toxins

Article
Influence of Two Garlic-Derived Compounds, Propyl
Propane Thiosulfonate (PTS) and Propyl Propane
Thiosulfinate (PTSO), on Growth and Mycotoxin
Production by Fusarium Species In Vitro and in
Stored Cereals
Kalliopi Mylona 1,† , Esther Garcia-Cela 1 , Michael Sulyok 2 , Angel Medina 1 and
Naresh Magan 1, *
1 Applied Mycology Group, Environment and AgriFood Theme, Cranfield University,
Cranfield MK43 0AL, UK
2 Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology (IFA-Tulln),
University of Natural Resources and Life Sciences, Vienna, Konrad Lorenzstr. 20, A-3430 Tulln, Austria
* Correspondence: [email protected]
† Present address: Centre for Agriculture, Food and Environmental Management (CAFEM) School of Life and
Medical Sciences, University of Hertfordshire College Lane, Hatfield, Hertfordshire AL10 9AB, UK.




Received: 29 July 2019; Accepted: 24 August 2019; Published: 27 August 2019


Abstract: Two garlic-derived compounds, Propyl Propane Thiosulfonate (PTS) and Propyl Propane
Thiosulfinate (PTSO), were examined for their efficacy against mycotoxigenic Fusarium species
(F. graminearum, F. langsethiae, F. verticillioides). The objectives were to assess the inhibitory effect of
these compounds on growth and mycotoxin production in vitro, and in situ in artificially inoculated
wheat, oats and maize with one isolate of each respectively, at different water activity (aw ) conditions
when stored for up to 20 days at 25 ◦ C. In vitro, 200 ppm of either PTS or PTSO reduced fungal
growth by 50–100% and mycotoxin production by >90% depending on species, mycotoxin and aw
conditions on milled wheat, oats and maize respectively. PTS was generally more effective than
PTSO. Deoxynivalenol (DON) and zearalenone (ZEN) were decreased by 50% with 80 ppm PTSO.
One-hundred ppm of PTS reduced DON and ZEN production in wheat stored at 0.93 aw for 20 days,
although contamination was still above the legislative limits. Contrasting effects on T-2/HT-2 toxin
contamination of oats was found depending on aw , with PTS stimulating production under marginal
conditions (0.93 aw ), but at 0.95 aw effective control was achieved with 100 ppm. Treatment of stored
maize inoculated with F. verticilliodies resulted in a stimulation of total fumonsins in most treatments.
The potential use of such compounds for mycotoxin control in stored commodities is discussed.

Keywords: Fusarium; mycotoxins; garlic-derived extracts; green chemistry; fungi; EU limits; abiotic
factors; storage; wheat; maize; oats

Key Contribution: PTSO was more effective than PTS in inhibiting Fusaria in vitro. In vitro efficacy
for control of growth/mycotoxin production was influenced by water activity. In situ efficacy by these
garlic-derived compounds was less effective as in in vitro studies. At times, lower doses triggered
mycotoxin production in situ.

1. Introduction
There has been interest in the use of essential oils (EOs) and extracts derived from plants to
control food spoilage microorganisms, especially mycotoxigenic moulds, as an alternative to traditional

Toxins 2019, 11, 495; doi:10.3390/toxins11090495 www.mdpi.com/journal/toxins

Toxins 2019, 11, 495 2 of 16

preservatives based on aliphatic acids [1]. However, an important aspect to consider when utilizing
natural plant extracts for control of mycotoxigenic spoilage fungi is whether they are classed as
food grade. In addition, many studies have studied effects on germination and growth of spoilage
mycotoxigenic fungi, while neglecting impacts on mycotoxin production, especially in situ.
Onion and garlic, both members of the Allium family, have received attention as extracts from these
two plant species have been found to have significant antimicrobial properties [1]. Their antifungal
efficacy has been studied despite the relative instability of some of their compounds or their strong
odour. Yin and Tsao [2] found garlic, out of seven Allium plants, to be the most effective against
three Aspergillus species. Some inter-species differences in efficacy were previously noted, with higher
concentrations of plant extracts required for control of Aspergillus flavus and Aspergillus fumigatus
than Aspergillus niger. Yoshida et al. [3] suggested that the antifungal activity of garlic was due to the
compounds allicin and ajoene. Benkeblia [4] observed growth inhibition of A. niger and Penicillium
cyclopium by red onion and garlic EOs, while higher concentrations (200–500 mL/L) of green and
yellow onion EOs were required for control the fungal pathogen Fusarium oxysporum. Singh and
Singh [5] studied the effect of Allium sativum extracts and other plant extracts on the growth of A. flavus
and aflatoxin production, but only in liquid cultures. Addition of the extract at the beginning of the
incubation period showed 85% inhibition in mycelial biomass and complete inhibition of aflatoxin
production. However, a later addition only gave marginal control of toxin biosynthesis. Liquid culture
systems are relatively artificial as the aim should be to try and develop intervention strategies to
control mycotoxigenic fungi under similar conditions found in different agrifood commodities or
under simulated nutritional conditions relevant to where the target control measures are going to
be instituted.
In the last decade, some compounds have been extracted from Allium sativus and been successfully
utilised as antimicrobial preservatives in a number of products, especially in cheese production and as
a fruit coating. The two extracted compounds being used are propyl propane thiosulfonate (PTS) an
organosulphate, and propyl propane thiosulfinate (PTSO). Formulations of these compounds are now
commercially available as Proallium, especially as a coating for extension of fruit shelf-life. While they
are considered effective anti-microbials, very little detailed information is available on the efficacy of
these compounds against mycotoxigenic fungi, either in vitro or in situ.
Previous studies to compare some EOs and antioxidants to control P. verrucosum and ochratoxin A
(OTA) production showed that of those tested only resveratrol was effective at controlling populations of
the mycotoxigenic species and inhibiting OTA production in stored wheat grain under different storage
conditions [6]. Environmental factors were also shown to have a significant influence on the relative
control achieved. In addition, this study showed that sometimes under sub-optimal concentrations of
these compounds, some stimulation of toxin production occurred despite growth being significantly
reduced. This has been suggested to be due to a combination of water and physiological stress caused
by the antifungal agent itself which may stimulate secondary metabolite production as a defence
response [7–10].
Some very early studies suggested thiosulfinates had potential applications as anti-microbials,
although very focused on their use in post-harvest grain preservation [11]. Some extracts of garlic and
onion have been shown to have promising efficacy against Aspergillus and Penicillium species and in
some cases mycotoxin production. However, very limited information is available on the efficacy of
such EOs on Fusarium species. In addition, such compounds have rarely been assessed on naturally
contaminated grains where a range of different species may be encountered.
The objectives of this study were to study the effect of two garlic-derived compounds (PTS, PTSO)
for the control of (a) fungal growth and (b) mycotoxin production by Fusarium graminearum
(Deoxynivalenol, Zearalenone; wheat), F. langsethiae (T-2/HT-2 toxins; oats) and F. verticilioides
(Fumonisins; maize) for the first time. Experiments were initially conducted in vitro for a preliminary
assessment of these two compounds to determine the effective concentrations for control of growth
and mycotoxin production. Subsequently, in situ experiments were carried out with inoculation of
Toxins 2019, 11, 495 3 of 16
Toxins 2019, 11, x FOR PEER REVIEW 3 of 16

wheat, oats and

compounds maize
based on with theresults
in vitro relevant
andmycotoxigenic
stored at 25 °Cspecies
underand treatment
different with content
moisture each of these two
conditions
compounds based on in vitro results and stored at 25 ◦ C under different moisture content conditions
for up to 20 days to examine effects on mycotoxin production.
for up to 20 days to examine effects on mycotoxin production.
2. Results
2. Results
2.1. In Vitro Efficacy of PTS and PTSO Garlic-Derived Compounds against Fungal Growth
2.1. In Vitro Efficacy of PTS and PTSO Garlic-Derived Compounds against Fungal Growth
Figure 1 shows the effect of 0–200 ppm PTS and PTSO on the in vitro radial growth rates of F.
Figure 1 shows the effect of 0–200 ppm PTS and PTSO on the in vitro radial growth rates of
graminearum, F. verticillioides and F. langsethiae. Both PTS and PTSO had very good inhibitory effects
F. graminearum, F. verticillioides and F. langsethiae. Both PTS and PTSO had very good inhibitory effects
on the growth of the isolate of each of these species studied and this increased with concentration.
on the growth of the isolate of each of these species studied and this increased with concentration.
Complete inhibition of the growth of F. langsethiae was observed with 100 ppm PTS and of F.
Complete inhibition of the growth of F. langsethiae was observed with 100 ppm PTS and of F. verticillioides
verticillioides with 200 ppm. Growth of F. graminearum was significantly inhibited, although complete
with 200 ppm. Growth of F. graminearum was significantly inhibited, although complete inhibition was
inhibition was not achieved in the range of concentrations examined. Two hundred ppm PTSO
not achieved in the range of concentrations examined. Two hundred ppm PTSO completely inhibited
completely inhibited the growth of F. langsethiae and was very effective against F. graminearum and
the growth of F. langsethiae and was very effective against F. graminearum and F. verticillioides. PTS was
F. verticillioides. PTS was always more effective than PTSO when applied at the same concentration
always more effective than PTSO when applied at the same concentration against the same fungal
against the same fungal species. Table 1 shows the concentration necessary for effective dose (ED) 50
species. Table 1 shows the concentration necessary for effective dose (ED) 50 and 90% control of growth
and 90% control of growth of the three Fusarium species.
of the three Fusarium species.

Figure 1. Effect of 0–200 ppm (a) Propyl Propane Thiosulfonate (PTS) and (b) Propyl Propane
Figure 1. Effect of 0–200 ppm (a) Propyl Propane Thiosulfonate (PTS) and (b) Propyl Propane
Thiosulphonate (PTSO) on the in vitro (2% wheat agar medium) on the radial growth rate (n = 6) of an
Thiosulphonate (PTSO) on the in vitro (2% wheat agar medium) on the radial growth rate (n = 6) of
isolate of F. graminearum, F. langsethiae and F. verticillioides. Vertical bars indicate the standard error of
an isolate of F. graminearum, F. langsethiae and F. verticillioides. Vertical bars indicate the standard error
the means. For PTS effects on growth: F. graminearum: H (4, N = 30) = 26.612, p < 0.001; F. langsethiae:
of the means. For PTS effects on growth: F. graminearum: H (4, N = 30) = 26.612, p <0.001; F. langsethiae:
H (4, N = 30) = 27.647, p < 0.001; F. verticillioides: H (4, N = 30) = 26.522, p < 0.001; for fungal species:
H (4, N = 30) = 27.647, p <0.001; F. verticillioides: H (4, N = 30) = 26.522, p <0.001; for fungal species: H
H (2, N = 90) = 3.176, p = 0.2004. For PTSO effects on growth: F. graminearum: H (4, N = 30) = 27.451,
(2, N = 90) = 3.176, p = 0.2004. For PTSO effects on growth: F. graminearum: H (4, N = 30) = 27.451, p
p < 0.001; F. langsethiae: H (4, N = 30) = 27.877, p < 0.001; fungal species: H (2, N = 90) = 12.926,
<0.001; F. langsethiae: H (4, N = 30) = 27.877, p <0.001; fungal species: H (2, N = 90) = 12.926, p = 0.002.
p = 0.002.

Table1.1.Effective
Table Effectivedose
doseofofPTS
PTSand
andPTSO
PTSOfor for50%
50%and
and90%90%control
control (ED
(ED ; ED
; ED
5050 values)of
9090values) ofgrowth,
growth,when
when
comparedtotothe
compared theuntreated
untreatedcontrol,
control,
forfor inhibition
inhibition of of F. graminearum,
F. graminearum, F. langsethaie
F. langsethaie andand F. verticillioides
F. verticillioides on
onmilled
2% 2% milled
wheatwheat
agaragar
medium at 25at◦ C.
medium 25 °C.

Treatment Treatment
(ppm) (ppm) PTS PTS PTSO
PTSO
Species SpeciesED ED50ED ED90 ED
ED50 ED90
50
ED90
50 90
F. graminearum 33 144 52 186
F. graminearum 33 144 52 186
F. langsethiaeF. langsethiae12 12 80 80 2121 113 113
F. 0verticilloides
F. verticilloides 64 0 64 172 172 >200 >200
>200 >200

The statistical analyses (ANOVA) showed that the effect of PTS and PTSO concentration was
highly significant on the growth of the isolate of each Fusarium species, with significant intra-isolate
differences for growth rate in the presence of the either of these two compounds.
Toxins 2019, 11, 495 4 of 16

The statistical analyses (ANOVA) showed that the effect of PTS and PTSO concentration was
highly significant
Toxins 2019, 11, x FORon growth of the isolate of each Fusarium species, with significant intra-isolate
theREVIEW
PEER 4 of 16
differences for growth rate in the presence of the either of these two compounds.
Additionalstudies
Additional studieswere
werecarried
carried out
out to to assess
assess thethe efficacy
efficacy of two
of the the compounds
two compounds at different
at different water
water activity
activity (aw) on
(aw ) levels levels on growth
growth of theFusarium
of the three three Fusarium
species.species.
Figure 2Figure
shows2an shows an example
example of the
of the effect of
effect of 0–100 ppm PTSO on the growth of F. graminearum and F. langsethiae
0–100 ppm PTSO on the growth of F. graminearum and F. langsethiae on wheat agar media modified on wheat agar media
modified
to to threeawdifferent
three different aw levels.
levels. The radial The radial
growth growth
of both of both
isolates isolates
of the of the was
two species two significantly
species was
inhibited as the PTSO concentration was increased and water stress was imposed (0.94, 0.92 aw ), (0.94,
significantly inhibited as the PTSO concentration was increased and water stress was imposed when
0.92 aw), to
compared when compared to
the unmodified the unmodified
control medium. Thecontrol
maximum medium. The (%)
percentage maximum percentage
inhibition was observed(%)
inhibition
at the highestwasPTSOobserved at the highest
concentration and PTSO
0.995 aconcentration and 0.995 aw (~83% for F. graminearum and
w (~83% for F. graminearum and ~90% for F. langsethiae).
~90% for F. langsethiae). ANOVA showed that the effects
ANOVA showed that the effects of either PTS or PTSO concentration, of either PTS or aPTSO concentration, aw and
w and fungal species were
fungal species were highly significant on the log-transformed radial
highly significant on the log-transformed radial growth rate data, while the effects growth rate
of data, while the
the interactions
effects ofthese
between the interactions
factors werebetween these factors were not significant.
not significant.

Figure
Figure2. 2. Effect
Effect of 0–100 ppm
of 0–100 ppm Propyl
PropylPropane
PropaneThiosulfinate
Thiosulfinate(PTSO)
(PTSO)ononthe
theradial
radial growth
growth rates
rates (n (n =
= 6)
6)
ofof
anan isolate
isolate of of F. F.
(a)(a) graminearum
graminearum and
and F. F.
(b)(b) langsethiae
langsethiae in in vitro
vitro (2%
(2% wheat
wheat agar
agar medium)
medium) in relation
in relation to
to water
water activity
activity (aw(a).wVertical
). Vertical bars
bars indicate
indicate thethe standard
standard error
error of the
of the means.
means. Statistical
Statistical analyses
analyses for
for (a)
(a) F. graminearum:
F. graminearum: H (4,HN 30)==30)
(4,=N = 27.451,
27.451, p < and
p <0.001; 0.001;
forand (b) F. langsethiae:
forlangsethiae:
(b) F. H (4, N H = 30) =
(4, =N27.877,
= 30) 27.877,
p <0.001.
p < 0.001.
Table 2 summarises the effective dose (ED50) concentrations of PTSO only for the isolates of F.
Table 2 summarises
graminearum, F. langsethiaetheand
effective dose (ED50used
F. verticillioides ) concentrations
in this study of grown
PTSO only for theagar
on wheat isolates
mediaof
F.modified
graminearum, F. langsethiae and F. verticillioides used in this study grown on wheat agar
to different aw levels and 25 °C. Overall, lower concentrations of each compound were media modified
to differentfor
required aw50%
levels and 25 ◦ of
inhibition C. growth
Overall,of lower concentrations
the isolate of each compound
of F. langsethiae than for thewere required
isolates of thefor 50%
other
inhibition of growth
two Fusarium species. of the isolate of F. langsethiae than for the isolates of the other two Fusarium species.

Table 2. Effective dose ED50 values (ppm) of PTSO for 50% inhibition of the growth of F. graminearum,
Table 2. Effective dose ED50 values (ppm) of PTSO for 50% inhibition of the growth of F. graminearum,
F. verticillioides and F. langsethiae on wheat agar media of different water activities (aw ) at 25 ◦ C.
F. verticillioides and F. langsethiae on wheat agar media of different water activities (aw) at 25 °C.
Treatments
Treatments F. graminearum
F. graminearum F. F.verticillioides
verticillioides F. langsethiae
F. langsethiae
PTSO (0.98(0.98
PTSO aw ) aw) 42
42 189
189 9.5 9.5
PTSO (0.94(0.94
PTSO aw ) aw) 36
36 >100
>100 50 50
PTSO (0.92 aw ) 37 >100 32
PTSO (0.92 aw) 37 >100 32

2.2.
2.2. Effects
Effects of
of the
the Compounds
Compounds on
on In
In Vitro
Vitro Mycotoxin
Mycotoxin Inhibition
Inhibition
Figure
Figure 33 shows
shows thethe effect
effect of
of different
different concentrations
concentrationsofofPTS PTSand
andPTSO
PTSOonon fumonisins
fumonisins B1 B 1 and
and B2
B(FB F. verticillioides ◦ C.
2 (FB and FB ) production by the isolate of on wheat-based medium
1 and FB2) production by the isolate of F. verticillioides on wheat-based medium at 25 °C. Two-
1 2 at 25
Two-hundred
hundred ppmppm of PTS
of PTS inhibited
inhibited FB1FB 1 and
and FB2FB 2 toxins
toxins bybyupuptoto 90%.For
90%. ForPTSO,
PTSO,the
theproduction
productionofofboth
both
toxins was slightly stimulated with up to 100 ppm. However, the production of the two fumonisins
was inhibited with 200 ppm. ANOVA showed that the effect of PTS concentration was highly
significant on the log-transformed data of both FB1 and FB2. For PTSO concentration, analyses showed
Toxins 2019, 11, 495 5 of 16

toxins was slightly stimulated with up to 100 ppm. However, the production of the two fumonisins was
inhibited with
Toxins 2019, 11, 200
x FORppm.
PEERANOVA
REVIEW showed that the effect of PTS concentration was highly significant 5 of 16
on the log-transformed data of both FB1 and FB2 . For PTSO concentration, analyses showed that
thatwas
there there was a significant
a significant effect oneffect on FB1 production
FB1 production by theofisolate
by the isolate of F. verticillioides
F. verticillioides in vitro butinitvitro
was but
not it was
not significant
significant for thefor the production
production of FB
of FB2 (see 2 (see Supplementary
Supplementary Table S1).Table S1).
The
The production
production of deoxynivalenol
of deoxynivalenol (DON)(DON)
by F.by F. graminearum
graminearum in response
in response to exposure
to exposure to either
to either of of
these
thesecompounds
compounds was reduced
was reduced whenwhencompared
compared to the
to untreated control
the untreated (Supplementary
control (Supplementary FigureFigure
S1). S1).
DON
DONproduction
production was below
was belowthethe
limit of detection
limit of detection in thein200
theppm PTS treatments.
200 ppm However,
PTS treatments. DON DON
However,
production
production in the 250 ppm PTSO was ~4 times more than in the control. ANOVA showedthe
in the 250 ppm PTSO was ~4 times more than in the control. ANOVA showed that that the
concentration
concentration of PTS significantly
of PTS affected
significantly DON DON
affected production. PTSO had
production. no significant
PTSO effect on ineffect
had no significant vitro on in
DON
vitroproduction by F. graminearum.
DON production by F. graminearum.
There was a decrease
There was a decrease in T-2 production
in T-2 production by F.bylangsethiae as the
F. langsethiae asPTS
the concentration
PTS concentrationwas increased,
was increased,
and
and this was below the limit of detection with 200 ppm concentration (data not shown). toxin
this was below the limit of detection with 200 ppm concentration (data not shown). HT-2 HT-2 toxin
in the same samples was not detected at ≥50 ppm PTS (Supplementary Figure S2; Supplementary
in the same samples was not detected at ≥50 ppm PTS (Supplementary Figure S2; Supplementary
Table S2). With PTSO, T-2 toxin production by F. langsethiae was inhibited compared to the control,
Table S2). With PTSO, T-2 toxin production by F. langsethiae was inhibited compared to the control,
although no specific pattern was observed with concentration. HT-2 toxin production was below
although no specific pattern was observed with concentration. HT-2 toxin production was below the
the limit of detection in all PTSO concentrations. Statistically, the effect of PTS concentration was
limit of detection in all PTSO concentrations. Statistically, the effect of PTS concentration was
significant on T-2 toxin production but not for HT-2 toxin. The effect of PTSO was not significant for
significant on T-2 toxin production but not for HT-2 toxin. The effect of PTSO was not significant for
either of these two related type A trichothecenes.
either of these two related type A trichothecenes.

Figure 3. Effect of 0–200 ppm of (a) PTS and (b) PTSO on the production of fumonisins B1 and B2 by
Figure 3. Effect of 0–200 ppm of (a) PTS and (b) PTSO on the production of fumonisins B1 and B2 by
F. verticillioides in vitro at 25 ◦ C. Vertical bars indicate the standard error of the means. For statistical
F. verticillioides in vitro at 25 °C. Vertical bars indicate the standard error of the means. For statistical
analyses see Supplementary Table S2.
analyses see Supplementary Table S2.
2.3. In Situ Mycotoxin Control in Stored Wheat, Oats and Maize Using PTS and PTSO
2.3. In Situ Mycotoxin Control in Stored Wheat, Oats and Maize using PTS and PTSO
Figures 4 and 5 show the effects of either PTS or PTSO on DON and ZEN production in wheat
modifiedFigure 4 and
to two 5 show
different aw the effects
levels, of either
inoculated withPTS or PTSO on and
F. graminearum DON and ZEN
stored for 10production
and 20 daysin at wheat
◦ C. The red
25modified to two
linesdifferent
show theaEU w levels, inoculated
regulatory with
limits for each F. of
graminearum
the toxins inand stored
wheat [12]for
(EC10 and 20 days at
1881/2006).
25 °C.
DON The red lines
production was show
reducedtheinEUallregulatory limits samples
the PTS-treated for each stored
of the toxins
at 0.93 in
awwheat
for up[12] (EC
to 20 1881/2006).
days at
◦ C compared to the control. Maximum inhibition was obtained with 100 and 200 ppm PTS after 10
25DON production was reduced in all the PTS-treated samples stored at 0.93 aw for up to 20 days at 25
and
°C 20 days storage
compared (76%
to the and 95%)
control. respectively
Maximum when compared
inhibition was obtainedwith the
withcontrol.
100 andIn the
200wetter wheat
ppm PTS after 10
samples
and 20 at
days0.95storage
aw stored(76%forand
10 days
95%)DON production
respectively when increased
comparedwithwith
increasing PTS concentration,
the control. In the wetter wheat
while afterat
samples 200.95
daysawirregular
stored for results wereDON
10 days obtained ranging increased
production from 90% inhibition of DON
with increasing production
PTS concentration,
with 200after
while ppm20 PTS to >100%
days irregular stimulation at 300
results were ppm. In
obtained the PTSO
ranging fromtreatments, a small
90% inhibition ofreduction in
DON production
DON contamination (~33%) was observed with 80 ppm PTSO in wheat stored
with 200 ppm PTS to >100% stimulation at 300 ppm. In the PTSO treatments, a small reduction in for 10 days at both
awDON
levels. With lower concentration
contamination of PTSO (40
(~33%) was observed ppm)
with 80 ppmtherePTSO
was ainstimulation
wheat stored of toxin
for 10production
days at both aw
when compared to the control. After 20 days storage stored wheat treated with PTSO had toxin levels
levels. With lower concentration of PTSO (40 ppm) there was a stimulation of toxin production when
compared to the control. After 20 days storage stored wheat treated with PTSO had toxin levels
higher than in the controls at both aw levels. ANOVA showed that the effects of PTS concentration,
aw and storage time significantly affected log(DON) production by F. graminearum in the stored wheat
treatments (see Supplementary Table S3). However, interactions between these factors was not
Toxins 2019, 11, 495 6 of 16

higher than in the controls at both aw levels. ANOVA showed that the effects of PTS concentration,
aw and storage time significantly affected log(DON) production by F. graminearum in the stored
wheat treatments (see Supplementary Table S3). However, interactions between these factors was not
significant. For PTSO, ANOVA showed that concentration and aw significantly affected log (DON)
production,
Toxins 2019, 11,while
x FOR the
PEEReffects
REVIEW of storage time and interactions between factors were not significant.
6 of 16

Figure
Figure 4. Effect of
4. Effect of 0–300
0–300 ppm
ppm of ofPTS
PTS(a)
(a)and
and0–80
0–80ppm
ppmPTSO
PTSO(b)
(b)onondeoxynivalenol
deoxynivalenol production
production bybyF.
F. graminearum in artificially inoculated wheat of 0.93 and 0.95 a stored ◦
graminearum in artificially inoculated wheat of 0.93 and 0.95 aw stored w for 10 and 20 days at 25 °C (nC=
for 10 and 20 days at 25
Toxins 2019, 11,3).
x FOR PEER REVIEW 7 of 16
2 ×=3).
(n 2× Vertical
Vertical barsbars indicate
indicate thethe standard
standard error
error of of the
the means.The
means. Theredredlines
linesshow
show the
the EU
EU legislative
legislative
limits
limits (EC
(EC 1881/2006)
1881/2006) for
for deoxynivalenol
deoxynivalenolin inunprocessed
unprocessedwheat
wheatfor
forfeed
feeduse
use(1750
(1750µg/kg).
µg/kg).

Figure 5 shows the effect of PTS and PTSO on ZEN production by F. graminearum in stored
wheat. The most effective control of ZEN production by PTS was 100ppm at 0.93 aw with 89% control
after 20 days storage. In the wetter 0.95 aw grain 100 ppm of PTS inhibited ZEN production by about
64% after 10 days storage. However, after 20 days storage, 200 ppm PTS was required for 96% ZEN
control. Overall, the most efficient PTS concentrations for the control of both toxins (DON, ZEN) was
100 ppm PTS in the 0.93 aw stored wheat, and 200 ppm for the wetter stored wheat (0.95 aw). PTSO at
40 and 80 ppm reduced ZEN contamination of stored wheat inoculated with F. graminearum at both
0.93 and 0.95 aw by 30–60% after 20 days storage. Statistically, the Kruskal–Wallis analyses (non-
parametric analyses) showed that there was no significant effect of PTS concentration or grain aw on
ZEN production, while storage time was highly significant. For PTSO, concentration or storage aw
were not significant for ZEN production, while storage time was highly significant.
Figure 6 shows the T-2 + HT-2 toxins in stored oats inoculated with F. langsethiae and treated
with 0–300 ppm PTS and 0–80 ppm PTSO for up to 20 days. The red line shows the indicative directive
withFigure
regard5.toEffect
limitsofmore commonly
(a) 0–300 ppm PTS established in Europe
and (b) 0–80 ppm PTSO for theonsum of T-2 + HT-2
zearalenone toxinsby[13]
production F. (EC
Figure 5. Effect of (a) 0–300 ppm PTS and (b) 0–80 ppm PTSO on zearalenone production by F.
165/2013). In all cases,
graminearum the toxin
in artificially levels produced
inoculated stored wheat wereat below
0.93 and the0.95
indicative
aw for 10levels
and 20suggested
days at 25 by the
graminearum in artificially inoculated stored wheat at 0.93 and 0.95 aw for 10 and 20 days at 25 °C (n =
C (n = 2 × 3).
EU. ◦Statistical analyses
Verticalshowed that the
bars indicate the standard
effects of PTSof concentration,
error the means. The and storage
red lines showtimethe EUhad no
2 × 3). Vertical bars indicate the standard error of the means. The red lines show the EU legislative
significant effect
legislative limits on log(T-2
(EC. +
1881/2006) HT-2) toxin
for zearalenone contamination
in unprocessed of stored
wheat foroats
limits (EC. 1881/2006) for zearalenone in unprocessed wheat for human use (100 µg/kg). Kruskal–
human by F.
use langsethiae
(100 µg/kg). (see
Kruskal–Wallis
Supplementary TableANOVA foreffect
Statistical weffects for PTS concentration: H×(3, N = 48) = 1.39, p =significant.
0.707,
Wallis ANOVA forS3) The
Statistical of afor
effects , PTS
andconcentration:
interaction between
H (3, N =aw48) storage
= 1.39, p =time were
0.707, grain a w: H
grain
For PTSO, aw : H
ANOVA (1, N = 48)
showed= 3.44,
that p = 0.064, storage
concentration time:
and a H (1, N = 48) = 12, p < 0.001; For PTSO
w had significant effects on the production of conc.:
(1, N = 48) = 3.44, p = 0.064, storage time: H (1, N = 48) = 12, p <0.001; For PTSO conc.: H (2, N = 36) =
theseH0.47, = 36) = 0.47, p =
(2, Ntoxins
two H (1, N = 36)
0.789, aw : of = 1.48, p =interactions
0.223, storagebetween
time: H (1, = 36) = 21.34,
N factors
p = 0.789, while the
aw: H (1, N =effect
36) = 1.48,storage
p = 0.223,time and
storage time: H (1, N = 36) = 21.34, the
p <0.001. were not
p < 0.001.
significant.
Figure 5 shows the effect of PTS and PTSO on ZEN production by F. graminearum in stored wheat.
The most effective control of ZEN production by PTS was 100ppm at 0.93 aw with 89% control after
20 days storage. In the wetter 0.95 aw grain 100 ppm of PTS inhibited ZEN production by about
64% after 10 days storage. However, after 20 days storage, 200 ppm PTS was required for 96% ZEN
control. Overall, the most efficient PTS concentrations for the control of both toxins (DON, ZEN)
Toxins 2019, 11, 495 7 of 16

was 100 ppm PTS in the 0.93 aw stored wheat, and 200 ppm for the wetter stored wheat (0.95 aw ).
PTSO at 40 and 80 ppm reduced ZEN contamination of stored wheat inoculated with F. graminearum
at both 0.93 and 0.95 aw by 30–60% after 20 days storage. Statistically, the Kruskal–Wallis analyses
(non-parametric analyses) showed that there was no significant effect of PTS concentration or grain aw
on ZEN production, while storage time was highly significant. For PTSO, concentration or storage aw
were not significant for ZEN production, while storage time was highly significant.
Figure 6 shows the T-2 + HT-2 toxins in stored oats inoculated with F. langsethiae and treated with
0–300 ppm PTS and 0–80 ppm PTSO for up to 20 days. The red line shows the indicative directive
with regard
Figure 5.toEffect
limitsofmore commonly
(a) 0–300 ppm PTS established
and (b) 0–80in Europe
ppm PTSO for on
thezearalenone + HT-2 toxins
sum of T-2production by F. [13]
(EC 165/2013).
graminearum in artificially inoculated stored wheat at 0.93 and 0.95 aw for 10 and 20 days at 25 °C (nby
In all cases, the toxin levels produced were below the indicative levels suggested = the
EU. Statistical analyses
2 × 3). Vertical barsshowed
indicatethat the effectserror
the standard of PTS concentration,
of the means. The red andlines
storage
show time
the had no significant
EU legislative
effect limits
on log(T-2 + HT-2) toxin
(EC. 1881/2006) for contamination
zearalenone in unprocessed by F. for
of stored oatswheat langsethiae
human (see Supplementary
use (100 Table S3)
µg/kg). Kruskal–
The effect of a
Wallis ANOVAw , and interaction between a × storage time were significant. For PTSO, ANOVA
for Statistical effects for PTSw concentration: H (3, N = 48) = 1.39, p = 0.707, grain aw: H
showed that
(1, N concentration
= 48) andstorage
= 3.44, p = 0.064, aw had significant
time: H (1, N =effects onpthe
48) = 12, production
<0.001; For PTSO ofconc.:
theseHtwo toxins
(2, N = 36) while
=
the effect
0.47, of
p =storage
0.789, awtime and
: H (1, N =interactions
36) = 1.48, p =between the factors
0.223, storage time: Hwere
(1, Nnot
= 36)significant.
= 21.34, p <0.001.

Figure
Figure6. 6. Effect
Effect of
of (a)
(a) 0–300
0–300 ppm
ppm aqueous
aqueous PTS
PTS and
and (b)
(b) 0–16
0–16 ppm
ppm PTSO
PTSO on
on T-2+HT-2
T-2+HT-2 toxin
toxin production
production
by F. langsethiae
by F. langsethiae in artificially inoculated oats of 0.93 and 0.95 aw and stored for 10 and 20
in artificially inoculated oats of 0.93 and 0.95 a w and stored for 10 and 20 days
days at
at 25
25
◦ C. Vertical bars indicate the standard error of the means. The red lines show the EU recommended
°C. Vertical bars indicate the standard error of the means. The red lines show the EU recommended
for
for maximum
maximum limits limits (EC.
(EC. 165/2013)
165/2013) for
for sum
sum T-2
T-2 and
and HT-2
HT-2toxin
toxin in
inunprocessed
unprocessed oat
oat for
for human
human useuse
(1000 µg/kg).
(1000 µg/kg).

Figure 7 shows the effect of treatments on the production of total fumonisins B1 + B2 (FUMs)
Figure 7 shows the effect of treatments on the production of total fumonisins B1 + B2 (FUMs) in
in maize rewetted to 0.91 and 0.94 aw and inoculated with F. verticillioides spores and treated with
maize rewetted to 0.91 and 0.94 aw and inoculated with F. verticillioides spores and treated with 0–300
0–300 ppm PTS, Only PTS was studied in these assays because of the limited efficacy of PTSO in
ppm PTS, Only PTS was studied in these assays because of the limited efficacy of PTSO in controlling
controlling FUMs production in vitro (see Figure 3). The red line shows the EU legislative limits
FUMs production in vitro (see Figure 3). The red line shows the EU legislative limits established for
established for the sum of FUMs in maize. At 0.91 aw PTS was effective with up to 80% control achieved
the sum of FUMs in maize. At 0.91 aw PTS was effective with up to 80% control achieved after 20 days
after 20 days with 200–300 ppm treatment. This was also below the relevant EU limit. However,
with 200–300 ppm treatment. This was also below the relevant EU limit. However, in wetter maize
in wetter maize stored at 0.94 aw most treatments were ineffective in controlling FUMs contamination.
stored at 0.94 aw most treatments were ineffective in controlling FUMs contamination. Indeed there
Indeed there was a stimulation in FUMs, especially with 100–200 ppm PTS, regardless of aw or storage
was a stimulation in FUMs, especially with 100–200 ppm PTS, regardless of aw or storage time. There
time. There was a statistically significant effect of PTS concentration, maize aw and storage time on the
was a statistically significant effect of PTS concentration, maize aw and storage time on the logarithm
logarithm of total FUMs production (see Supplementary Table S3). The interaction of aw ×storage time
of total FUMs production (see Supplementary Table S3). The interaction of aw×storage time and aw ×
and aw × PTS concentration were also significant. However, the interactions of PTS × storage time
and interaction between all three factors was not significant. For PTSO, none of the single factors or
interacting factors were significant except for aw × storage time was significant.
Toxins 2019, 11, x FOR PEER REVIEW 8 of 16

PTS concentration were also significant. However, the interactions of PTS × storage time and
Toxins 2019, 11,between
interaction 495 8 of or
all three factors was not significant. For PTSO, none of the single factors 16

interacting factors were significant except for aw × storage time was significant.

Figure 7. Effect of 0–300 ppm aqueous PTS on total fumonisins production by F. verticillioides in
Figure 7. Effect of 0–300 ppm aqueous PTS on total fumonisins production by F. verticillioides in
artificially inoculated maize stored at 0.91 and 0.94 aw for 10 and 20 days at 25 ◦ C. Vertical bars indicate
artificially inoculated maize stored at 0.91 and 0.94 aw for 10 and 20 days at 25 °C. Vertical bars indicate
the standard error of the means. The red lines show the EU legislative limits (EC. 1881/2006) for
the standard error of the means. The red lines show the EU legislative limits (EC. 1881/2006) for
fumonisins (FB1 + FB2 ) in unprocessed maize for human use (2000 µg/kg).
fumonisins (FB1 + FB2) in unprocessed maize for human use (2000 µg/kg).
3. Discussion
3. Discussion
3.1. In Vitro Efficacy of PTS and PTSO
3.1. In Vitro Efficacy of PTS and PTSO
This is the first study to evaluate the efficacy of the garlic-derived compounds PTS and PTSO for
controlThisof is the first
fungal growthstudy andto mycotoxin
evaluate the efficacy of
production bythe garlic-derived
isolates of the three compounds
important PTS and PTSO
mycotoxigenic
for control
Fusarium of in
species fungal
vitro. growth
The present andstudymycotoxin
has shown production
that solutions by ofisolates
either PTS of or the
PTSOthree
were important
effective
mycotoxigenic
at inhibiting theFusarium species of
in vitro growth in each
vitro.isolate
The present
of the study has shown
three Fusarium that solutions
species studied.ofThe either PTS or
inhibitory
PTSO were effective at inhibiting the in vitro growth of each isolate
effect increased with concentration and generally, the level of inhibition was species-dependent. F. of the three Fusarium species
studied. The
langsethiae wasinhibitory
the mosteffect increased
sensitive Fusariumwithspecies
concentration
to both and generally,
compounds the complete
with level of inhibition
inhibition was
of
species-dependent.
growth with 100 andF.200 langsethiae
ppm of PTS wasand the PTSO
most sensitive
respectively.Fusarium species to both compounds with
complete inhibition
Our results of growth
suggest with 100
that there and 200 ppm
is differential of PTS and
sensitivity PTSO respectively.
of Fusarium species to PTS and PTSO.
Thus,Ourhigherresults
ED50suggest that there
concentrations is differential
of the sensitivity
two garlic-derived of Fusarium
compounds werespecies
required to for
PTStheand PTSO.
isolate of
Thus,
F. higher ED
verticilloides 50 concentrations
when compared to of the two garlic-derived
F. gaminearum compounds
and F. langshetiae. were required
Previously, 200 ppmfor ofthe
starisolate
anise
of F. verticilloides
extract was neededwhen compared
to inhibit growthto ofF. gaminearum completely
F. verticillioides and F. langshetiae. Previously,
when compared 200 ppm
to effects on F.ofsolani,
star
anise
F. extract and
oxysporum wasF.needed
graminearumto inhibit
wheregrowthonly of
100F.ppmverticillioides
was requiredcompletely when compared
[14]. Similarly, Morcia et to al.
effects
[15]
on F. solani,
observed F. oxysporum
lower ED50 values and F.
ofgraminearum
seven EOs were where only 100
needed ppm
in the wasofrequired
case [14]. Similarly,
F. langsethiae when comparedMorcia
et al.
to [15] on
effects observed lower EDand
F. graminearum 50 values of seven EOsHowever,
F. sporotrichioides. were needed most inofthe casestudies
these of F. langsethiae
did not assesswhen
compared towith
interactions effects
wateron F. graminearum
availability whichandisF.critical
sporotrichioides. However,
for determining most especially
efficacy, of these studies did not
in relation to
assesscontrol
toxin interactions
[14–22]. with water
It is availability
also difficult which isthe
to compare critical for determining
dose-effect relationships efficacy, especially
obtained between in
relationacross
species to toxin thecontrol
studies[14–22].
becauseItdifferent
is also difficult
times and to compare
doses, and thedifferent
dose-effect relationships
matrices were used, obtained
often
between species
nutritionally acrosstothe
unrelated thestudies
commodity because different
in which times
in situ and was
control doses, and different matrices were
required.
used,Inoften nutritionally
addition, differentunrelated
EO extracts to have
the commodity in which
different active in situ control
ingredients and thus was dorequired.
not have the same
In addition,
efficacy against the different EO extracts
same species. For have different
example, activeofingredients
the effect EOs belonging and thus do botanical
to five not have the same
families
efficacy against the same species. For example, the effect of EOs belonging
(Umbeliferae, Labiateae, Compositeae, Rosaceae and Lauraceae) against F. moniliforme (=F. verticillioides) to five botanical families
(Umbeliferae,
was evaluated [16]. Labiateae,
Growth Compositeae,
was completely Rosaceae
inhibited and Lauraceae)
with 500 ppm of against
anise, F. thymemoniliforme ( = F.
and cinnamon,
verticillioides)
2000 was evaluated
ppm of marigold, and 3000 [16].
ppm Growth was completely
of the other inhibited
extracts examined. with 500
Overall, ppm of anise,
no significant thyme
differences
and cinnamon,
between 2000 ppm
these botanical of marigold,
families were found.and 3000Also,ppm of theinhibition
complete other extracts
of thisexamined. Overall,was
Fusarium species no
significant
achieved differences
with 600 ppm between
of thyme,these 800 ppm botanical
of basilfamilies were found.
and lemongrass andAlso, complete
1000–2500 ppminhibition of this
of ginger [17,18].
Fusarium
These species was
differences couldachieved
be duewith to the 600 ppm of tolerances
different thyme, 800between
ppm of basil andbut
strains, lemongrass
also becauseand of 1000–
the
2500 ppmcompositions
different of ginger [17,18]. These of
and purity differences
EOs obtained couldfrombe duethe to the different
different plants.tolerances
Elhouiti etbetween strains,
al. [23] studied
the chemical composition of leaves and flowers of Rhanterium adpressum, harvested at different times
over three years. They observed that the percentage of the leading chemical groups changed according
Toxins 2019, 11, 495 9 of 16

to the month of extraction. Also, the EOs produced by the flower had better inhibitory activity than
the leaf extracts (MICs, 6–10 ppm vs 11–14 ppm) against F. culmorun and F. graminearum respectively.
Kurita and Koike et al. [24] pointed out that the magnitude of the antifungal effect was related to
the functional groups and they proposed a scale of antifungal potency of chemical groups from the
best to worst being phenols > alcohols > aldehydes > ketones > ethers > hydrocarbons. However,
these previous studies did not evaluate the impact that changing water availability might have on the
relative control achieved.
In terms of control of mycotoxin production, both PTS and PTSO reduced DON, ZEN, T-2, HT-2
and FUMs production by the relevant Fusarium species when compared to the controls. There were
some differences in efficacy between PTS and PTSO. The latter compound only controlled FB1 and
FB2 production at 200 ppm, with >4 times the amount of PTS required, compared to PTSO to obtain
the same inhibitory effects. Generally, PTS was more effective in controlling mycotoxin production
than PTSO, except in the case of T-2 toxin where the latter compound was more effective at <200 ppm.
Similarly, HT-2 toxin was completely inhibited at all PTSO concentrations, while for PTS ≥50 ppm
was required. It was also generally observed that higher PTS concentrations (200 ppm) completely
inhibited the production of all toxins, except in the case of the FUMs.
Previous studies have suggested that complete inhibition of FUMs could be achieved with
6 ppm of 3-carene, D-limonene and B-ocimene [21]. Also, ginger EOs inhibited FB1 (2500 ppm),
FB2 (2000 ppm) and DON (2000 ppm) production [17,22]. Lower doses of extracts of R. adpressum
(0.25 ppm) significantly inhibited production of type B tricothecenes (3-acetyl deoxynivalenol, 15-acetyl
deoxynivalenol and fusarenon X) [23]. Morcia et al. [15] reported that the seven EOs (cuminaldehyde,
cinnamaldehyde, lemon oil, citral, limonene, bergamot and citroneall) had a variable effect on the
biosynthesis of T-2 and HT-2 by F. langsethiae and F. sporotrichioides. 0.1 mL of bergamoil/mL reduced
T-2 and HT-2 toxin produced by the former species but stimulated production by the latter species.
However, again, few of these studies included the impact of aw stress on the efficacy of the EOs.

3.2. In Situ Efficacy of PTS and PTSO against Mycotoxin Production in Stored Grain
The aw levels chosen for the wheat, oats and maize storage studies was based on the marginal and
optimum aw levels for growth and mycotoxin production by the isolates of these three species from
previous studies [25–27]. Drier conditions of <0.90 aw are considered very marginal for colonisation by
Fusarium species of wheat, oats or maize. Treatment of wheat with 100 ppm of PTS resulted in 76–94%
control of DON, depending on the storage aw . Better results were obtained in the 0.93 aw treatment
where colonisation was slower than in the 0.95 aw treatment. PTS was also effective in controlling
ZEN production, by reducing the production to below or near the applicable EU limits after both 10
and 20 days storage respectively. However, it should be noted that under some storage conditions
intermediate PTS concentrations stimulated mycotoxin production.
Overall, PTS was not efficient in controlling the production of T-2 and HT-2 toxins in artificially
inoculated oats at 0.93 aw , particularly in samples stored for 20 days. In contrast, at 0.95 aw reasonably
good control with 100 ppm PTS for T-2 and HT-2 production was achieved after 10 days storage,
and with 200 ppm after 20 day storage. For control of FUMs, 300 ppm PTS was necessary in stored
maize at 0.91 aw and 0.94 aw resulting in 39–80% inhibition when compared to the control samples.
The control achieved was below the EU legislative limits but only in the 0.91 aw treatments. In moist
maize, much higher concentrations would be required to try and control FUMs to below the EU
legislative limits, especially if destined for feed use.
Treatment of wet grain with PTSO was generally capable of reducing mycotoxin contamination
after storage at 0.93–0.95 aw for 10–20 days compared to the untreated control samples. Thus, where
water ingress might occur in a silo this compound would be effective at controlling toxin contamination
in stored cereals produced by Fusaria, in the short to medium term. DON contamination in wheat
treated with 80 ppm PTSO and stored for 10 days had 1/3rd less toxin than the control, which was also
below the relevant EU limits. However, this treatment was not efficient for extended storage beyond
Toxins 2019, 11, 495 10 of 16

20 days with the exception of ZEN, which was more effectively reduced, even after 20 days storage.
Better control was observed with 40 ppm PTSO (48–60%) in relatively moist wheat stored at 0.95 aw .
However, all the treatments contained ZEN levels above the applicable EU limits.
Overall, PTSO was more efficient in controlling the production of T-2/HT-2 toxins by F. langsethiae
in oats, with as little as 16 ppm required. This treatment was generally more effective against HT-2
toxin (8–78%) than T-2 toxin (18–42%) and this may be important, as often T-2 toxin is converted rapidly
to HT-2 toxin and thus control of this toxin is important.
Previously, Soliman et al. [16] examined the efficacy of low concentrations (0.1–2.0 ppb) of the
more efficient EOs tested in vitro (anise, cinnamon, spearmint and thyme) on FUMs contamination
of stored wheat over 8 week storage periods. They claimed that low doses (0.1 ppb) completely
inhibited the biosynthesis of FUMs after 2 weeks. Thyme EO was shown to have the highest
anti-mycotoxigenic activity and the best control of growth. Although water availability was not
considered, the concentrations appear to be very low for achieving control. In addition, F. verticillioides
colonisation is more important in maize where it is primarily responsible for FUM contamination.
Venkatesh et al. [21] suggested that use of guggul EO at 10 ppm treatment of maize at 28 ◦ C for 10 days
reduced FUMs from 42.5 to 2.6 ppm; with complete inhibition of FB1 achieved with 200 ppm of star
anise or 50 ppb of allyl isothiocyanate [14,28]. Allyl isothiocyanate was found to inhibit the production
of FB1 by F. verticilloides [2,28].
These cereals are naturally contaminated with a mixture of toxigenic fungi as part of the mycobiota.
Thus, the differential effect on different Fusarium species would also apply to other toxigenic species
such as Penicillium verrucosum (ochratoxin A producer) or Aspergillus species. Thus, consideration
should be given to changes in the ratio of mycotoxins which might occur when treated with different
preservatives. Recently, Giorni et al. [29] showed that in ripening maize co-inoculated with mixtures of
F. graminearum, F. verticillioides and Aspergillus flavus influenced the relative contamination of the maize
cobs with deoxynivalenol, FUMs and aflatoxins. Interactions between non-toxigenic mycobiota and
has also been shown to influence the relative contamination with different mycotoxins in both wheat
grain and in grape-based matrices [30].
The in situ storage studies have been done for a maximum of 20 days. For longer term storage
periods of 6–9 months it may be necessary to use a slightly increased treatment concentration of such
compounds for ensuring that control can be maintained. This would have an impact on the relative
economic costs of treatment which would have to be considered in the context of the overall inputs
into management of grain for food and feed use post-harvest.

4. Conclusions
This was the first detailed examination of these two compounds for control of growth and
mycotoxin contamination by Fusarium species in vitro and in artificially inoculated stored wheat, oats
and maize under different temporal and water availability conditions. Overall, in vitro efficacy should
include important parameters such as water availability, temperature and perhaps pH stress to identify
the most effective candidates for control of colonisation, and more importantly, mycotoxin production.
Potential efficacy was demonstrated and identified against isolates of three different Fusarium species.
However, efficacy in situ was not as effective as that observed in vitro. In addition, the efficacy of the
treatments depended on the specific “Fusarium species-toxin” pathosystem. Differences were observed
with regard to the production of different mycotoxins by an isolate of a single fungal species when
inoculated into naturally contaminated cereals. However, the right concentrations need to be used
for effective control to be achieved. This depended on the water availability and the mycotoxigenic
species involved.
Both garlic-derived compounds tested in this study (PTS and PTO) are liquids and water soluble
and can thus be applied to grain prior to storage for post-harvest control of spoilage mycotoxigenic fungi.
Garlic extracts have been approved for use as a pesticide (although currently under re-evaluation) and
commercial products are available based on such extracts. Certainly, the use of odourless versions of
Toxins 2019, 11, 495 11 of 16

these compounds could be effectively used for food applications. In addition, the existing compounds
could also be used for animal feed applications where often moist grain needs to be preserved for the
short to medium term prior to use.

5. Materials and Methods

5.1. Preparation of Stock Solutions of Chemical Compounds

Stock solutions of the following compounds were prepared in sterile distilled water.
(a) Propyl propane thiosulfonate (PTS): This organosulfonate compound is obtained from the
decomposition of initial compounds present in garlic bulbs (Allium sativum) and was kindly provided
by DOMCA SA, Granada, Spain. A stock solution of 5000 ppm was prepared by dissolving 1.1 g PTS
(90% PTS, Domca, S.A., Granada, Spain) (Mousala SL., 2006) into a 200 mL container containing 200 mL
of sterile distilled water and vigorously shaking. Due to the oily nature of PTS the stock solution had
the appearance of a stable water emulsion. A second stock solution of 20000 ppm was prepared by
dissolving 2.2 g PTS into 100 mL of a mixture of ethanol:H2 O (80:20). This solution was clear.
(b) Propyl propane thiosulfinate (PTSO): A stock solution of 10000 ppm was prepared by dissolving
1.1 g PTSO (90% PTSO, Domca, S.A., Granada, Spain) (Mousala SL., 2006) into a 100 mL container
containing 100 mL sterile distilled water and vigorously shaking. Due to the oily nature of PTSO the
stock solution had the appearance of a water emulsion. A second stock solution of 20,000 ppm was
prepared as for PTS.

5.2. In Vitro Studies: Fungal Species, Media, Inoculation and Measurements of Growth and Mycotoxin Production
Fusarium graminearum isolate L1-2/2D (wheat; DON, ZEN), F. langsethiae strain 2004/59 (oats;
T-2 + HT-2) and F. verticillioides isolate MPVP 294 (maize; FUMS) were used in this study. The strains
were all maintained on Malt Extract Agar (MEA) media (OXOID, malt extract, 30; mycological peptone,
5; agar, 15 g/L). These isolates were kindly supplied by Prof. S. Edwards, Harper Adams University
and Prof. P. Battilani, Catholic University of Italy, Piacenza, Italy). They have all been examined
previously for mycotoxin production and shown to be high producers of the respective mycotoxins in
previous studies [31,32].
For the in vitro trials a 2% milled wheat medium was prepared by adding 2% milled wheat and 2%
agar (OXOID Ltd, Basingstoke, England) to water to obtain the basal medium. For the initial screening,
concentrations of PTS and PTSO in the range 10–200 ppm were used by adding the necessary stock
solutions to the molten cooled medium, shaking vigorously and then pouring the media into 9 cm
Petri plates (15 mL per plate). The basal 2% media had a water activity (aw ) value of 0.995. This basal
medium was modified by replacing water with different amounts of mixtures of glycerol/water solution
to the milled cereal + agar to obtain the target aw values of 0.92, 0.94 and 0.98 (20.7, 15.4 and 4.9 g
glycerol/50 mL of water, respectively) without diluting the nutritional status of the media. These aw
levels represent the range over which these Fusaria can effectively grow [33]. The media were all
checked with an Aqualab TE4 to confirm the actual aw levels were achieved.
Agar plugs (4 mm diameter) cut from the margin of 10-day-old cultures with a sterile cork borer,
were used as an inoculum for the in vitro trials. Three replicate per treatment and replicate plates were
centrally inoculated with the inoculum agar plugs. All experiments were repeated once. Each aw
treatment and replicates were stored in separate polyethylene bags to maintain the environmental
conditions over the experimental period.
The treatments and replicates were all incubated at 25 ◦ C for 10 days, or until the Petri dishes
were completely colonised by the fungi. Each concentration of the PTS and PTSO treatments were kept
separately in polyethylene bags to avoid cross-contamination. Two diameters of the colonies formed
(at right angles of each other) were measured daily and compared against the diameters of the controls.
From these data the relative growth rates were calculated and the effect of different concentrations was
Toxins 2019, 11, 495 12 of 16

calculated. The percentage inhibition of mycelial growth of the Fusarium species was determined at
each different chemical compound concentration and the different water activities.
For mycotoxin analyses on the tenth day of incubation, agar plugs (5, 5-mm diameter) were cut
out from each of the replicate plates diagonally across the colony. The agar plugs were placed in 2-mL
safe-lock Eppendorf®tubes (Eppendorf AG, Hamburg, Germany), their weight was recorded and
frozen at −40 ◦ C for subsequent toxin analysis. The extraction and analysis of the relevant toxins for
each fungal species were performed according to the methods described later.

5.3. In Situ Studies with Stored Cereal Grain

Fungal inoculum: Cultures of the above fungal species were prepared on MEA and incubated
at 25 ◦ C for 10 days. A Tween 80 solution was prepared by addition of one drop of Tween 80
(ACROS organics) in 100 mL sterile water. Spore suspensions were prepared by gently scraping the
culture surface with a sterile spatula and transferring the spores into sterile 25 mL Universal glass
vials containing the water + Tween 80 solution. The spore suspensions were filtered through glass
wool in order to remove any mycelial fragments. The spore concentration was determined using a
haemocytometer (Olympus BX40 microscope, Microoptical Co.; slide Marienfeld superior, Germany;
microscope glass cover slips, No 3, 18 × 18mm, Chance Proper LTD, Smethwick, UK) and adjusted by
dilution to 107 spores/mL. Naturally contaminated wheat, oats and maize were artificially inoculated
with these suspensions for the in situ storage experiments.
Grain equilibration: Water adsorption curves were prepared for each grain type. The amount of
water required to accurately modify these cereals to 0.91, 0.94 (maize) and 0.93 and 0.95 aw (wheat, oats)
was determined from these curves. Initially, each grain type (approx. 1 kg) was taken from a 25-kg
bag and placed in a Duran bottle (2.5 L). The initial moisture content was known from the moisture
adsorption curves. The grain was randomly divided into batches of 100 g in Duran flasks (1 L). These
were labeled for each treatment condition and the required amounts of sterile water added to each one
including the treatment PTS or PTSO stock solution, shaken vigorously and sealed. They were placed
at 4◦ C to equilibrate overnight. The treatments were then inoculated with 1-mL spore suspension
containing ~105 spores/mL of the individual Fusarium species and thoroughly mixed using a roller
mixer in order for the spores to become dispersed throughout the grain mass. For each grain type, 15 g
was weighed into surface sterilized 40 mL vials (Chromacol Ltd., London, UK) with microporous lids to
obtain six replicates per aw treatment and stored in sandwich boxes for up to 20 days. The equilibrium
relative humidity (ERH) was maintained by including 2 × 500 mL of a glycerol-water solution in
beakers to maintain the treatment aw levels. The experiments were carried out twice with six replicates
per treatment.
For each experiment, after 10 and 20 days storage, three replicates were destructively removed
from storage chambers and frozen at −20 ◦ C for subsequent toxin analysis. Grain samples were
oven-dried for 24–48 h at 60 ◦ C, milled and then extracted and analysed as described later.

5.4. Mycotoxin Analyses

5.4.1. Equipment Description

High performance liquid chromatography (HPLC) were used to quantify DON, T-2 and HT-2
from media. In addition, liquid chromatography tandem mass spectrometry (LC-MS/MS) were
used to quantify FUMS from media and Fusarium toxins from grains. HPLC used consisted of an
Agilent 1200 Series system equipped with a UV diode array detector (DAD) set at 220.4 nm (Agilent
Technologies, Palo Alto, CA, USA). The column used for the chromatographic separation was a
Phenomenex® Gemini C18 , 150 mm × 4.6 mm, 3 µm (Phenomenex, Macclesfield, UK) preceded by a
Phenomenex® Gemini 3 mm guard cartridge and the column temperature was set at 25 ◦ C. LC-MS/MS
used consisted of an QTrap 5500 LC-MS/MS System (Applied Biosystems, Foster City, CA, USA)
equipped with a TurboIonSpray electrospray ionization (ESI) source and an 1290 Series HPLC System
Toxins 2019, 11, 495 13 of 16

(Agilent, Waldbronn, Germany). Chromatographic separation was performed at 25 ◦ C on a Gemini®

C18 -column, 150 × 4.6 mm i.d., 5 µm particle size, equipped with a C18 4 × 3 mm i.d. security guard
cartridge (all from Phenomenex, Torrance, CA, USA).

5.4.2. In Vitro Extraction and Analysis

Agar plugs were removed from media and placed in the 2-mL safe-lock Eppendorf®tubes. After
that, the weight of the agar plugs were register.

Deoxynivalenol
The extraction was performed using 1-mL acetonitrile:water (AcN:H2 O) (84:16), the mixture more
commonly used for trichothecenes extraction [34] and the tubes were shaken in an orbital shaker at
200 rpm in the dark for 60 min at 25 ◦ C. The extract was transferred to a new tube and oven-dried
overnight at 60 ◦ C. Subsequently, it was redissolved in 1 mL 90:10 (H2 O: AcN) and vortexed for a few
seconds. The cleaning step involved the addition of 150 mg/mL Alumina directly into the redissolved
extract followed by vortexing the mixture for 15 s. The treated extract was then filtered through a
0.22 µm Millipore filter (Minisart, Sartorius, Germany) into an amber silanised LC vial and inserted
into the LC-DAD for analysis. The chromatographic analysis was performed in the gradient mode,
using water (solvent A) and acetonitrile (solvent B). The starting composition of the mobile phase was
5% B, at a flow rate of 0.5 mL/min held for 2 min. The composition was then gradually changed to
25% B over 15 min and maintained for further 3 min. Then it increased gradually to 30% B over 3 min
at the same flow rate. The composition was then changed to 99% B during 1 min with a flow rate of
1 mL/min in this case, in order to achieve a fast cleaning step and maintained at 99% B for 4 more
minutes. Afterwards the composition of the mobile phase was changed linearly to 5% B in 1 min at a
flow rate of 1 mL/min and held for 4 min for further cleaning. In the last step, the composition was
maintained at 5% B, but the flow rate changed to 0.5 mL/min for 1 min, in order to be the same as the
starting composition of the mobile phase for the following chromatographic run. The injection volume
was 50 µL. The total time for the analysis of each sample was 35 min. DON was eluted from the column
at 16.2 min. The LOD was 4 µg/kg. The mean recovery for DON using this method was 63.2 ± 2.8%.

T-2 and HT-2

Extraction method used was Medina et al. [32] with modifications. The extraction was performed
using 1 mL acetonitrile:water (AcN:H2 O) (84:16) and the tubes were shaken for 1 h at 150 rpm at 25 ◦ C
in the dark in an orbital shaker. The samples were then centrifuged at 1150× g for 15 min. The extract
was filtered through a 0.2 µm Millipore filter (Minisart, Sartorius) directly into an HPLC silanised
amber vial and injected in the chromatograph. The analysis was performed in the gradient mode
with a mobile phase of AcN:H2 O at a flow rate of 1 mL/min and the conditions were 3 min 30% AcN,
changed linearly to 55% AcN over 18 min, changed to 99% AcN in 1 min and held to 99% AcN for
5 min. The LOD was 4 and 5 µg/kg for T-2 and HT-2. The mean recoveries for this method were
99 ± 1.53% for T-2 toxin and 101.28 ± 3.11% for HT-2 toxin.

Fumonisins
The extraction was performed using 1 mL of extraction solvent AcN:H2 O:Acetic acid (79:20:1)
and the tubes were shaking 250 rpm in the dark for 1 h. The extracts were filtered through a 0.22 µm
Millipore filter (Minisart, Sartorius) into new tubes, and dried in an oven at 60 ◦ C for 24 h. The dried
extracts were redissolved in a mixture of AcN:H2 O (1:1) containing 1% acetic acid. The individual
fumonisins were quantified using LC-MS/MS according to the method of Vishwanath et al. [35]. LOD
was 25 µg/kg with a recovery rate of 57% (FB1 ) and 70% (FB2 ).
Toxins 2019, 11, 495 14 of 16

5.4.3. In Situ Extraction and Analysis

The initial mycotoxin contamination levels were quantified and this was taken into account when
calculating the results of the treatments and appropriately corrected. The mean contamination levels
were: DON, 0.233 µg/kg; T-2 toxin, 9.07 µg/kg (no HT-2 toxin present); ZEN, 8.42 µg/kg, and FB1
0.14 µg/kg. The grain samples were oven-dried at 60 ◦ C for 24–48 h and then milled in a small
laboratory blender (Waring Commercial, Christison, UK). Samples were analysed for Fusarium toxins
by LC-MS/MS at the Centre for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln,
Tulln, Austria), University of Natural Resources and Life Sciences, Vienna, Austria. The analysis
was performed according to the methods described by Sulyok et al. and Vishwanath et al. [35,36].
The accuracy of the method was externally checked by participation in proficiency testing organised
by the Bureau Inter Professionel d’Etudes Analytiques (BIPEA; Gennevilliers, France). Z-scores were
0.4 and 0.62 for DON in two wheat samples, −0.8 and −1.09 for ZON in two wheat samples and 1.36
and 1.55 for FB1 and FB2 , respectively in a sample of maize.

5.5. Statistical Analysis

All experiments have been performed in triplicate and repeated once. Data were analysed with
Microsoft Office Excel 2007 and with the package STATISTICA 9 (StatSoft® , Inc. 2010. STATISTICA
(data analysis software system), version 9.1. www.statsoft.com, (Tulsa, OK, USA). The standard error
of the mean was calculated in all trials and it is denoted with vertical bars in the figures.
Datasets were tested for normality and homoscedasticity using the Shapiro–Wilk and Levene test,
respectively. When data failed the normality test, variable transformation was performed to try to
improve normality or homogenise the variances. If still not normally distributed, it was analysed
using the Kruskal–Wallis test by ranks.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6651/11/9/495/s1,

Figure S1: Effect of 0–250 ppm PTSO and PTS on in vitro DON production by F. graminearum in wheat agar media
at 25 ◦ C, Figure S2: Effect of 0–250 ppm (a) PTSO and (b) PTS on the production of T-2 and HT-2 toxins by
F. langsethiae in vitro at 25 ◦ C, Table S1: One-way ANOVA for the effect of different PTS and PTSO concentrations
on in vitro fumonisins (B1 and B2 ) production by F. verticillioides, Table S2: Kruskal-Wallis ANOVA by ranks for the
effect of PTSO concentration and substrate water activity on the production of T-2 and HT-2 toxins by F. langsethiae,
Table S3: Summary of statistical P-values of effects of (a) PTS and (b) PTSO on mycotoxin contamination of
stored cereals.
Author Contributions: K.M. and E.G.-C. carried out the practical work and statistical analyses, M.S. carried
out some of the mycotoxin analyses in naturally contaminated cereals and with preservatives. A.M. and N.M.
supervised the research work and wrote the draft and final manuscript.
Funding: Parts of this work were funded by the European Union via the FP7 MYCORED Project (Grant Agreement
No. 222690).
Acknowledgments: The authors would like to thank S. Edwards, Harper Adams University and P. Battilani,
Catholic University of Italy, Piacenza, Italy) for supplied the strains.
Conflicts of Interest: The authors declare no conflict of interest.

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