Journal of

Fungi
Article
Trichoderma asperellum T76-14 Released Volatile Organic
Compounds against Postharvest Fruit Rot in Muskmelons
(Cucumis melo) Caused by Fusarium incarnatum
Warin Intana 1 , Suchawadee Kheawleng 2,3 and Anurag Sunpapao 3, *

1 School of Agricultural Technology and Food Industry, Walailak University, Tha Sala,
Nakhon Si Thammarat 80161, Thailand; [email protected]
2 Graduate School, Prince of Songkla University, Hatyai 90112, Thailand; [email protected]
3 Agricultural Innovation and Management Division, Faculty of Natural Resources, Prince of Songkla
University, Hatyai 90112, Thailand
* Correspondence: [email protected]; Tel.: +66-74-28-6103

Abstract: Postharvest fruit rot caused by Fusarium incarnatum is a destructive postharvest disease of
muskmelon (Cucumis melo). Biocontrol by antagonistic microorganisms is considered an alternative
to synthetic fungicide application. The aim of this study was to investigate the mechanisms of action
involved in the biocontrol of postharvest fruit rot in muskmelons by Trichoderma species. Seven
Trichoderma spp. isolates were selected for in vitro testing against F. incarnatum in potato dextrose
agar (PDA) by dual culture assay. In other relevant works, Trichoderma asperellum T76-14 showed
a significantly higher percentage of inhibition (81%) than other isolates. Through the sealed plate
method, volatile organic compounds (VOCs) emitted from T. asperellum T76-14 proved effective
at inhibiting the fungal growth of F. incarnatum by 62.5%. Solid-phase microextraction GC/MS
analysis revealed several VOCs emitted from T. asperellum T76-14, whereas the dominant compound





 was tentatively identified as phenylethyl alcohol (PEA). We have tested commercial volatile (PEA)
against in vitro growth of F. incarnatum; the result showed PEA at a concentration of 1.5 mg mL−1
Citation: Intana, W.; Kheawleng, S.;
Sunpapao, A. Trichoderma asperellum
suppressed fungal growth with 56% inhibition. Both VOCs and PEA caused abnormal changes in the
T76-14 Released Volatile Organic fungal mycelia. In Vivo testing showed that the lesion size of muskmelons exposed to VOCs from
Compounds against Postharvest Fruit T. asperellum T76-14 was significantly smaller than that of the control. Muskmelons exposed to VOCs
Rot in Muskmelons (Cucumis melo) from T. asperellum T76-14 showed no fruit rot after incubation at seven days compared to fruit rot in
Caused by Fusarium incarnatum. J. the control. This study demonstrated the ability of T. asperellum T76-14 to produce volatile antifungal
Fungi 2021, 7, 46. https://doi.org/ compounds, showing that it can be a major mechanism involved in and responsible for the successful
10.3390/jof7010046 inhibition of F. incarnatum and control of postharvest fruit rot in muskmelons.

Received: 21 December 2020 Keywords: muskmelon; biocontrol agent; volatile compounds; Trichoderma
Accepted: 11 January 2021
Published: 12 January 2021

Publisher’s Note: MDPI stays neu-

1. Introduction
tral with regard to jurisdictional clai-
ms in published maps and institutio- The muskmelon (Cucumis melo) is a species of melon that has been bred into several
nal affiliations. cultivated varieties worldwide. In Thailand, the cultivation of muskmelons has increased
due to market demand, as observed by the Department of Agricultural Extension, Ministry
of Agriculture and Cooperative, Thailand. However, Thailand is located in tropical and
subtropical zones, in which the weather is favorable for pathogen germination and disease
Copyright: © 2021 by the authors. Li- dispersal. The cultivation of muskmelons is complicated by several diseases that reduce
censee MDPI, Basel, Switzerland.
both the quality and quantity of pre-harvest and postharvest production. Postharvest fruit
This article is an open access article
rot is considered one of the most destructive diseases that negatively impact saleable stock.
distributed under the terms and con-
This disease has recently been reported to be caused by Fusarium incarnatum [1] in Thailand.
ditions of the Creative Commons At-
The application of high doses of chemical fungicide has effectively controlled plant
tribution (CC BY) license (https://
fungal diseases. However, they cause a negative effect in terms of the pesticide resistance
creativecommons.org/licenses/by/
4.0/).
of pathogens [2], and can have harmful side effects for humans [3]. Biological control is

J. Fungi 2021, 7, 46. https://doi.org/10.3390/jof7010046 https://www.mdpi.com/journal/jof

J. Fungi 2021, 7, 46 2 of 13

considered an alternative way to control postharvest fruit rot disease. It is well known that
Trichoderma species have many helpful qualities, including antibiosis [4–7], competition for
nutrients and space [8], mycoparasitism [9], and induction of systemic resistance [10–12] to
control disease pathogens, as well as to promote plant growth ability [13].
Endophytes are well known as a source of novel biologically active secondary metabo-
lites that confer major ecological benefits on their host plants [14]. Antibiosis is one of
the strongest mechanisms of the Trichoderma species, and its endophytic relationship in
nature provides a better combination of defense responses against several plant pathogenic
fungi [11,15]. The compounds released by endophytic fungi may benefit the host plant
by contributing antifungal activity [16], and offer a defense against plant pathogens [17].
Endophytes are a successful biological control agent (BCA) also capable of secreting volatile
organic compounds (VOCs) and cell-wall-degrading enzymes to inhibit the fungal growth
involved in antibiosis. Some Trichoderma species produce nonvolatile and volatile com-
pounds responsible for inhibiting pathogens [4–6,16,18–21], and may directly trigger the
defense system in host plants [6,11,22].
Trichoderma species play an important role as a BCA controlling diseases in a wide
variety of plants, but the mode of action for this antagonism remains unclear. It is known
that several Trichoderma species produce volatile organic compounds (VOCs) responsible
for inhibiting pathogens, activating a defense response as well as increased plant growth.
Therefore, the present study focuses on the potential of VOCs emitted by endophytic
Trichoderma as a biofumigant against postharvest fruit rot disease in muskmelons caused
by F. incarnatum. Endophytic fungi Trichoderma spp. were screened for their antifungal
activities, and attempts were made to determine the antagonistic mechanisms that may be
responsible for suppressing F. incarnatum and controlling postharvest fruit rot.

2. Materials and Methods

2.1. Trichoderma Species and Pathogen Isolates
In this study, seven Trichoderma species were tested for their ability to inhibit F. incar-
natum [1]. Six endophytic fungal isolates of the Trichoderma species, Trichoderma asperellum
T1 [11]; T. asperellum T76-14 (LC158827); Trichoderma sp. PSU-P1; Trichoderma sp. T76-1
(LC158829), T76-12/2 (LC158830), and V76-12 (LC158826); one soil-borne T. harzianum
TM2/1; and the fungal pathogen F. incarnatum, were obtained from the Culture Collection
of Pest Management, Faculty of Natural Resources, Prince of Songkla University, Thailand.
The Trichoderma species and F. incarnatum were cultured on potato dextrose agar (PDA)
(Himedia, Mumbai, India) for three days prior to this study.

2.2. In Vitro Screening of Trichoderma spp. against F. incarnatum by Dual-Culture Assay

Seven isolates of Trichoderma spp. were screened for their inhibitory effects on the
growth of F. incarnatum through a dual-culture assay on PDA plates [23]. The dual-culture
plates were then incubated at an ambient temperature (28 ± 2 ◦ C) for seven days. The
experiment was subjected to complete randomized design (CRD) with five replicates, and
the experiment was repeated twice. Colony radii of F. incarnatum on the control and test
plates were measured, and the percentage of inhibition was calculated by the formula
previously described by Rahman et al. [24]:

R1 − R2
Percentage inhibition (%) = × 100, (1)
R1
where R1 = radial growth of F. incarnatum in control and R2 = radial growth of F. incarnatum
with treatment.

2.3. Volatile Antifungal Bioassay

In order to test the volatile effects of the Trichoderma species on the growth of F. incarna-
tum, the sealed plate method was conducted [25], with some modifications. The Trichoderma
species were grown on potato dextrose agar (PDA) in 9-cm Petri dishes (Citotest, Jiangsu,
J. Fungi 2021, 7, 46 3 of 13

China) for three days. An agar plug (0.5 cm in diameter) was cut from the culture plate,
inserted centrally, and the lid of each petri dish was removed. We replaced the bottom with
one containing PDA inoculated with tested fungi, and the two bottom plates were then
sealed together with Parafilm and incubated at 28 ± 2 ◦ C for five days. For the control,
the lids of the control plates without Trichoderma inoculation were replaced in the same
manner. The tested plates were also incubated at ambient temperature (28 ± 2 ◦ C) for five
days. Colony diameters of F. incarnatum were measured and converted to the percentage of
inhibition through the following formula:

Dc − Dt
Percentage inhibition (%) = × 100, (2)
Dc
where Dc = mycelial growth of F. incarnatum on control plate, and Dt = mycelial growth of
F. incarnatum on the test plate. Each treatment was performed in five replicates, and the
results are presented as the mean ± standard error (n = 5).

2.4. GC/MS Analysis

In order to examine the volatile and semi-volatile antifungal compounds within the
Trichoderma species, gas chromatography spectrophotometry (GC/MS) was conducted.
The Trichoderma species was cultured in a 20-mL chromatography vial, 20 mm in diameter
(PerkinElmer, Waltham, MA, USA), and incubated at ambient temperature (28 ± 2 ◦ C)
for 14 days according to the method of Wonglom et al. [6] Solid-phase microextraction
(SPME) was performed to extract the volatile organic compounds (VOCs), as previously
described by Arthur et al. [6,26] SPME fiber (DVB/CAR/PDMS fiber) was exposed to the
vapors above each Trichoderma culture for 30 min, and inserted into the injection port of
the gas chromatograph SQ8 (PerkinElmer, Waltham, MA, USA), equipped with a DB-Wax
capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness). The oven temperature
was initially maintained at 40 ◦ C, then increased to 230 ◦ C, at a rate of 7 ◦ C min−1 . The
injector temperature was 230 ◦ C. The carrier gas was ultra-high-purity helium with an
initial column head pressure of 60 kPa, at a flow rate of 1 mL min−1 . Electron impact (EI)
mass spectra were collected at 70 eV ionization voltage over the m/z range 29–550. The ion
source and quadrupole temperatures were both set to 200 ◦ C. Volatiles and semivolatiles
were identified based on the computer searches dictated by The National Institute of
Standards and Technology (NIST) Mass Spectral Library Search Chromatogram.

2.5. Sealed Plate Method of Commercial Volatile against Mycelial Growth of F. incarnatum
To test the effect of dominant volatile compound participated in antifungal activity
against F. incarnatum, the sealed plate method was conducted as shown in Section 2.3.
The compound phenylethyl alcohol (PEA) was purchased from Sigma-Aldrich (St. Louis,
MO, USA). The effect of commercial PEA versus the other volatile antifungal compounds
2-ethylhexanol, 1-nonanol, 6-PP, and 2-methyl-1-butanol (Sigma-Aldrich, St. Louis, MO,
USA) [6] was tested through the sealed plate method. PEA was dissolved in 95% ethanol
and we adjusted the dilution to 10−1 , 10−2 , and 10−3 . Each volatile compound was
applied on a sterile cotton pad (20 µL) and subjected to the method of Wonglom et al. [6]
Application of 95% ethanol served as a negative control. The tested plates were then
incubated at 28 ± 2 ◦ C for seven days. Each treatment was composed of five replicates and
the experiment was repeated twice. Colony diameters of F. incarnatum were measured and
the percentage inhibition was calculated as described in Section 2.2.

2.6. In Vivo Test of Volatile against Postharvest Fruit Rot

To test the effect of VOCs and commercial volatiles against postharvest fruit rot in
muskmelon fruits, an in vivo test was conducted. F. incarnatum was cultured on PDA and
incubated at ambient temperature (28 ± 2 ◦ C) for seven days. Conidia of F. incarnatum were
harvested and we adjusted the concentration to 1 × 106 conidia mL−1 . A conidia suspen-
sion (20 µL) was applied to muskmelon fruit, and each experiment, consisting of three fruits
 2.6. In Vivo Test of Volatile against Postharvest Fruit Rot
To test the effect of VOCs and commercial volatiles against postharvest fruit rot in
muskmelon fruits, an in vivo test was conducted. F. incarnatum was cultured on PDA and
J. Fungi 2021, 7, 46
incubated at ambient temperature (28 ± 2 °C) for seven days. Conidia of F. incarnatum4were of 13

harvested and we adjusted the concentration to 1 × 106 conidia mL−1. A conidia suspension
(20 μL) was applied to muskmelon fruit, and each experiment, consisting of three fruits
(threereplicates),
(three replicates),waswasrepeated
repeatedthree
threetimes.
times.Inoculated
Inoculatedmuskmelon
muskmelonfruits
fruitswere
wereincubated
incubated
with VOCs from Trichoderma (eight petri dishes per treatment) or commercial
with VOCs from Trichoderma (eight petri dishes per treatment) or commercial volatile at volatile at
10 − 1
−1
dilutioninin5050μLµL
10 dilution applied onon
applied a cotton padpad
a cotton (four cotton
(four pads)
cotton in a in
pads) 24 a× 35
24 ××18
35cm
× plastic
18 cm
box (Figure
plastic 1) to share
box (Figure 1) to the atmosphere
share for three
the atmosphere days. days.
for three Inoculated muskmelon
Inoculated fruitsfruits
muskmelon incu-
bated withwith
incubated PDAPDA alone or 95%
alone ethanol
or 95% alone
ethanol served
alone as controls.
served Lesion
as controls. development
Lesion development was
measured and compared between the treatment and the
was measured and compared between the treatment and the control. control.

Figure1.
Figure 1. Overview
Overviewofofexperimental
experimentalsetup
setupand
andVOC
VOCeffect
effectagainst
againstFusarium
Fusariumincarnatum
incarnatumononmuskmel-
musk-
melons.
ons. Schematic
Schematic overview
overview of plate-within-a-box
of the the plate-within-a-box system.
system.

2.7.
2.7. Morphological
Morphological Study
Study of
of F.
F.incarnatum
incarnatumExposed
ExposedtotoVolatiles
Volatiles
To test the
To test the effect of VOCs or commercial volatiles
or commercial volatiles on onthe
themorphology
morphologychanges
changesofof
F.
F.incarnatum
incarnatummycelia,
mycelia,the
thesealed
sealedplate
platemethod
methodwaswasconducted
conducted as as described in Section 2.3.
2.3.
Changes
Changes in in morphology
morphology between
between VOCs
VOCs and
and commercial
commercial volatiles
volatilestreated
treatedand
anduntreated
untreated
with
with mycelia
mycelia from
from this
this study
study were
wereobserved
observedbybyaaLeica
LeicaDM750
DM750compound
compoundmicroscope
microscope
(Leica
(LeicaMicrosystems,
Microsystems,Wetzlar,
Wetzlar,Germany).
Germany).
2.8.
2.8. Effect
Effect of
of VOCs
VOCs Emitted
Emitted from
from T.
T.asperellum
asperellumT76-14
T76-14ononFruit
FruitRot
Rot
To test the effect of VOCs emitted from T. asperellum T76-14 on fruit rot in muskmel-
To test the effect of VOCs emitted from T. asperellum T76-14 on fruit rot in muskmel-
ons, five muskmelon fruits were disinfected with 70% ethanol prior to VOC exposure.
ons, five muskmelon fruits were disinfected with 70% ethanol prior to VOC exposure.
Muskmelon fruits were incubated with VOCs from Trichoderma or commercial volatiles, as
Muskmelon fruits were incubated with VOCs from Trichoderma or commercial volatiles,
described in Section 2.6, in a 24 × 35 × 18 cm plastic box to share the atmosphere for seven
as described in Section 2.6, in a 24 × 35 × 18 cm plastic box to share the atmosphere for
days. Fruit rot was observed from the day after treatment and recorded.
seven days. Fruit rot was observed from the day after treatment and recorded.
2.9. Statistical Analysis
2.9. Statistical Analysis
Data including fungal growth, percentage inhibition, and lesion size were subjected
Data including
to one-way analysis fungal growth,
of variance percentage
(ANOVA). inhibition,
Statistically and lesiondifferences
significant size were subjected
between
to one-way analysis of variance (ANOVA). Statistically significant differences
treated samples and the untreated control were analyzed by Tukey’s test with a threshold between
treated samples
of p < 0.05 [27]. and the untreated control were analyzed by Tukey’s test with a threshold
of p < 0.05 [27].
3. Results
3.1. Trichoderma Species Inhibit Mycelial Growth of F. incarnatum
Based on the primary screening for BCA by dual-culture assay, Trichoderma spp.
inhibited the fungal growth of F. incarnatum on the PDA plates. The percentage of inhibition
ranged from 62% to 81%. Among the seven tested isolates, T. asperellum T76-14 effectively
 3. Results
J. Fungi 2021, 7, x FOR PEER REVIEW 5 of 14

3.1. Trichoderma Species Inhibit Mycelial Growth of F. incarnatum

Based on the primary screening for BCA by dual-culture assay, Trichoderma spp. in-
3. Results
J. Fungi 2021, 7, 46 hibited the fungal growth of F. incarnatum on the PDA plates. The percentage of inhibition 5 of 13
3.1. Trichoderma Species Inhibit Mycelial Growth of F. incarnatum
ranged from 62% to 81%. Among the seven tested isolates, T. asperellum T76-14 effectively
Based on the primary screening for BCA by dual-culture assay, Trichoderma spp. in-
suppressed the growth of F. incarnatum by 81% (Figures 2 and 3), statistically higher than
hibited the fungal growth of F. incarnatum on the PDA plates. The percentage of inhibition
thoseranged
of the from
other62%
isolates (p < 0.05), and was therefore selected for further analysis.
to 81%. Among the seven tested isolates, T. asperellum T76-14 effectively
suppressed
suppressed the growththe
of growth of F.byincarnatum
F. incarnatum 81% (Figuresby 81%
2 and (Figures 2higher
3), statistically and 3),
thanstatistically higher than
those
those of of the
the other other
isolates (p <isolates
0.05), and(pwas
< 0.05), and
therefore was therefore
selected selected for further analysis.
for further analysis.

Figure 2. Percentage inhibition of Trichoderma spp. against Fusarium incarnatum in PDA by dual-
Figure 2. Percentage inhibition of Trichoderma
Figure 2. Percentage inhibition spp. againstspp.
of Trichoderma Fusarium incarnatum
against Fusarium in PDA
incarnatum in PDAbyby
dual-assay
dual- plates. Different
assay assay
plates. Different
plates. letters
Different indicate
letters statistically
indicate statisticallysignificant differences
significant differences among
among treatments
treatments (p < (p <
letters indicate statistically significant
0.05) using Tukey’s test. differences among treatments (p < 0.05) using Tukey’s test.
0.05) using Tukey’s test.

Figure 3. In vitro test antagonistic Trichoderma against Fusarium incarnatum, Trichoderma asperellum
T76-14, and F. incarnatum in dual culture plate (a), F. incarnatum in PDA plate alone (b), F. incar-
natum (c) and T. asperellum T76-14 (d) in sealed plate method and F. incarnatum in PDA plate alone
Figure 3. In vitroFigure 3. In vitro test
(e).
test antagonistic antagonistic
Trichoderma Trichoderma
against against
Fusarium Fusarium incarnatum,
incarnatum, TrichodermaTrichoderma
asperellumasperellum
F. incarnatum
T76-14, and
T76-14, and F. incarnatum in dual culture plate (a), F. incarnatum in PDA plate alone (b), F. incar-
in dual culture plate (a),
natum3.2.
F. incarnatum
(c) and
in PDA plate
T. asperellum
alone (b), F. incarnatum (c) and T. asperellum T76-14 (d) in sealed plate
Volatiles Emitted byT76-14 (d) in sealed
T. asperellum T76-14plate method
Inhibit andGrowth
Mycelial F. incarnatum in PDA plate alone
of F. incarnatum
method and F. incarnatum
(e). in PDA plate alone (e).
To test the effects of the VOCs emitted by T. asperellum T76-14 on the fungal growth
of F. incarnatum, the sealed plate method was conducted. After five days of incubation in
3.2. Volatiles Emitted by T. asperellum T76-14 Inhibit Mycelial Growth of F. incarnatum
3.2. Volatiles Emitted by T. asperellum T76-14 Inhibit Mycelial Growth of F. incarnatum
To test the effects of the VOCs emitted by T. asperellum T76-14 on the fungal growth
of F. incarnatum, To
thetest theplate
sealed effects of the
method VOCs
was emitted
conducted. by T.
After asperellum
five T76-14 on
days of incubation in the fungal growth
of F. incarnatum, the sealed plate method was conducted. After five days of incubation
in ambient temperature, the colony diameters of F. incarnatum in the tested plates were
smaller than those of the control bioassay plates (Figure 3). In the presence of VOCs
emitted by T. asperellum T76-14, the growth of F. incarnatum was slower than without VOCs
on PDA (Figure 3). The colony diameters of F. incarnatum in the control and test plates
were measured and converted to represent the percentage of inhibition of fungal growth.
The results demonstrated suppressed mycelial growth of F. incarnatum, with the greatest
percentage of inhibition being 62.5%.

3.3. Identifying Volatile Organic Compounds

The results above demonstrate that the VOCs emitted by T. asperellum T76-14 inhibited
the fungal growth of F. incarnatum, which suggests the antifungal activity of the volatile
J. Fungi 2021, 7, 46 6 of 13

organic compounds. To characterize the VOCs of T. asperellum T76-14, SPME was performed
to collect the VOCs, and they were analyzed by GC/MS. The results shown in Table 1 and
Figures 4 and 5 are from the preliminary screening of the VOCs produced by T. asperellum
T76-14 that were responsible for inhibiting F. incarnatum. The chromatogram is presented
in Figure 4. A total of 17 compounds with >70% were tentatively identified using the
NIST library (Table 1). The compounds detected in T. asperellum T76-14 are members of
the compound classes alcohols, alkane, and terpene, as illustrated in Table 1. The most
dominant volatile compounds detected in this study were phenylethyl alcohol (PEA) at
6.52 min with 23.188% peak area, followed by fluorotrinitromethane, at 1.578 min with
8.778% peak area, and β-Curcumene, a sesquiterpene, at 11.641 min with 8.488% peak area.
Figure 5 shows the mass spectrum of some of the major compounds, and the structure of
the most dominant compound, PEA.

Table 1. Volatile compounds produced by Trichoderma asperellum T76-14 tentatively identified through SPME GC/MS analysis.

RT (min) Volatile Compounds Formula % Math % Area

1.58
J. Fungi 2021, 7, x FOR PEER REVIEW Fluoro(trinitro)methane * CFN3 O6 95 8.78 7 of 14
2.29 Pentan-1-ol C5 H12 O 86.4 2.98
2.31 3-Methylpentane C6 H14 89.2 4.10
5.66 2,4,6-Trimethyloctane C11 H24 83.7 1.34
6.28 5,7-Dimethylundecane
3.4. Effect C13 HActivity
of Commercial Volatile Compounds on Antifungal 28 84.4 F. incarnatum
against 1.60
6.52 Phenylethyl alcohol C8 H10 O 94.8 23.19
7.45 Commercial volatile compounds that have been
Octadec-9-yne C18reported
H34 as antifungal
74.9 compounds
3.09
7.65 (2-ethylhexanol, 1-nonanol, 6-PP, and 2-methyl-1-butanol)
1-Methylideneindene C10 H10 and PEA were tested
92.8 against
1.34
10.27 F. incarnatum growth in vitro on PDA plates. The results
3-Isopropyl-6,8a-dimethyl-1,2,4,5,8,8a-hexahydroazulene C15 H24showed that 85.8all commercial
1.41 vol-
11.64 β-Curcumene
atile compounds C15 H24
inhibited the mycelial growth of F. incarnatum 84.4
(Table 8.49
2). High percentage
11.83 inhibition of volatile compounds depended on high concentrations. A dilution1.69
α-Bisabolene C 15 H 24 83.8 of 10−1 of
12.04 Sesquisabinene B C15 H24 83.5 1.74
all volatile compounds strongly inhibited F. incarnatum, with the percentage inhibition
13.10 Zingiberenol C15 H26 O 81.8 3.11
13.30 ranging from 21.68% to 74.29%. 2-ethylhexanol was
Cubenol C15found
H26 O to be the 83.1 best volatile
6.04 com-
13.61 pound for inhibiting F. incarnatum,
Undeca-3,4-diene-2,10-dione, 5,6,6-trimethyl with a percentage
C14 H22 O2inhibition of
75.1 74.28%. The PEA
1.44
14.03 showed a percentage inhibition of F. incarnatum of 56%
Allyldimethyl(prop-1-ynyl)silane C8 H14 atSia dilution71.4
of 10 , equivalent
−1 0.77 to
15.12 cis-Z-α-Bisabolene
a concentration of 1.5epoxide C15 His
mg mL−1. This confirmed that PEA 24 O
a volatile 71.3 1.73
antifungal compound
* The result of three replicates.
against F. incarnatum.

Figure4.
Figure 4. Total
Totalion
ionchromatogram
chromatogramof
ofvolatile
volatilecompounds
compoundsidentified
identifiedfrom
fromT.T.asperellum
asperellumT76-14.
T76-14.
J. Fungi 2021, 7, 46 7 of 13

Figure 4. Total ion chromatogram of volatile compounds identified from T. asperellum T76-14.

Figure
Figure 5.
5. Mass
Mass spectrum
spectrum for
for the
the compound
compound at
at 6.52
6.52 min
min (a)
(a) and
and phenylethyl
phenylethyl alcohol (PEA) with its structure (b).

3.4. Effect of Commercial Volatile Compounds on Antifungal Activity against F. incarnatum

Commercial volatile compounds that have been reported as antifungal compounds
(2-ethylhexanol, 1-nonanol, 6-PP, and 2-methyl-1-butanol) and PEA were tested against
F. incarnatum growth in vitro on PDA plates. The results showed that all commercial volatile
compounds inhibited the mycelial growth of F. incarnatum (Table 2). High percentage
inhibition of volatile compounds depended on high concentrations. A dilution of 10−1
of all volatile compounds strongly inhibited F. incarnatum, with the percentage inhibition
ranging from 21.68% to 74.29%. 2-ethylhexanol was found to be the best volatile compound
for inhibiting F. incarnatum, with a percentage inhibition of 74.28%. The PEA showed
a percentage inhibition of F. incarnatum of 56% at a dilution of 10−1 , equivalent to a
concentration of 1.5 mg mL−1 . This confirmed that PEA is a volatile antifungal compound
against F. incarnatum.

Table 2. Comparison of phenylethyl alcohol (PEA) and other commercial volatiles against Fusar-
ium incarnatum.

Percentage Inhibition (%) a

Commercial Volatiles
10−1 10−2 10−3
1-Nonanol 55.42 ± 13.69 b 29.71 ± 3.32 b 19.67 ± 5.69 c
2-Ethylhexanol 74.29 ± 11.12 a 29.71 ± 5.01 b 24.89 ± 9.04 a
2-Methyl-1-butanol 39.12 ± 8.43 c 20.88 ± 4.86 d 16.86 ± 1.20 d
6-Pentyl-2H-pyran-2-one 21.68 ± 5.25 d 32.53 ± 14.65 a 20.08 ± 6.18 b
Phenylethyl alcohol 56.00 ± 5.25 b 24.00 ± 6.92 c 12.00 ± 4.00 e
a Percentage inhibition of mycelial growth was calculated using 95% ethyl alcohol as a negative control. Means

within the same column followed by the same superscript letters are not significantly different at p < 0.05 using
Tukey’s test.

Fungal mycelia among the VOCs released from T. asperellum T76-14, PEA, and the
control was observed by a compound microscope. The fungal mycelia of F. incarnatum
exposed to VOCs and PEA showed an abnormal shape, whereas the control showed no
change of mycelia and remained a normal shape (Figure 6).
 control. Means within the same column followed by the same superscript letters are not signifi-
cantly different at p < 0.05 using Tukey’s test.

Fungal mycelia among the VOCs released from T. asperellum T76-14, PEA, and the
J. Fungi 2021, 7, 46control
was observed by a compound microscope. The fungal mycelia of F. incarnatum 8 of 13
exposed to VOCs and PEA showed an abnormal shape, whereas the control showed no
change of mycelia and remained a normal shape (Figure 6).

Figure 6. Morphological
Figure 6. Morphological observationobservation
of Fusariumof Fusariummycelia,
incarnatum incarnatum mycelia,
in control (a),inexposed
control to
(a), exposed
VOCs to by T. asperellum
emitted
VOCs emitted by T. asperellum T76-14 (b), and PEA (c), black arrows indicate normal
T76-14 (b), and PEA (c), black arrows indicate normal hyphal tips and red arrows indicate abnormal hyphalhyphal tips tips.
and red arrows indicate abnormal hyphal tips.

3.5. VOCsT76-14
3.5. VOCs of T. asperellum of T. asperellum T76-14 Reduced
Reduced Postharvest FruitPostharvest Fruit Rot
Rot in Muskmelon in Muskmelon Fruits
Fruits
As seen above, As the seen
VOCs above, thefrom
emitted VOCs T.emitted
asperellumfrom T. asperellum
T76-14 and PEAT76-14showed and PEA showed anti-
anti-
fungal F.
fungal activity against activity against
incarnatum, theF. pathogen
incarnatum,of the pathogenfruit
postharvest of postharvest fruit rot in muskmelons.
rot in muskmelons.
Therefore, they Therefore,
were selectedtheytowere selected
test in to test fruit
vivo against in vivo
rotagainst fruit rot in
in muskmelons bymuskmelons
observing by observing
the inoculation
the lesion size after lesion size after
with inoculation with a spore
a spore suspension of F. incubation
suspensionAfter
of F. incarnatum. incarnatum. After incuba-
tionlesions
for three days, the for three days, the
of VOCs- andlesions of VOCs-
PEA-treated and PEA-treated
muskmelons muskmelons
were statistically were statistically
sig-
J. Fungi 2021, 7, x FOR smaller than in the control (Figures 7 and 8). The lesion sizes of the control,sizes of the con-
PEER REVIEW
nificantly significantly smaller than in the control (Figures 7 and 8). The lesion 9 of 1
VOCs-treated, andtrol,PEA-treated
VOCs-treated, and PEA-treated
muskmelons were 1.5,muskmelons
0.5, and 0.5were 1.5, 0.5, and (Fig-
cm, respectively 0.5 cm, respectively
ure 8). (Figure 8).

Figure7.7.Lesion
Figure Lesion development
development after
after inoculation
inoculation with
with F. F. incarnatum,
incarnatum, control control
without without
VOCs and VOCs
PEA, and
PEA, muskmelons
muskmelons exposedexposed
to VOCstoofVOCs of Trichoderma
Trichoderma asperellumasperellum
T76-14 andT76-14 and phenylethyl
phenylethyl alcohol
alcohol (PEA).
(PEA). indicate
Arrows Arrowsthe indicate the inoculation
inoculation points. points.
J. Fungi 2021, 7, 46 9 of 13
Figure 7. Lesion development after inoculation with F. incarnatum, control without VOCs and
PEA, muskmelons exposed to VOCs of Trichoderma asperellum T76-14 and phenylethyl alcohol
(PEA). Arrows indicate the inoculation points.

Figure
Figure 8. Lesion
8. Lesion diameterdiameter on muskmelons
on muskmelons in themuskmelons
in the control versus control versus
exposedmuskmelons
to Tricho- exposed to Tricho-
derma asperellum T76-14 (T76-14) and phenylethyl alcohol (PEA). Different letters indicate statisti-
derma asperellum T76-14 (T76-14) and phenylethyl alcohol (PEA). Different letters indicate statistically
cally significant differences among treatments (p < 0.05) using Tukey’s test.
significant differences among treatments (p < 0.05) using Tukey’s test.
3.6. VOCs of T. asperellum T76-14 Suppressed Fruit Rot in Muskmelons
3.6. VOCs of T. asperellum T76-14 Suppressed Fruit Rot in Muskmelons
Based on the results above, we hypothesized that the VOCs emitted from T. asperel-
lum may Based on muskmelons
preserve the results from
above,fruitwe
rothypothesized
during postharvestthat the VOCs
storage. to test from T. asperellum
emitted
In order
may
the preserve
effect muskmelons
of VOCs on from postharvest,
muskmelons during fruit rot during
fruit rot postharvest
was observed fromstorage.
the In order to test
day
theafter exposure
effect with on
of VOCs VOCs or PEA. The results
muskmelons duringshowed that, after fruit
postharvest, incubation at anobserved from the
rot was
ambient temperature for seven days, rotten tissue was observed in control muskmelons
day after exposure with VOCs or PEA. The results showed that, after incubation at an
and muskmelons exposed to PEA, whereas VOC-treated muskmelons did not have rot
ambient
(Figure temperature
7). Fungal for seven
mycelia-covered tissuesdays, rottenwere
at peduncle tissue
mostwas observed
prominent in theincon-
control muskmelons
andfollowed
trol, muskmelons exposed
by PEA-treated to PEA, whereas
muskmelons, VOC-treated
causing tissue rot inside the muskmelons
muskmelon. did not have rot
Muskmelons
(Figure 7).exposed
Fungaltomycelia-covered
VOCs from T. asperellum T76-14
tissues showed less
at peduncle fungal
were mycelia
most on
prominent in the control,
the fruit peduncle and no rotten tissue inside seven days after incubation.
followed by PEA-treated muskmelons, causing tissue rot inside the muskmelon. Muskmel-
4.ons exposed to VOCs from T. asperellum T76-14 showed less fungal mycelia on the fruit
Discussion
peduncle and no
In the present rotten
study, tissue inside
T. asperellum T16-14seven days
exhibited after incubation.
antifungal activity against the
mycelial growth of F. incarnatum, the causal microorganism of postharvest fruit rot in
4. Discussion
muskmelons. T. asperellum T16-14 effectively inhibited mycelial growth of F. incarnatum
In the present study, T. asperellum T16-14 exhibited antifungal activity against the
mycelial growth of F. incarnatum, the causal microorganism of postharvest fruit rot in
muskmelons. T. asperellum T16-14 effectively inhibited mycelial growth of F. incarnatum
in vitro by a dual-culture assay, which suggested a competition mechanism (Figure 2). The
sealed plate method also revealed the antibiosis mechanism of T. asperellum T16-14 against
F. incarnatum (Figure 3). The dominant volatile compound released by T. asperellum T16-14,
tentatively identified as PEA based on SPME GC/MS, was involved in antifungal activity
against F. incarnatum (Figures 4 and 5). VOCs released by T. asperellum T16-14 caused
abnormal changes in the F. incarnatum mycelia (Figure 6), reducing the postharvest fruit rot
in muskmelons (Figures 7 and 8).
In a primary screening by dual-culture assay, T. asperellum T76-14 grew faster than
F. incarnatum on PDA (Figure 3), suggesting a competition mechanism. This mechanism
is common in several species of Trichoderma against plant pathogens [4,5,28]. In the study
above, T. asperellum T76-14 was found to produce VOCs (Figure 3 and Table 1) that slow
the fungal growth of F. incarnatum. This production involved an antibiosis mechanism
within the Trichoderma species. It has been established that several Trichoderma species
emit volatile antifungal compounds that inhibit pathogen growth [4–6,25]. In this study,
volatiles emitted from T. asperellum T76-14 inhibited the fungal growth of F. incarnatum by
62.5% (Figure 2), as determined through the sealed plate method. The VOCs emitted by
T. asperellum T76-14 slow the fungal growth of F. incarnatum, similar to PEA. Both VOCs
and PEA caused abnormal changes in the fungal mycelia (Figure 6), which may result in
inhibiting the fungal growth of F. incarnatum. As shown in Figure 4 and Table 1, GC/MS
revealed some minor peaks, which may have contributed to antifungal activity due to a
low percent peak area. This revealed that the VOCs emitted by T. asperellum T76-14 were
responsible for the antifungal activity against F. incarnatum. The GC/MS results presented
17 compounds; however, according to the literature, the only antifungal compound found
in this study was PEA (Figures 4 and 5).
J. Fungi 2021, 7, 46 10 of 13

PEA, also known as an aromatic alcohol, is an organic compound found as a colorless

liquid with a rose-like odor. This compound occurs widely in nature as an essential oil
from several plant species such as roses, jasmine, carnations, and hyacinths [29]. PEA
has been used to modify certain flavor compositions of foods with low toxicity [30,31].
It has been shown that PEA has antimicrobial properties [29,30,32–34]. An in vitro study
revealed that PEA inhibits fungal growth and the synthesis of RNA, DNA, and the protein
of Neurospora crassa [32]. PEA has been reported to show strong antifungal activity against
Ganoderma boninense [35], significantly reducing fungal growth to prolong the postharvest
life of strawberries [34], and controlling the blue mold decay caused by Penicillium digitatum
and P. italicum in citrus fruits [36]. Our result is in agreement with previous reports that
PEA inhibited the fungal growth of F. incarnatum in vitro (Table 2) and reduced lesions on
muskmelon fruits (Figures 7 and 8). This also proved that PEA in VOCs released from
T. asperellum T76-14 had antifungal activity against F. incarnatum.
PEA has been reported to be produced by several Trichoderma species. For instance,
PEA emitted by Trichoderma sp. [29], T. atroviride [37], T. harzianum [38], T. spirale [5], and
T. virens [36] displayed antifungal ability against several plant pathogens. In this study,
PEA emitted by T. asperellum T76-14, is a dominant compound with 23.188% peak area
(Table 2), and has been reported to have antifungal activities [34,35]. Therefore, the ability
of T. asperellum T76-14 to produce volatile antifungal compounds would further promote
antifungal activity against F. incarnatum. In this study, we investigated the effect of VOCs
emitted by T. asperellum T76-14 on fungal growth, morphology changes of fungal mycelia,
and the reduction in postharvest fruit rot, as well as the prolonged postharvest life of
muskmelons. In comparison with commercial PEA, VOCs of T. asperellum T76-14 prevented
fruit rot in muskmelons, which may be due to a complex mix of VOCs with other volatiles,
working synergistically to inhibit fungal growth. Commercial volatile PEA can limit lesion
development at three days after treatment (Figure 7); however, at seven days it showed
fruit rot similar to the control (Figure 9). This may result from PEA vapor after incubation
in a long period in a shared-atmosphere plastic box. The use of fresh Trichoderma11may
J. Fungi 2021, 7, x FOR PEER REVIEW of 14
produce VOCs continuously; however PEA was applied once so the concentration may
reduce after a long period of incubation.

Figure 9.
Figure 9. Effect
Effect of
of VOCs
VOCs and
and PEA
PEA on
on postharvest
postharvest fruit
fruit rot
rot in
in muskmelons,
muskmelons, T76-14
T76-14 == Trichoderma
Trichoderma as-
asperellum T76-14 and PEA = phenylethyl alcohol. Arrows indicate the rotten
perellum T76-14 and PEA = phenylethyl alcohol. Arrows indicate the rotten tissue. tissue.

Several reports have clarified the effect of microbial volatiles as a biofumigant con-
trolling postharvest diseases and prolonging postharvest life. For instance, Li et al. [39]
used VOCs produced by Ceratocystis fimbriata as biofumigation on postharvest disease of
J. Fungi 2021, 7, 46 11 of 13

Several reports have clarified the effect of microbial volatiles as a biofumigant con-
trolling postharvest diseases and prolonging postharvest life. For instance, Li et al. [39]
used VOCs produced by Ceratocystis fimbriata as biofumigation on postharvest disease
of fruit. The authors showed that VOCs inhibited the growth of Monilinia fructicola and
Penicilium digitatum (causing peach brown rot and citrus green mold, respectively) [39].
Volatile compounds produced by Aureobasidium pullulans increased fruit waxes’ complexity,
reducing pathogen attacks on stone fruit [40]. Furthermore, VOCs emitted from T. atroviride
inhibited the mycelial growth of Phytophthora infestans in postharvest potato tubers [41].
The authors investigated VOCs by GC/MS and found abundant compounds, namely
3-mrthyl-1-butanol, p-6-pentyl-2-pyrone, 2-mthyl-1-propanol, and acetone, which were re-
sponsible for serious morphological and ultrastructural damage of the pathogens [41]. Our
findings are in agreement with [41]: T. asperellum T76-14 produced VOCs, which contained
dominant volatiles of PEA. VOCs of T. asperellum T76-14 are responsible for inhibiting
postharvest fruit rot pathogens and could prolong the postharvest life of muskmelons.
During postharvest storage, some fungi or mold can infest the fruit surface and penetrate
into tissues, causing fruit rot. This finding indicates that VOCs released from T. asperellum
T76-14 contain PEA to clean the fruit surface and reduce fungal infection, which may
prolong the postharvest life of muskmelons.

5. Conclusions
Herein, we described the ability of VOCs emitted by T. asperellum T76-14 involved in
antifungal activity against the postharvest fruit rot pathogen (F. incarnatum) of muskmel-
ons to reduce fruit rot during postharvest storage by inhibiting postharvest pathogens.
However, the extraction of valuable compounds, and the quality and quantity of volatiles
emitted by this strain, have yet to be investigated. In order to apply VOCs from T. asperellum
T76-14 as a biofumigant to control postharvest disease and maintain postharvest quality
on a large scale, further experiments need to be conducted.

Author Contributions: Conceptualization, W.I. and A.S.; methodology, W.I. and S.K.; software, W.I.
and A.S.; validation, A.S.; formal analysis, A.S.; investigation, W.I. and S.K.; resources, A.S.; data
curation, A.S.; writing—original draft preparation, W.I. and A.S.; writing—review and editing, W.I.,
S.K. and A.S.; visualization, A.S.; supervision, A.S.; project administration, A.S.; funding acquisition,
A.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Research Council of Thailand (NRCT) via grant
NRCT5-RSA63022-01.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Special thanks go to Prince of Songkla University and the Center of Excellence
in Agricultural and Natural Resources Biotechnology (CoE-ANRB) phase 3 for facilities and Kitsada
Pitija, PerkinElmer Co. Ltd., Bangkok, Thailand for the GC/MS analysis and MDPI’s English editing
service for English editing.
Conflicts of Interest: The authors declare no conflict of interest.

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