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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:xdzheng@zju.edu.cn(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:xdzheng@zju.edu.cnhttp://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:xdzheng@zju.edu.cn

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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.

W. Feng et al. / Food Control 22 (2011) 78e81 79

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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