

Peak 2 Fusaric acid

RT 4.3 min
m/z 194.0810

Peak 4
RT 5.25 min
m/z 178.0877

Fig. 5 UHPLC-MS-IT-TOF chromatogram of an F. foetens extract showing ions which present neutral losses of water in MS1 spectra

difference in exact mass (− 36.6 mDa) is too high to identify the
detected compound as a methyl ester derivate of FA. The corre-
spondence of the fragmentation patterns tentatively identifies this
molecule as an analog of FA, and the predicted formula just
differed in two hydrogen atoms with regard to hydroxylated
fusaric acid (C10H13NO3). Therefore, this compound might be
a hydroxylated unsaturated fusaric acid. Next, peak 3 was stud-
ied, and the most probable formula for the ion m/z 196.0962 (Fig.
6c) was C10H13NO3, which corresponds to a hydroxylated ana-
log of FA. Up to now, the analogs 8-hydroxyfusaric acid, 9-
hydroxyfusaric acid (fusarinolic acid), and 10-hydroxyfusaric
acid have been described (Burmeister et al. 1985; Crutcher
et al. 2017). However, the employed technique does not allow
us to identify which of the analogs was in the sample. Finally,
peak 4 was studied, and the ion at m/z 178.0877 (Fig. 6d) has a
probable formula of C10H11NO2, which corresponds to 9,10-
dehydrofusaric acid (dehydrofusaric acid). This compound, to-
gether with FA, is the main metabolite produced by some
Fusarium species such as F. nygamai (Capasso et al. 1996;
Zonno et al. 1996).
In order to provide a more stable basis for the production of
these compounds, the strain was grown in different culture media
including PDA, PDAs, PDAcs, MEA, YES, Komada, DRBC,
and SNA. As shown in Table 3, BEA and FA were produced in
all media with the exception of SNA. FA analogs were detected
in PDA without and with antibiotics, MEA, and YES. In addi-
tion, the analog m/z 194.1176 was produced in Komada medium.
Therefore, these compounds are produced in media containing
simple sugars together with yeast extract (YES) or malt extract
(MEA), while low nutrient media, specially SNA, avoid the pro-
duction of mycotoxins. On the other hand, the presence of anti-
biotics does not affect the production of the studied toxins.
The presence of other mycotoxins was evaluated in all
culture media, and no further toxins were detected. As it was
previously mentioned, F. oxysporum-like species, the sister
species of F. foetens, produce BEA, enniatins, FA, and
MON (Gruber-Dorninger et al. 2017; Rabie et al. 1982).
However, under the employed cultured conditions, the isolate
examined in this work does not produce enniatins nor MON.
FA is a potent phytotoxin for several plant species, and it
has been related to the development of basal rot illness, dis-
coloration of calli, reduction in the chlorophyll content of
seedlings, leaf wilting, and necrosis (Curir et al. 2000;
Löffler and Mouris 1992; Wu et al. 2008). Moreover, this

Fig. 6 Accurate MSn spectra of Fusarium compounds. Peak 1: a MS1 scan, e MS2 of 212.0924. Peak 2: b MS1 scan, f MS2 of 194.0810. Peak 3: c MS1
scan, g MS2 of 196.0962. Peak 4: d MS1 scan, h MS2 of 178.0877
 Mycotoxin Res

Table 2 Data for MSn

compounds: mass stage, Compound RT 1.2 MS Elemental Predicted m/z Measured m/z Error
elemental composition, composition (Da) (Da) (mDa)
accurately predicted m/z, [M+H]+ 1 C10H13NO4 212.0917 212.0924 0.7
accurately measured m/z, the [M+H-H2O]+ 2 • C10H11NO3 194.0812 194.0816 0.4
mass errors expressed as milli-
Daltons (mDa) [M+H-CO]+ 2 • C9H13NO3 184.0999 183.0926 3.1
[M+H-CH2O2]+ 2 • C9H11NO2 166.0863 166.0862 − 0.1
Compound RT 4.3 MS Elemental Predicted m/z Measured m/z Error
composition (Da) (Da) (mDa)
[M+H]+ 1 C10H11NO3 194.0812 194.0810 − 0.2
[M+H-H2O]+ 2 • C10H11NO2 176.0706 176.0705 − 0.1
[M+H-CO]+ 2 • C9H11NO2 166.0863 166.0868 0.5
[M+H-CH2O2]+ 2 • C9H9NO 148.0757 148.0774 1.7
Compound RT 4.4 MS Elemental Predicted m/z Measured m/z Error
composition (Da) (Da) (mDa)
[M+H]+ 1 C10H13NO3 196.0968 196.0962 − 0.6
[M+H-H2O]+ 2 • C10H11NO2 178.0863 178.0879 1.6
[M+H-CO]+ 2 • C9H13NO2 168.1019 168.1036 1.7
[M+H-CH2O2]+ 2 • C9H11NO 150.0913 150.0920 0.7
Compound RT 5.25 MS Elemental Predicted m/z Measured m/z Error
composition (Da) (Da) (mDa)
[M+H]+ 1 C10H11NO2 178.0863 178.0877 1.4
[M+H-H2O]+ 2 • C10H9NO 160.0757 160.0763 0.6
[M+H-CO]+ 2 • C9H11NO 150.0913 150.0933 2.0
[M+H-CH2O2]+ 2 • C9H9N 132.0808 132.0802 − 0.6

toxin has an important role in the virulence of fungal infec-
tions since the fungal capacity to cause mortality is reduced when
it is unable to produce FA (Lopez-Diaz et al. 2018). Therefore,
phytotoxic effects caused by FA are similar to those symptoms
observed in begonia plans infected by F. foetens. Consequently,
the high pathogenicity of F. foetens to begonia could be related to
the production of FA and their analogs. However, only one strain
was analyzed; therefore, these findings do not necessarily apply
to the species as secondary metabolite production may be vari-
able within a species and could be related to transposable genetic
elements (Li-Jun et al. 2010).
In this study, we develop an accurate method for the iden-
tification of the FA and their analogs on the basis of the accu-
rate mass measurement and fragmentation patterns. The
proposed UHPLC-MS-IT-TOF method allows the separation
and identification of fusaric acid and four analogs (10,11-
dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric
acid, and a hydroxylated unsaturated fusaric acid analog) in
a F. foetens extract. In addition, the production of BEA is
reported. These results identify, at least, one strain of
F. foetens as a mycotoxin producer. Moreover, the detected
toxins are highly phytotoxic to several plants which could
explain the mortality of Begonia plants infected by this
fungus.
Acknowledgments The research leading to these results has received
funding from the following FEDER cofunded grants: from Conselleria
de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia,
2017 GRC GI-1682 (ED431C 2017/01); from CDTI and Technological

Table 3 Mycotoxin production in

different culture media. Culture BEA FA Compound m/z Compound m/z Compound m/z Compound m/z
Production of BEA, FA, media 212.0924 196.0962 178.0877 194.0810
compound m/z 212.0924
10,11-dihidroxyfusaric acid), PDA + + + + + +
compound m/z 196.0962 PDAs + + + + + +
(hydroxyfusaric acid), compound PDAcs + + + + + +
m/z 178.0877 (dehydrofusaric
acid), compound m/z 194.0810 MEA + + + + + +
(hydroxylated unsaturated FA). YES + + + + + +
ND not detected Komada + + ND ND ND +
DRBC + + ND ND ND ND
SNA ND ND ND ND ND ND
Mycotoxin Res
Funds, supported by Ministerio de Economía, Industria y
Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/
FEDER, UE), ISCIII/PI16/01830, RTC-2016-5507-2, and ITC-
20161072; from European Union POCTEP 0161-Nanoeaters-1-E-1,
Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-
2018, and H2020 778069-EMERTOX. Jesús M. González-Jartín was
supported by a fellowship from Programa de Formación de Profesorado
Universitario (FPU14/00166), Ministerio de Educación, Cultura y
Deporte, Spain.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest. The au-
thors have full control of all primary data and allow the journal to review
the data if requested.
Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
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