3 Thyme Oil to Control Alternaria Alternata in Vitro and in Vivo as Fumigant and Contact Tratments
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Thyme oil to control Alternaria alternata in vitro and in vivo as fumigant and
contact treatments
Wu Feng a,b, Jiaping Chen a, Xiaodong Zheng b,*, Qing Liu a
a College of Food Science and Technology, Huazhong Agricultural University, 430070 Wuhan, Hubei, People s Republic of Chinab Department of Food Science and Nutrition, Zhejiang University, 310029 Hangzhou, Zhejiang, People s Republic of China
a r t i c l e i n f o
Article history:
Received 12 March 2010
Received in revised form
3 May 2010
Accepted 11 May 2010
a b s t r a c t
This study was conducted to evaluate the efcacy of thyme oil in suppressing Alternaria alternata(Fr.:Fr.)
Keissl in vitro and in vivo as fumigant and contact treatments. Thyme oil possessed great fumigant and
contact toxicity against A. alternata at different concentrations in vitro. Irreversible inhibition of fungal
growth could be caused by exposure to 500 mL/L thyme oil as a fumigant for 6 and 12 days. The spores of
A. alternatawere completely suppressed 6 h after incubation in potato dextrose broth (PDB) by 2000 mL/L
of thyme oil. The thyme oil as a fumigant at 66.7 mL/L showed a signicant inhibition effect onA. alternata
of cherry tomatoes stored at 25 C for 5 days. Thyme oil at 500 mL/L showed a signicant contact inhi-
bition effect on A. alternata of cherry tomatoes stored at 25 C for 3 days.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Fruits and vegetables are highly perishable products, especially
during the postharvest phase, when considerable losses can occur
(Janisiewicz & Korsten, 2002; Spadaro & Gullino, 2004). Cherry
tomatoes (Lycopersicon esculentum) are one of the most widely
produced and consumed horticultural crops in the world, both for
the fresh produce market and the processed food industries.
Postharvest rots of cherry tomatoes are mainly caused by fungal
pathogens such as Alternaria alternata, Botrytis cinerea and Alter-
naria solani (El Ghaouth, Ponnampalam, Castaigne, & Arul, 1992).
Synthetic chemical fungicides are the primary means to control
postharvest diseases of fruits or vegetables. But repeated use of
certain synthetic chemical fungicide in packinghouses has led to
the appearance of fungicide-resistant populations of storage
pathogens (Spotts & Cervantes, 1986; Stange & Eckert, 1994).
Furthermore, the use of synthetic chemicals is becoming more
difcult to justify, because of the increasing concerns for humanhealth as well as environmental considerations (Okigbo &
Ikediugwu, 2001). Thus there has been considerable interest in
exploring new alternatives in order to reduce the use of synthetic
fungicides.
In recent years, many researchers have shown that natural
sources such as essential oils ofMonarda citriodora var. citriodora,
Melaleuca alternifoliaand citrus fruit could develop as a promising
alternative to chemical control (Bishop & Thornton, 1997; Caccioni,
Guizzardi, Biondi, Renda, & Ruberto, 1998). Essential oils and their
components are gaining increasing interest because of their rela-
tively safe status, their wide acceptance by consumers, and their
exploitation for potential multi-purpose functional use (Ormancey,
Sisalli, & Coutiere, 2001; Sawamura, 2000). There are hundreds of
essential oils available for use, many with known antifungal
properties (Ahmet, Saban, Hamdullah, & Ercan, 2005; Montes-
Belmont & Convajal, 1998; Nielsen & Rios, 2000).
Fumigants are ideal for postharvest treatment because they
allow minimal handling of the fruits or vegetables. But there are
few fumigants available for postharvest disease control. The
toxicity of essential oils upon solution contact as measured by the
broth dilution and agar dilution methods has been studied by many
researchers (Rasooli & Abyaneh, 2004). However, the toxicity by
vapour contact has been reported more rarely.
The objective of this study was to assess antifungal effects of
thyme oil against A. alternata in vitro and in vivoby fumigant andcontact treatments.
2. Materials and methods
2.1. Essential oil
Pure-grade (not containing synthetic chemicals and/or
nonnatural components) thyme oil was obtained from Interna-
tional Flavors & Fragrances Inc., Shanghai, China. Thyme oil was
stored in bottles at 4 C.* Corresponding author. Tel.:86 571 86971167; fax: 86 571 871167.
E-mail address:[email protected](X. Zheng).
Contents lists available at ScienceDirect
Food Control
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . co m / l o c a t e / f o o d c o nt
0956-7135/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.05.010
Food Control 22 (2011) 78e81
mailto:[email protected]://www.sciencedirect.com/science/journal/09567135http://www.elsevier.com/locate/foodconthttp://dx.doi.org/10.1016/j.foodcont.2010.05.010http://dx.doi.org/10.1016/j.foodcont.2010.05.010http://dx.doi.org/10.1016/j.foodcont.2010.05.010http://dx.doi.org/10.1016/j.foodcont.2010.05.010http://dx.doi.org/10.1016/j.foodcont.2010.05.010http://dx.doi.org/10.1016/j.foodcont.2010.05.010http://www.elsevier.com/locate/foodconthttp://www.sciencedirect.com/science/journal/09567135mailto:[email protected] -
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2.2. Fungi and cultures
A. alternata was obtained from Institute of Microbiology,
Chinese Academy of Sciences. A. alternatawas cultured on potato
dextrose agar medium (PDA) at 28 C for 7 days.
2.3. In vitro antifungal assay
2.3.1. Fumigation bioassay
The experiments were conducted in petri plates (90 mm e in
diameter) of PDA (15 ml/plate), inoculated with 6 mm plugs from
cultures (7-days-old). Plates were kept in an inverted position.
A sterilized lter paper disc (diameter 6.0 mm) was placed in the
center of the lid and different volumes (1 ml/platee5 ml/plate) of
thyme oil were added to the paper. Blank served as control. Three
replicates were used per treatment. Plates were tightly sealed with
paralm and incubated for 7 days at 28 C. The fungi growth
recorded after 7 days. Growth inhibition was calculated as the
percentage of inhibition of radial growth relative to the control.
Experiments were performed three times.
2.3.2. Contact bioassayThe experiments were conducted based on the method of
Soliman and Badeaa (2002).Potato dextrose agar (PDA) was auto-
claved and cooled in a water bath to 40 C. The thyme oil was mixed
with sterile molten PDA to obtain nal concentrations of 0, 100,
200, 300, 400 and 500 mL/L. The PDA was poured into 90 mm petri
plates (15 ml/plate) that were then inoculated with 6 mm plugs
from 7-days-old cultures. Threereplicates were used per treatment.
Plates were incubated for 7 days at 28 C. Fungal growth was
recorded after 7 days. Growth inhibition was calculated as the
percentage of inhibition of radial growth relative to the control.
Experiments were performed three times.
2.3.3. Transfer experiment
Tomake a distinction between fungistatic or fungicidal effects of
the essential oil on the target organism, transfer experiments were
done. Plugs that did not grow were transferred to fresh PDA to
assess their viability after one, three, six and twelve daysexposure
at 28 C. The residual fungal growth was monitored by measuring
the radial growth of the fungi.
2.3.4. Fungicidal kinetics of thyme oil
Thyme oil was added to a 10 ml glass tube containing 5 ml PDB
to obtain nal concentrations of 0, 100, 300, 500,1000 and
2000 mL/L. At the same time, aliquots (100 ml) of spore suspensions
(1 107 spores/ml) of A. alternata were added to each tube.
Samples were taken after the time intervals and were cultured on
PDA for 48 h at 30 C. 100 ml of dilution solvent was added to
control tubes instead of essential oil. Microbial colonies were
counted after incubation period and the total number of viable
spores per ml was calculated. The calculation was converted to
percent dead spores using routine mathematical formulae.
2.4. In vivo antifungal assay
2.4.1. Control rot of cheery tomatoes by essential oil as a fumigant
Experiments were conducted with commercially grown toma-
toes from Fujian, China. Fruits were selected for free of injuries then
infections placed in plastic boxes (22 16.2 5.5 cm). Fruits were
disinfected with 2% sodium hypochlorite for 2 min, rinsed with tap
water, and air-dried before wounding.
A uniform 2-mm deep and 5-mm wide wound was made on
their peel at the equatorial of each fruit using a sterile puncher.
Then, 10 ml of conidial suspension ofA. alternata(5 104/ml) was
pipetted into each wound.
The essential oil was selected in the preliminary experiment.
Different doses (25 ml, 50 ml and 100 ml) of essential oil were added
to sterilized lter papers that were placed in the lids of Petri plates.
The concentrations were 16.7 ml/l, 33.3 ml/l and 66.7 ml/l. Filterpaper
was added to each box immediately after inoculation and the boxes
were sealed with lms. Treated fruits were stored at 25 C. The
percentage of infected fruits was recorded after 5 days of incuba-
tion. There were 20 fruits in each treatment and the treatments
were replicated three times. The experiment was repeated two
times.
2.4.2. Effects of thyme oil on decay development in articially
inoculated and wounded fruits
Cheery tomatoes were wounded with a sterile puncher to make
one uniform 2-mm deep by 5-mm wide wound on their peel at the
equatorial region. Aliquots of 20 ml of 100, 200, 300, 400, 500 mL/L
thyme oil and sterile distilled water (control) were pipetted into
each wound site. After 0.5 h, 10 ml of conidial suspension of
A. alternata(5 104/ml) was pipetted into each wounds. Treated
tomatoeswere stored at 20 C. The percentage of infected fruits was
recorded after 5 days of incubation. Each treatment was replicatedthree times with 20 fruits per replicate and the entire experiment
was repeated twice.
3. Statistical analyses
Statistical analyses of the data were performed with SPSS
statistical software (SPSS for Windows v.11.5).
4. Results
4.1. In vitro antifungal assay
4.1.1. Fumigation bioassay
The thyme oil vapour was toxic to A. alternata at all concentra-tions. The inhibition ratio was proportional to its concentration.
Mycelial growth ofA. alternata was inhibited 48.0% at 1 ml/plate and
100% inhibited at 5 ml/plate (Fig. 1).
4.1.2. Contact bioassay
The thyme oil had good contact inhibitory effect onA. Alternata,
the radial growth of them was inhibited above 62.0% at 500 mL/L.
The results shown that the inhibition ratio was proportional to its
concentration (Fig. 2).
A B C D E
0
20
40
60
80
100
dc
b
aa
Treatments
Inhibition(%)
Fig. 1. Fumigant antifungal activity of thyme oil on A. Alternata A. 1 mL/plate B. 2 mL/
plate C. 3 mL/plate D. 4 mL/plate E. 5 mL/plate.
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4.1.3. Transfer experiment
To make a distinction between fungistatic or fungicidal effects ofthe essential oil on the target organism, transfer experiments were
done. Thyme oil acted as fungistatic agentsat 5 ml/plate after 3 days
exposure and as fungicidal agents after 6 and 12 days exposure.
Thyme oil presented weak fumigant toxicity toA. alternataafter 1
days exposure (Fig. 3).
4.1.4. Fungicidal kinetics of thyme oil
The fungistatic effect of thyme oil at 100e500 mL/L was insig-
nicant. The Spore germination ofA. alternatawas inhibited 100%
at 2000 mL/L after 6 hcontact (Fig. 4).
4.2. In vivo antifungal assay
4.2.1. Control rot of cheery tomatoes by essential oil as a fumigant
The results shown in Fig. 5indicated that in cherry tomatoes
fumigated by thyme oil, the percentage of infected fruits in all
treated cherry tomatoes was signicantly lower than that in the
control cherry tomatoes (p < 0.05). The percentages of decayed
cherry tomatoes fumigated by thyme oil at 16.7 ml/l, 33.3 ml/l and
66.7 ml/l were reduced by 14.2%, 26.6% and 42.7% compared to the
control, respectively. Fumigation with thyme oil did not cause any
visible disorders to the fruits after 5 days of incubation.
4.2.2. Effects of thyme oil on decay development in articially
inoculated and wounded fruits
The results shown in Fig. 6indicated that in cherry tomatoes
treated by thyme oil, the percentage of infected fruits in all treated
cherry tomatoes was signicantly lower than that in the control
cherry tomatoes (p < 0.05). The percentages of decayed cherry
tomatoes treated by thyme oil at 500 mL/L were reduced by about
40%. Treated with thyme oil did not cause any visible disorders and
off-avor to the fruits after 3 days of incubation.
5. Discussion
This work highlights the antifungal properties of thyme oil and
the potential for using thyme oil by different treatments for post-
harvest disease control of fresh tomatoes.
Results of the present work showed that thyme oil was seen toexert good antifungal activities both in vitroandin vivoby different
treatments. Under in vitro conditions, the percentage of fungal
inhibition was dependent on thyme oil concentration by different
treatments: the more signicant the decrease in the mycelial
growth was, the higher the increase in thyme oil concentration. But
in vivoconditions, the inhibition effect of thyme oil on A. alternata
in cherry tomatoes by different treatments was not as dramatic as
A B C D E
0
10
20
30
40
50
60
70
d
c
bb
a
)%(noitibihnI
Treatments
Fig. 2. Contact antifungal activity of thyme oil on A. Alternata A.100 mL/L B.200 mL/L
C.300 mL/L D.400 mL/L E.500 mL/L.
A B C D E F G
0
10
20
30
40
50
60
70
80
)
mm(retemaid
days
0 days
1 days
3 days
6 days
12 days
Fig. 3. The inhibitory effects of thyme oil on A. alternata The plugs ofA. alternata were transferred to PDA plates after 1, 3, 6 or 12 days of exposure to 5 mL thyme oil.
0 2 4 6 8 1 0 1 2
-1
0
1
2
3
4
5
6
)lm/ufcgol(stnuocelbaiV
Time (h)
Fig. 4. Time-kill plots of thyme oil against spores ofA. Alternata -control C100mL/L
:300 mL/L;500 mL/LA1000 mL/L+2000 mL/L.
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that in plates. Arras and Usai (2001) also showed that thyme
essential oil was less effective on orange fruits thanin vitroagainst
Penicillium digitatum. Because the lter paper discs containing
essential oils did not contact the fungi tested directly in our study,
the fumigant toxicity of the volatile portion of the essential oils
could be evaluated. It is necessary to mention that how to differ-entiate the direct effect of vapour on fungi from the indirect effect
of vapour acting after absorption by the medium during a long
incubation period. Due to the hydrophobic nature of compounds
that constitute the volatile fraction of essential oil, it could be
expected that their dilution in the agar medium during incubation
period was not so important. Because the high lipophilic nature of
mycelia coupled with a large surface area relative to the volume of
a fungus, vapours of essential oils may act mainly by accumulation
on mycelia than in the agar (Inouye et al., 2000). Some studies
(Alvarez-Castellanos, Bishop, & Pascual-Villalobos, 2001; Feng &
Zheng, 2007) suggest that the time of exposure is also a critical
parameter for the susceptibility of conidia, germinated conidia and
hyphae to antifungal compounds. Our experiments also revealed
that thyme oil was proved to have fungistatic action at lowerconcentration or for short exposure time and fungicidal action at
higher concentration or for long exposure time.
The results suggested that a maximal vapour level of 5 mL/plate
it completely 100% suppress the growth of A. alternata. The level
was lower than that required (7.5 mL/plate) for the suppression of
A. alternata by contact treatment. The result further supports the
results of Lehtijrvi (2006)who found that both the contact and
volatile assays signicantly reduced the fungi in comparison with
the control, the volatile assay was more effective than the contact
assay in vitro. The fungitoxic activity of the essential oils was
probably due to volatile phenolic compounds such as the mono-
terpenes (Faid, Charai, & Mosaddak, 1996). Non-phenolic volatile
compounds, such as AITC, citral and limonene, were most effective
when applied through the air phase (Suhr & Nielsen, 2003). Our
results showed that thyme more effective as fumigant treatments
than by contact treatments. Thyme oil was proved to have fungi-
static action at lower concentration or for short exposure time and
fungicidal action at higher concentration or for long exposure time.
Alvarez-Castellanos et al., (2001) mentioned that the results from
the agar diffusion plate assay might also include some effects of the
oil vapours besides the contact action.
Application of thyme oil via the vapourphase should alsomake its
use more effective, rapid and convenient than dipping. Therefore, it
can be used as a potential sourceof sustainable eco-friendly botanical
fungicides, after successful completion of wide range trials.
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674.
A B C D0
20
40
60
80
100
d
c
b
a
)%(stiurfdetcef
nI
Treatments
Fig. 5. Inhibition of A. alternata on cherry tomatoes by thyme oil as a fumigant
A. Control B. 16.7 mL/L C. 33.3 mL/L D. 66.7 mL/L Signicant differences (p < 0.05)
between means are indicated by letters above histogram bars. Where the letters are
the same, there is no signicant difference between different treatments.
A B C D E F0
20
40
60
80
100
e
dc
bb
a
)%(stiurFdetcefnI
Treatments
Fig. 6. Inhibition ofA . alternata on articially inoculated and wounded fruits cherry
tomatoes by thyme oil (A) Control (B) 100 mL/L (C) 200 mL/L (D) 300 mL/L (E) 400 mL/L
(F) 500 mL/L Signicant differences (p < 0.05) between means are indicated by letters
above histogram bars. Where the letters are the same, there is no signi cant difference
between different treatments.
W. Feng et al. / Food Control 22 (2011) 78e81 81