Received: 4 December 2019 | Revised: 25 February 2020 | Accepted: 26 February 2020

DOI: 10.1111/jph.12889

ORIGINAL ARTICLE

Assessment of glucosinolate-derived isothiocyanates as

potential natural antifungal compounds against citrus sour rot
disease agent Geotrichum citri-aurantii

Merve Kara | Emine Mine Soylu

Department of Plant Protection, Faculty

of Agriculture, Hatay Mustafa Kemal
University, Hatay, Turkey
Correspondence
Merve Kara, Department of Plant
Protection, Faculty of Agriculture, Hatay
Mustafa Kemal University, 31034 Hatay,
Turkey.
Email: mervekara@mku.edu.tr
Funding information
Mustafa Kemal Üniversitesi, Grant/Award
Number: MKU BAP 13381
Abstract
In this study, the antifungal effects of six different isothiocyanate (ITCs) compounds
(methyl, allyl, butyl, ethyl, benzyl and 2-phenylethyl ITCs) were investigated to be use
against the citrus sour rot disease caused by Geotrichum citri-aurantii in vitro and semi-
commercial (in vivo) conditions. Antifungal activities of the vapour phases of different
ITC compounds were examined on the arthroconidia germination and mycelial growth
of G. citri-aurantii. Mycelial growth of G. citri-aurantii was inhibited in a concentra-
tion-dependent way. The minimum inhibitory concentrations of benzyl, methyl, allyl
and ethyl ITCs on mycelial growth were 0.06, 0.08, 0.10 and 0.10 µl/L, respectively.
Arthroconidia germination of G. citri-aurantii was completely inhibited by benzyl, me-
thyl, allyl and ethyl ITCs at concentrations of 0.05, 0.07, 0.07 and 0.07 µl/L, respec-
tively. Light microscopy observations revealed that the ITC compounds, at completely
inhibiting concentrations, caused considerable morphological changes in the fungal
hyphae. Under in vivo conditions, the average rotting area caused by G. citri-arantii
was inhibited 100% by ethyl, methyl and allyl ITC compounds at concentrations of
8.0, 12.0 and 12.0 µl/L, respectively. Results suggest that ITC’s may be useful and
effective natural antifungal compounds to control the citrus sour rot disease agent.

KEYWORDS
antifungal activity, citrus, Geotrichum citri-aurantii, isothiocyanates

1 | I NTRO D U C TI O N
Sour rot is caused by Geotrichum citri-aurantii and has been reported
as an important postharvest disease of citrus fruits found widely
in orchards (Brown, 1988). Arthroconidia of the pathogen, which
belong to the Saccharomycetes class of the Ascomycotina order,
constitute by infecting the fruit surface via wind or splashing rain.
Also, the fungus generates the most infections on injured fruits and
on fruit located close to the ground. Dropped fruits are suitable for
severe infections due to contact with the soil. Arthroconidia of the
pathogen are also an important source of inoculum for the next year.
The pathogen can enter the fruits from wounds opened by mechan-
ical means or insects (Brown, 2003). Fruit pallets, washing brushes
and other equipment in fruit packing facilities can infect healthy
fruits. The mycelium and arthroconidia which are located in rotten
and soft tissues allow the transmission of the disease from infected
fruits to healthy fruits (Brown, 2003). The characteristic sour smell
of sour rot attracts fruit flies which causes fungi to spread to other
healthy fruits. The first symptoms of sour rot are similar to green
and blue mould rots. Lesions are first water-soaked, later to become
clear-dark yellow in pigment and have a lightly swollen appearance.
The pathogen causes the fading of colour and degrades the fruit to
a slimy and aqueous mass (Brown, 2003; Liu et al., 2009).
Various fungicides have been widely used for a long time to mini-
mize losses occurring in postharvest fruits. The control measures em-
ployed to manage other important postharvest decays are usually not

Journal of Phytopathology. 2020;00:1–11. wileyonlinelibrary.com/journal/jph © 2020 Blackwell Verlag GmbH | 1

2 |
effective against G. citri-aurantii. This pathogen cannot be controlled
by any of the conventional fungicides (e.g. Imazalil and Thiabendazole)
registered for use on citrus fruit (Liu et al., 2009). In most commercial
packing facilities in Turkey, guazatine is commonly applied by posthar-
vest drenchers to reduce the incidence of sour rot in citrus fruits before
processing, or before degreeing operations. However, this fungicide is
not registered in several countries. The adverse effects of these chem-
icals on human health and the environment, and the acquired resis-
tances against some fungicides by disease agents, have brought about
the need for the development of environmentally friendly control al-
ternatives and crop protection methods with or without reduced use
of conventional fungicides (Lima, Curtis, & Cicco, 2008; Sivakumar &
Bautista-Banos, 2014; Soylu & Kose, 2015; Soylu, Kurt, & Soylu, 2010;
Soylu, Özdemir, Ertürk, Şahinler, & Soylu, 2008).
Recent studies showed that Brassica vegetables such as cabbage,
broccoli, cauliflower and radish are rich in numerous health-promoting
compounds such as carotenoids, polyphenols, flavonoids and gluco-
sinolates (GLS), as well as isothiocyanates (ITCs) which can be used for
human health. ITCs are breakdown products of GLS and are typically
used in the food industry as a food preservative and colouring agent
(Jaiswal & Abu-Ghannam, 2016). They are also used in the pharma-
ceutical industry due to their several pharmacological properties
such as antibacterial, antifungal, antiprotozoal, anti-inflammatory and
chemoprotective effects (Mari, Bautista-Baños, & Sivakumar, 2016;
Pal Vig, Rampal, Singh Thind, & Arora, 2009). ITC compounds have
been reported to have antifungal capabilities against soil-borne fun-
gal pathogens (Kurt, Güneş, & Soylu, 2011; Smolinska & Horbowicz,
1999). Although there are few reports on the antifungal activities of
ITC’s against postharvest plant pathogenic fungi such as Botrytis cine-
rea, Penicillium expansum, Alternaria alternata and Monilinia laxa (Mari,
Leoni, Iori, & Cembali, 2002; Mari, Leoni, Bernardi, Neri, & Palmeri,
2008; Troncoso-Rojas, Sánchez-Estrada, Ruelas, García, & Tiznado-
Hernández, 2005; Wu, Zhang, Zhang, Zeng, & Lin, 2011), to our knowl-
edge the in vitro and in vivo antifungal efficacies of the ITC’s against
the postharvest citrus sour rot disease agent have not yet been studied.
The aims of the present study were to determine the antifungal
activities of individual pure aliphatic and aromatic ITC compounds
to different fungal growth parameters such as, mycelial growth
and arthroconidia germination of G. citri-aurantii in in vitro condi-
tions. Moreover, effects of different pure ITC products on disease
incidence were investigated in in vivo conditions. Finally, the mor-
phological changes in hyphae exposed to ITC application were also
determined. To the best of our knowledge, this is the first report on
the use of ITC’s to control the postharvest sour rot of citrus.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Isolation of Geotrichum citri-aurantii and
pathogenicity
Isolation was carried out on mandarin fruits (cv. Owari Satsuma)
which were brought from packaging houses and thought to be
KARA and SOYLU
infected. G. citri-aurantii was isolated from naturally decayed man-
darin (Citrus reticulata cv. Owari Satsuma) fruits that showed typical
sour rot symptoms. Cultures with elongated cells that fragmented
into numerous conidia with no mycelial clusters present were puta-
tively considered to be G. citri-aurantii (McKay, Förster, & Adaskaveg,
2012). A pure culture of this fungal isolate obtained from a single
spore was grown on Potato Dextrose Agar (PDA, Merck). The isolate
was maintained on PDA medium at 4°C.
To confirm the identity of the fungus, pathogenicity tests
were conducted using the susceptible mandarin fruits (cv. Owari
Satsuma). The inoculum consisted of aqueous arthroconidia suspen-
sion obtained from 7-day-old culture plates containing PDA grown
at 25°C. The arthroconidia concentration was determined by a hae-
mocytometer and concentrations were adjusted to 106 conidia/ml in
sterile distilled water. Fruits were inoculated by wounding the equa-
tor (three wounds per fruit) with a sterile cork borer, dipping them
in the pathogen conidial suspension for 1–2 min and allowing them
to air dry for 60 min at room temperature (25°C). As a control, only
sterilized pure water treated fruits were used. Control and inocu-
lated fruits were placed inside disinfected 5-L plastic jars to provide
high relative humidity (±85% RH). Fruits were allowed to incubate
for 5 days at 21°C. A total of 15 fruits were used for each application
in the experiment having three replications and five fruits in each
replicate. Finally, the pathogenicity test was assessed 5 days after
inoculation; and the average disease severity (%), mean softening de-
velopment diameter (mm) and mean mycelial growth diameter (mm)
were recorded.
2.2 | Isothiocyanates (ITCs)
The pure ITCs were obtained from Sigma-Aldrich Chemical Co.,
Switzerland. Methyl ITC (MITC), allyl ITC (AITC), butyl ITC (BUITC)
and ethyl ITC (EITC) represent a series of alkenyl aliphatic ITCs with
increasing side chain length, while benzyl ITC (BITC) and 2-pheny-
lethyl ITC (PEITC) have an aromatic side chain (Table 1). Stock solu-
tions of each ITC were prepared in ethanol.
2.3 | Effects of ITCs on mycelial growth of
Geotrichum citri-aurantii
The antifungal properties of ITCs on the pathogen were tested, as-
sessing their vapour-phase effects. A series of concentrations of the
compounds tested were used to determine the effects of ITCs on
the mycelial growth of G. citri-aurantii by inoculating a 5 mm diam-
eter agar disc from the margins of a one-week-old actively grow-
ing culture of G. citri-aurantii. Agar discs were placed in the centre
of Petri dishes containing 10 ml of fresh PDA, offering 90 ml of air
space as described by Soylu et al. (2010). Different concentrations
of ITCs (0.0–0.14 μl/L air) were added to the filter paper attached
to the lid using a micropipette. Inoculated dishes were sealed im-
mediately with two layers of parafilm and incubated at 24–25°C for
KARA and SOYLU | 3

TA B L E 1 Characteristics of synthetic ITCs used in the experiments

Trivial name of parent Molecular weight

ITC Chemical group glucosinolate (g/mol) Purity (%) Common occurrence

Methyl Aliphatic Glucocapparin 73.11 ˃97 Capparales, metam sodium

(synthetic fumigant)
Allyl Aliphatic Sinigrin 99.15 ˃98 Brassica juncea, B. carinata
B. nigra
Butyl Aliphatic Gluconapin 113.18 ˃99 B. napus, B. campestris,
B. juncea, B. oleracea
Ethyl Aliphatic Glucobrassinapin 127.20 ≥95 B. napus, B. campestris
Benzyl Aromatic Glucotropaeolin 149.20 ≥97 Sinapis alba
2-Phenylethyl Aromatic Gluconasturtiin 163.23 ≥98 B. campestris, B. juncea

5–7 days. Control consisted of unamended Petri plates containing
PDA medium. All tests were performed in triplicate, and each experi-
ment was repeated twice.
The antifungal activity was expressed in terms of percentage of
mycelial growth inhibition and calculated according to the following
formula:
( ) [( ) ]
% Mycelial growth inhibition MGI = FGC −FGA ∕FGC x 100
FGC = Fungal growth diameter in control Petri dishes (mm).
FGA = Diameter of fungal growth in applied Petri dishes (mm).
To determine the fungicidal or fungistatic effects of ITC com-
pounds, lids of the Petri dishes that did not show mycelial growth
were replaced with sterile Petri lids and allowed to incubate again at
24–25°C for 4 days. Activity of the each concentration of the ITCs
was considered as fungicidal if the pathogen did not grow, or fungi-
static if pathogen growth occurred.
2.5 | Determination of the morphological changes
caused by ITC compounds in fungal structures
The morphological changes occurring on mycelium and germina-
tion tubes were investigated under the Olympus B × 51 microscope
equipped with Nomarski DIC optics. The discs from the fungus cul-
tures were transferred to Petri dishes containing PDA and then al-
lowed to incubate for 2 days at 25°C for mycelial growth. When the
fungus grew approximately 2 cm from the disc, different concentra-
tions of the compounds were dropped onto the Petri lids as described
above, and the Petri dishes were sealed with parafilm and allowed to
incubate again. The treated fungal structures were observed by prep-
arations of very fine agar blocks from Petri dishes for light microscopy.
2.6 | Determination of control potentials of ITC
compounds on disease development in vivo trials

The effects of different concentrations of ITC compounds on sour rot

2.4 | Effects of ITCs on arthroconidia germination of
Geotrichum citri-aurantii
The inhibitory effects of ITCs on the arthroconidia germination of
Geotrichum citri-aurantii were determined as described by Soylu et al.
(2005). Spore suspension (103 spores/ml) of Geotrichum citri-aurantii
was prepared from actively growing culture (5 days old) in distilled
sterile water. Three different 50 µl aliquots of the spore suspension
drops were spread onto the surface of the PDA medium, and dif-
ferent concentrations of ITCs (0.0–0.11 μl/L air) were added to the
interior of each Petri lid using a micropipette and incubated at 25ºC
for 8 hr in the dark. Sterile distilled water was used instead of ITCs as
a control. Germinated and non-germinated conidia were examined
under a light microscope. Germination was defined as the point at
which the germ tube length exceeded the spore diameter. The per-
centage of spore germination was evaluated under the microscope
by counting 100 arthroconidia for each, and repeating this at each
concentration.
disease development were investigated in vivo trials. Mandarin fruits
(cv. Owari Satsuma), which are susceptible to sour rot disease and
have a short life after harvest, were hand-harvested at commercial
maturity (a total water-soluble dry matter/titratable acidity (WSDM/
TA) ratio of 6.5/1, a juice ratio of 30%–35%, and 8.3 ml CO2/kg hour
of respiration rate) the 3rd week of September from the local or-
chard in the Erzin province of Hatay and used in vivo experiments
as suggested by Ozdemir et al. (2010). Freshly hand-harvested unin-
jured mature mandarin fruits were chosen as to be homogeneous in
shape, colour and size. All fruits were rinsed with tap water before
treatments and left in 2% sodium hypochlorite (NaOCl) for 2 min,
then rinsed with sterile distilled water. Disinfected fruits were then
dried in a laminar flow cabinet. In the equatorial region of the air-
dried mandarin fruits, three uniformly spaced cuts about 2 mm in
depth and 5 mm in width were made with a sterile cork borer.
To determine the therapeutic effects of ITC compounds, inocu-
lations were carried out immersing fruits in fungal inoculum suspen-
sion (106 conidia/ml) for 1–2 min. After the inoculation process, the
4 |
fruits were dried for 1–2 hr for fungal penetration. ITC compounds
(methyl, allyl and ethyl ITCs), which have the greatest effect on the
pathogen in in vitro conditions, were used. Five inoculated mandarin
fruits were placed in plastic jars (having 5 L air capacities), and dif-
ferent concentrations of ITCs (0.0, 4.0, 6.0, 8.0, 10.0 and 12.0 μl/L)
were applied. Subsequently, all jars were tightly closed and wrapped
with two layers of parafilm and kept at 25°C for five days. During
the experiments, all jars were kept in horizontal positions to prevent
the fruits from coming into contact with each other. At the end of
this period, the fruits were removed from the jars and the average
lesion diameters (mm) were measured. All tests were performed in
triplicate, and each experiment was repeated twice. Positive control
consisted of fruits which were inoculated with fungal suspension;
negative control consisted of uninoculated healthy fruits.
2.7 | Statistical analysis
Effective concentrations for a 50% and 90% reduction of myce-
lial growth and conidial germination (EC50 and EC90 values) for
the ITC compounds were calculated by probit analysis (Lesaffre &
Molenberghs, 1991) applied to the percentage of conidial germina-
tion and mycelial growth inhibition, respectively. All data regarding
the infected fruit percentages were subjected to one-way analysis
of variance (ANOVA) using SPSS statistical software (SPSS Inc.).
Comparison of means was performed using the Duncan test at
p ˂ .05.
3 | R E S U LT S
3.1 | Geotrichum citri-aurantii isolates and
pathogenicity
White, circular, rapidly developing and relatively sparse colonies at
the end of the nutrition medium were observed by microscope, and
based on morphological and taxonomic characters such as hyphae
and conidial structures, the fungus was identified as Geotrichum citri-
aurantii (Hernandez-Montiel, Holguin-Pena, & Latisnere-Barragan,
2010; McKay et al., 2012). Once single spore cultures were obtained,
inoculation tests were conducted to verify the pathogenicity of each
fungal isolate. According to the results from pathogenicity test,
the average disease rate of sour rot in mandarin fruits (cv. Owari
Satsuma) was 96%, the average softening development diameter was
30.83 mm, and the average mycelial growth diameter was 18.23 mm.
3.2 | Effects of ITCs on mycelial growth of
Geotrichum citri-aurantii
The results obtained from the studies investigating the vapour ef-
fects of ITC compounds on mycelial growth and % inhibition of
G. citri-aurantii are given in Table 2 and Figure 1.
KARA and SOYLU
As seen in the Table 3; methyl, allyl, butyl, ethyl, benzyl and 2-pheny-
lethyl ITC compounds showed antifungal activity at varying concen-
trations of 0.0–0.14 μl/L. The strongest antifungal activity was shown
by benzyl, methyl, allyl and ethyl ITC compounds, followed by butyl
and 2-phenylethyl ITC compounds. At the lowest concentration used
(0.01 µl/L), the highest and the lowest inhibitory effects were obtained
with benzyl ITC (54.4%) and allyl ITC (35.8%). Benzyl ITC at 0.06 µl/L,
methyl ITC at 0.08 µl/L, allyl and ethyl ITCs at 0.10 µl/L, butyl ITC at
0.13 µl/L and 2-phenylethyl ITC at 0.14 µl/L concentrations completely
inhibited mycelial growth of G. citri-aurantii (Table 2 and Figure 1).
Based on the inhibition rates of mycelial growth of fungus at
different concentrations of ITC compounds, effective concentra-
tions for a 50% and 90% reduction (EC50 and EC90 values) of myce-
lial growth and conidial germination for each ITC compound were
determined by Probit analysis (Table 2). The EC90 values of benzyl,
methyl, ethyl, allyl, butyl and 2-phenylethyl ITC compounds were
determined as 0.044, 0.061, 0.072, 0.084, 0.10 and 0.170 µl/L, re-
spectively (Table 2).
In the experiments conducted to determine the fungistatic or
fungicidal effects of the ITC compounds, benzyl ITC at 0.06 µl/L,
methyl ITC at 0.08 µl/L, allyl and ethyl ITC at 0.10 µl/L concentra-
tions were determined to have fungistatic effects.
3.3 | Effects of ITCs on arthroconidia germination of
Geotrichum citri-aurantii
The results obtained from studies investigating the vapour effects
of ITC compounds on the germination and the % inhibition of the
asexual spores (conidia) of G. citri-aurantii, known as arthrospores,
are given in Table 3 and Figure 2.
It was observed that ITC compounds inhibited the conidial ger-
mination of fungi below the concentrations that inhibit mycelial
growth (Table 3 and Figure 2). According to the results obtained,
conidial germination rates of fungus decreased with increasing ITC
concentrations.
At the lowest concentration used (0.005 µl/L), the highest and
the lowest inhibitory effects were obtained with methyl ITC (25.6%)
and ethyl ITC (2.3%). The complete inhibition of conidial germination
by ITC compounds was recorded at the concentrations of 0.05 μl/L
of benzyl ITC, 0.07 μl/L of methyl, allyl and ethyl ITCs, 0.8 μl/L of
butyl and 0.10 μl/L of 2-phenylethyl ITC (Table 3 and Figure 2).
According to statistical analyses, the antifungal activities of ITC
compounds on conidial germination significantly increased depend-
ing on the concentrations, and the difference between the concen-
trations was statistically significant (p ≤ .05).
The inhibition rates of 50% and 90% (EC50 and EC90, respec-
tively) of different concentrations of ITC compounds on arthroco-
nidia germination are given in Table 3. According to values estimated
by Probit analysis, EC50 and EC90 values of benzyl, allyl, methyl, ethyl,
butyl and 2-phenylethyl ITC compounds were determined as 0.013–
0.036, 0.018–0.050, 0.015–0.052, 0.024–0.053, 0.018–0.064 and
0.023–0.089 μl/L, respectively.
KARA and SOYLU | 5

TA B L E 2 Activity of pure ITC compounds at different concentrations in the vapour phase on mycelial growth (mm) of Geotrichum
citri-aurantiia

Effects of different ITCs used on mycelial growth (mm)

Concentrations (µl/L) Methyl ITC Allyl ITC Butyl ITC Ethyl ITC Benzyl ITC 2-Phenylethyl ITC

0.0 85.3h 83.0j 83.3l 83.7k 84.0g 82.0l

0.01 53.0gD 53.3iD 43.0kBC 41.7jB 38.3fA 44.3kC
0.02 42.7fD 47.7hE 34.0jBC 28.3iA 33.3eB 37.3jC
0.03 40.7fD 39.0gD 30.7iB 26.0jA 26.0dA 33.7iC
0.04 28.3eCD 31.0fDE 26.0hBC 24.3gB 19.7cA 32.3iE
0.05 21.3dB 26.0eC 22.0gB 21.7fB 13.7bA 29.7hD
0.06 13.0cB 22.7dD 21.0gD 18.0eC 0.0aA 27.3gE
0.07 6.0bB 19.7dD 18.0fD 13.3dC 0.0aA 26.0fgE
0.08 0.0aA 12.7cC 15.0eD 10.3cB 0.0aA 24.0fE
0.09 0.0aA 7.0bB 11.7dC 7.7bB 0.0aA 20.7eD
0.10 0.0aA 0.0aA 9.0cB 0.0aA 0.0aA 12.7dC
0.11 0.0aA 0.0aA 5.3bB 0.0aA 0.0aA 10.6dC
0.12 0.0aA 0.0aA 2.3aB 0.0aA 0.0aA 7.3cC
0.13 0.0aA 0.0aA 0.0aA 0.0aA 0.0aA 3.3bB
0.14 0.0a 0.0a 0.0a 0.0a 0.0a 0.0a
b
EC50 0.020 0.023 0.016 0.014 0.014 0.019
EC90 0.061 0.084 0.101 0.072 0.044 0.170
a
The values obtained are the average of fungus colony diameter (mm) values of 3 Petri dishes. Similar small letters next to average values in the same
column or similar large letters next to average values in the row indicate that the difference between the applications is not statistically significant
(Duncan's multiple range test, p ≤ .05).
b
Effective concentrations of ITC compounds, inhibiting mycelial growth by 50% (EC50) and 90% (EC90), were determined by Probit analysis with the
help of SPSS statistical program (version 11.5, SPSS Inc.) using the values of mycelium diameters obtained at different concentrations for each ITC
compound.

3.4 | Effects of ITC compounds on morphologies of
hypha of Geotrichum citri-aurantii
It has been observed that ITC compounds, at the concentration
which ensures complete inhibition of the fungus, cause quite severe
morphological deterioration on the mycelium (Figure 3). Fungicidal
MIC of vapour of ITC caused cytoplasmic clotting, discharge out
of hyphae, darkening in hyphae with increasing intensity of cyto-
plasmic structure and the cytoplasm became empty afterwards
becoming fully darkened and deformed into a necrotic mycelium
(Figure 3c,d) compared to control hyphae. While the thickness of
healthy hyphae is about 7.5–10 μm in diameter, ITC applied/exposed
hyphae's thickness is about 5–7 μm in diameter with a thinning of
about 20%–33%.
3.5 | Effects of ITC compounds on disease
development in in vivo trials
The results obtained from the studies in which the effect of the ITC
compounds of different concentrations on the average lesion area of
G. citri-aurantii in semi-commercial conditions are shown in Table 4.
ITC compounds (methyl, allyl and ethyl ITCs) which have the greatest
effect on the pathogen, as a result of mycelial and conidial germina-
tion experiments in in vitro conditions, were used. Benzyl ITC has
not been applied to fruit because it is not effective in preliminary
experiments in semi-commercial conditions.
As seen in Table 4, methyl, allyl and ethyl ITC compounds showed
antifungal activity at a varying concentration range of 0.0–12.0 μl/L.
The strongest antifungal activity is shown by the ethyl ITC com-
pound, followed by methyl and allyl ITC compounds, respectively.
Regarding to the complete inhibition concentrations of the average
lesion diameter of the fungus, ethyl ITC was the best at a concentra-
tion of 8.0 μl/L, methyl and allyl ITCs also completely inhibited each
at the concentration of 12.0 μl/L (Table 4). Methyl, allyl and ethyl
ITCs were found to cause phytotoxicity in the fruits at the concen-
tration of 16.0 μl/L (data not shown).
Based on the inhibition rates of the average lesion diameter of
different concentrations of ITC compounds, the effective concen-
trations for inhibition of mycelial growth of each ITC compound EC50
and EC90 (50% and 90%, respectively) were determined by Probit
analysis (Table 4). According to the results obtained, the EC90 val-
ues of the ethyl, methyl and allyl ITCs were determined to be 7.386,
13.269 and 13.416 μl/L, respectively.
Evaluation of the average % inhibition of the lesion diameter of
G. citri-aurantii treated by pure ITCs was given in Figure 4. Ethyl ITC
6 | KARA and SOYLU

100 F I G U R E 1 Activity of pure ITC

compounds at different concentrations
90 in vapour phase on inhibition of mycelial
80 growth of Geotrichum citri-aurantii (%)

70
Inhibition (%)

60
50
40
30
20
10
0
0.01 0.02 0.04 0.06 0.08 0.1 0.13 0.14
Concentrations (µl/L)
Methyl ITC Allyl ITC Butyl ITC
Ethyl ITC Benzyl ITC 2-Phenylethyl ITC

TA B L E 3 Activity of pure ITC compounds at different concentrations in the vapour phase on arthroconidia germination of Geotrichum
citri-aurantiia

Effects of different ITCs used on arthroconidia germination

Concentrations (µl/L) Methyl ITC Allyl ITC Butyl ITC Ethyl ITC Benzyl ITC 2-Phenylethyl ITC

0.0 87.3h 88.0h 86.7j 85.0g 82.3g 85.0k

0.005 65.0gA 78.7gC 71.0iB 83.0gD 66.0fA 72.0jB
0.01 55.7fB 62.0fCD 58.3hBC 72.7fE 51.3eA 63.7iD
0.02 41.3eBC 43.3eC 40.3gB 56.3eE 35.7dA 51.0hD
0.03 35.0dB 38.0dC 35.0fB 42.3dD 22.7cA 40.7gD
0.04 23.3cB 22.7cB 29.0eC 22.3cB 6.3bA 33.3fD
0.05 12.7bBC 10.7bB 24.7dD 14.7bC 0.0aA 26.3eD
0.06 4.0aB 2.7aB 11.0cC 3.3aB 0.0aA 21.3dD
0.07 0.0aA 0.0aA 5.0bB 0.0aA 0.0aA 15.3cC
0.08 0.0aA 0.0aA 0.0aA 0.0aA 0.0aA 9.3bB
0.09 0.0aA 0.0aA 0.0aA 0.0aA 0.0aA 2.3aB
0.10 0.0aA 0.0aA 0.0aA 0.0aA 0.0aA 0.0aA
b
EC50 0.015 0.018 0.018 0.024 0.013 0.023
EC90 0.052 0.05 0.064 0.053 0.036 0.089
a
The values obtained are the average of germinated arthroconidia values of 3 Petri dishes. Similar small letters next to average values in the same
column or similar large letters next to average values in the row indicate that the difference between the applications is not statistically significant
(Duncan's multiple range test, p ≤ .05).
b
Effective concentrations of ITC compounds, inhibiting mycelial growth by 50% (EC50) and 90% (EC90), were determined by Probit analysis with the
help of SPSS statistical program (version 11.5, SPSS Inc.) using the values of mycelium diameters obtained at different concentrations for each ITC
compound.

had the highest effect (18.9%) at the 4.0 μl/L concentration, and
methyl ITC showed the lowest effect (6.3%). At the concentration of
8.0 μl/L, a 100% effect of ethyl ITC on the pathogen was observed;
it was followed by methyl and allyl ITC compounds, at 36.0% and
28.3%, respectively. When the concentration was increased three-
fold (12.0 μl/L), methyl and allyl ITC compounds inhibited mycelial
growth of the pathogen by 100%.
4 | D I S CU S S I O N
Postharvest decay of fruits and vegetables presents a major fac-
tor causing postharvest losses and limits the duration of the stor-
age and shelf life of produce. There is a growing interest in the use
of natural antimicrobials for pathogen control due to the gener-
ally accepted safe status of these compounds. Among the most
KARA and SOYLU | 7

F I G U R E 2 Activity of pure ITC 100

compounds at different concentrations
in vapour phase on inhibition of 90
arthroconidia germination of Geotrichum 80
citri-aurantii (%)
70

Inhibition (%)
60

50

40

30

20

10

0
0.005 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1
Concentrations (µl/L)
Methyl ITC Allyl ITC Butyl ITC
Ethyl ITC Benzyl ITC 2-Phenylethyl ITC

F I G U R E 3 The effect of volatile (a) (b) (c)

vapours of ITC on hyphal morphology.
(a–c) Apparently healthy hyphal growth
(arrow) in control plates. (d–f) The
effected hyphae exhibited abnormalities
(arrows) including vacuolation (d), leakage
of cytoplasm (e) and vacuole-mediated
organelle degradations (f) especially at
the hyphal tips. (a) Bar: 20 µm; (b–f) Bar:
40 µm

(d) (e) (f)

potent antimicrobials from plant origin, isothiocyanates (ITCs) are
hydrolysed products of sulphur containing compounds called glu-
cosinolates, which occur naturally in cruciferous vegetables, such
as broccoli and cabbage (Mari et al., 2016). Strong bioactivities
of glucosinolates such as antifungal, antibacterial, bioherbicidal,
antioxidant, antimutagenic and anticarcinogenic have been re-
ported in several researches (Mari et al., 2016; Zhang, 2004). The
isothiocyanates (ITCs) were found to inhibit the growth of fungi,
yeast and bacteria at very low concentrations. Recent reviews
reported a wide range of phytopathogenic fungi susceptible to
8 | KARA and SOYLU

different ITCs. Among several ITCs, the literature reports that the the lowest concentration of benzyl ITC (0.06 μl/L) had the high-
allyl, benzyl, butenyl, propenyl, 2-phenylethyl and methyl ITCs have est activity in inhibiting mycelial growth of the pathogen. These
remarkable antimicrobial activities (Mari et al., 2016; Pal Vig et al., results are in harmony with Drobnica et al.’s (1967) findings that
2009). benzyl ITC was one of the most effective ITC against Aspergillus
Similar to other studies (Smolinska & Horbowicz, 1999), the in- niger, Penicillium cyclopium and Rhizopus oryzae. In another similar
hibitory effect of the ITCs on the G. citri-aurantii isolate tested in study, benzyl ITC at a concentration of 0.05 mg/ml was reported
our investigation was usually dependant on the concentration used. to inhibit mycelial growth of Alternaria alternata by 80% isolated
When the effects of ITC compounds in the vapour phase on my- from tomato fruit and was inhibited by 100% at a concentration of
celial growth of G. citri-aurantii in vitro conditions were examined, 0.1 mg/ml (Troncoso-Rojas et al., 2005). Allyl (AITC), phenyl (PITC)
and benzyl isothiocyanates (BITC), which were employed in the
TA B L E 4 The effect of pure ITC compounds at different study conducted by Azaiez, Meca, Manyes, and Fernández-Franzón
concentrations on the average lesion diameter (mm) of sour rot (2013), inhibited the growth of three mycotoxigenic Fusarium re-
caused Geotrichum citri-aurantii on mandarin fruitsa ducing 2.1%–89.7% of the mycelial growth and toxin production by
ITC compounds used the fungal agent depending on the dose and time of exposure. The
lowest antifungal effect was observed in the application of phenyl-
Concentrations (µl/L) Methyl ITC Allyl ITC Ethyl ITC
ethyl ITC. It has been noted that even at the highest concentration
0.0 25.7e 25.7e 25.7d of phenylethyl ITC (0.1 mg/ml) inhibition was 50% (Troncoso-Rojas
4.0 24.1eA 24.0deA 20.8cA et al., 2005). Kurt et al. (2011) reported that volatiles of ITC com-
6.0 19.5dB 22.4dB 12.2bA pounds (methyl, allyl, butyl, ethyl, phenyl, benzyl and 2-pheny-
8.0 16.5cB 18.5cB 0.0aA lethyl ITCs) were highly effective in inhibiting mycelial growth of
10.0 10.9bB 11.3bB 0.0aA Sclerotinia sclerotiorum. Methyl ITC inhibited mycelial growth by

12.0 0.0a 0.0a 0.0a 100% at the concentrations of 732, 1,024 and 1,460 μmol/L. In
b addition, these data were supported by Mari et al. (2008) who re-
EC50 8.139 8.582 5.331
ported that ITCs (allyl, butyl, benzyl, 2-phenylethyl and 4-methylth-
EC90 13.269 13.416 7.386
iobutyl-ITCs) in the vapour phase significantly inhibited conidial
a
The values obtained are the average of lesion diameter (mm) values
germination and mycelial growth of Monilinia laxa in in vitro condi-
of the disease developed in 3 different mandarin fruits. Similar small
letters next to average values in the same column or similar large letters
tions. Lewis and Papavizas (1971) also reported that vapours of the
next to average values in the row indicate that the difference between ITC’s (allyl, methyl, butyl, phenyl ethyl), sulphides and disulphides
the applications is not statistically significant (Duncan's multiple range caused appreciably reduced mycelial growth, zoospore motility and
test, p ≤ .05). germination of root rot disease of peas caused by Aphanomyces eu-
b
Effective concentrations of ITC compounds, inhibiting lesion diameter
teiches. According to in vitro test, ITC vapours were more effective
of disease by 50% (EC50) and 90% (EC90), were determined by Probit
analysis with the help of SPSS statistical program (version 11.5, than those of sulphides in inhibiting mycelial growth, zoospore mo-
SPSS Inc.) using the values of lesion diameters obtained at different tility and germination. Among treatments, allyl ITC and methyl ITC
concentrations for each ITC compound. were more effective than butyl ITC or phenyl ethyl ITC.

100

90

80

70
Inhibition (%)

60

50

40

30

20

10
F I G U R E 4 Effect of pure ITC
0 4, 0 6 8 10 12 compounds at different concentrations
Concentrations (µl/L) on inhibition (%) of formation of lesion
diameter of sour rot caused by Geotrichum
Methyl ITC Allyl ITC Ethyl ITC citri-aurantii on mandarin fruits
KARA and SOYLU
The EC50 values of the benzyl, ethyl and butyl ITCs, which were
showing the strongest antifungal activity, were determined as 0.014,
0.014 and 0.016 μl/L, respectively. These results are similar to those
of Wu et al.’s (2011) findings which reported that the combination of
allyl and ethyl ITCs had a fungitoxic effect against spore germination
and mycelial growth of P. expansum and Botrytis cinerea. Authors also
pointed out that the optimized combination showing the lowest EC50
values at 0.08 and 0.14 μg/ml air for inhibition of conidial germination
and mycelial growth of P. expansum, respectively. It showed the low-
est EC50 values at 0.07 and 0.12 μg/ml air for conidial germination and
mycelial growth of B. cinerea, respectively. Mycelial growth of both
fungal species was inhibited by higher ITC concentrations than conid-
ial germination. In our study, the different reaction of G. citri-aurantii
mycelia and conidia to ITC exposure is, to some extent, probably due
to the morphology involved in mycelia and conidia. The hyphal wall
is perhaps thicker than the conidial wall; hence, the volatile penetra-
tion in these structures was easier. Similar results were reported with
conidia of the fungus Metarhizium anisopliae (Inyang, Butt, Doughty,
Todd, & Archer, 1999). In that study, the exposure of 1 μl of 100% v/v
of the ITCs of phenylethyl, 2-chlorophenyl, and phenyl consistently
inhibited conidia germination during the incubation period of this fun-
gus while mycelium growth always took place.
The current results revealed that MITC (0.08 μl/L), AITC and
EITC (0.10 μl/L) together with BITC (0.06 μl/L) showed a fungistatic
effect on mycelial growth of G. citri-aurantii according to the inhibi-
tion tests. These results are in accordance with the studies which
reported that the ITC compounds have fungistatic and fungitoxic
effects against Gaeumannomyces graminis var. tritici (Sacc.) Arx &
DL Olivies, Rhizoctonia solani JG Kühn, Gibberella zeae (Schwein)
Petch (Syn. Fusarium graminearum), Fusarium oxysporum, Cochliobolus
sativus (S Ito & Kurib) Drescher ex Das (syn. Bipolaris sorokiniana)
and various soil-borne plant pathogens such as Pythium irregu-
lar Buisman (Angus, Gardner, Kirkegaard, & Desmarchelier, 1994;
Dhingra, Costa, & Silva, 2004; Motisi, Montfort, Dore, Romillac,
& Lucas, 2009; Ramos et al., 2012; Sarwar, Kirkegaard, Wong, &
Desmarchelier, 1998; Smolinska et al., 2003) and sclerotia forming
fungi (Rahmanpour, Backhouse, & Nonhebel, 2009).
Different glucosinolates hydrolysis products respond differ-
ently to the microbial population but ITCs are the major inhibi-
tors of microbial activity (Mayton, Oliver, Vaughn, & Loria, 1996).
The antimicrobial activities of ITCs may be associated to their
chemical structure. The toxicity and range of activity vary with
changes in the isothiocyanate R-group. Greater toxicity is often
related to increased volatility (Lewis & Papavizas, 1971). Previous
studies reported that aromatic forms are more biologically active
than aliphatic forms due to their higher penetrability and increas-
ing volatility (Kurt et al., 2011; Pal Vig et al., 2009). In our study,
the strongest antifungal activity was also displayed by aromatic
benzyl ITC with an EC 50 value of 0.013 μl/L based on the inhibi-
tion rates of the conidial germination of ITCs. These results are in
agreement with Kurt et al.’s (2011) findings reporting that benzyl
ITC showed the highest inhibitory effect on sclerot germination
of Sclerotinia sclerotiorum with an EC 50 of 75.1 μmol/L. Kojima and
| 9
Oawa (1971) tried to elucidate the particular biochemical mech-
anism of fungicidal activity of several ITCs using three different
strains of Saccharomyces cerevisiae (yeast). They reported that
ITCs act by inhibiting the oxygen uptake by yeast through the
uncoupler action of oxidative phosphorylation in mitochondria of
yeast, that is inhibiting the coupling between the electron trans-
port and phosphorylation reactions and thus eventually hindering
the ATP synthesis.
It was also observed that the ITCs caused severe morphological
deformations on the mycelium of fungus at concentrations that would
provide complete inhibition. It is thought that this damage caused by
different ITCs at the level of cell membrane and cytoplasmic content
on G. citri-aurantii probably resulted from the ITCs causing cytotoxic
effects by destroying the fungus membrane. The activity of plant orig-
inated volatile compounds, such as essential oils or their components,
results in structural modifications and alterations of hyphal morphol-
ogy mainly by partitioning the lipid layer of the cell membrane due to
their hydrophobic nature. In addition, this kind of compound can effect
the permeability of the cell membrane, subsequently causing leakage of
the cell components (Laird & Phillips, 2011; Shao, Wang, Xu, & Cheng,
2013; Soylu et al., 2010; Soylu, Soylu, & Kurt, 2006). Morphological
changes within the fungal cell wall and cytoplasm were also reported
as a result of the direct impact of these kind of compounds with the
enzymes responsible for the cell wall synthesis (Rasooli, Rezaei, &
Allameh, 2006). Oxygenated monoterpenes and terpene hydrocar-
bon such as citral, thymol, carvacrol and p-cymene were reported to
assist the entry of oxygenated monoterpenes into the cell by modi-
fying the cell membranes (Nguefack et al., 2012). Vapours from the
decomposition of cabbage tissue, an amendment which suppressed
root rot disease of pea, adversely effected the morphology of the
fungus, the development of oospores and the mycelial growth (Lewis
& Papavizas, 1971) . Furthermore, the application of plant originated
volatile was shown to act as a signalling compound to stimulate the
induced defence mechanism in avocado, the gene expression and the
enzyme activities of pathogenesis related proteins such as chitinase
and 1,3-b-glucanase (Bill, Sivakumar, Korsten, & Thompson, 2014;
Sellamuthu, Mafune, Sivakumar, & Soundy, 2013). Although several re-
ports were available on the morphological changes caused by essential
oils, to the best of our knowledge, our study is showing for the first
time that ITCs resulted in structural modification and alterations of hy-
phal and cytoplasmic morphologies. These results coincide with Lin’s ,
Preston, and Wei (2000) findings which reported fumigation with allyl
ITC causes metabolite leaks in bacterial cells.
Numerous studies have evaluated the vapour-phase activity of
AITC in vitro tests, obtaining important information on the growth
inhibition of some food borne pathogens and on the duration of
treatment (Manyes, Luciano, Manes, & Meca, 2015; Sotelo, Lema,
Soengas, Cartea, & Velasco, 2014; Wilson, Bergaentzie, Bindler,
Marchioni, & Lintz, 2013). The best conditions of AITC application
in in vitro experiments are not always comparable with those ob-
tained in in vivo experiments. In semi-commercial conditions, ethyl
ITC (8 μl/L) had the highest activity at the lowest concentration in
inhibiting the average lesion diameter of sour rot disease caused
10 | KARA and SOYLU
by G. citri-aurantii. Depending on the increase of concentrations,
this compound was followed by methyl and allyl ITCs (12 μl/L),
respectively. Our findings are supported by encouraging results
of previously published studies reporting the in vivo antifungal
activity of allyl ITC in controlled conditions such as the artificial
inoculations of pears (Mari et al., 2002), apples (Wu et al., 2011),
peaches (Mari et al., 2008) and netted melons (Troncoso-Rojas
et al., 2009).
In conclusion, the different ITCs used in the current experiment
have a high level of antifungal activity in the vapour phase against
the fungal agent G. citri-aurantii, which causes postharvest sour rot
disease in citrus. Since ITCs have a general biocidal character, they
can be tested against both postharvest and soil-borne pathogens
which can be controlled by biofumigation.
AC K N OW L E D G E M E N T S
This research was supported by the Scientific Research Projects
Coordinatorship (BAP) of Mustafa Kemal University (MKU BAP-
13381). This article was submitted as a poster presentation in the
VI. Plant Protection Congress of Turkey (5-8 September 2016,
Konya). The author would like to sincere thank Prof. Dr. Soner Soylu
for statistical analyses and constructive scientific criticism of the
manuscript and Sarah Celik Welch for diligent proofreading of this
manuscript.
C O N FL I C T O F I N T E R E S T
The authors declare no conflicts of interest.
ORCID
Merve Kara https://orcid.org/0000-0001-7320-3376
Emine Mine Soylu https://orcid.org/0000-0001-5961-0848
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How to cite this article: Kara M, Soylu EM. Assessment of
glucosinolate-derived isothiocyanates as potential natural
antifungal compounds against citrus sour rot disease agent
Geotrichum citri-aurantii. J Phytopathol. 2020;00:1–11.
https://doi.org/10.1111/jph.12889