Appl Microbiol Biotechnol (2013) 97:7483–7492
DOI 10.1007/s00253-012-4460-y
ENVIRONMENTAL BIOTECHNOLOGY
Antifungal effects of citronella oil against Aspergillus niger
ATCC 16404
Wen-Ru Li & Qing-Shan Shi & You-Sheng Ouyang &
Yi-Ben Chen & Shun-Shan Duan
Received: 6 May 2012 / Revised: 14 September 2012 / Accepted: 20 September 2012 / Published online: 19 October 2012
# Springer-Verlag Berlin Heidelberg 2012
Abstract Essential oils are aromatic oily liquids obtained from
some aromatic plant materials. Certain essential oils such as
citronella oil contain antifungal activity, but the antifungal effect
is still unknown. In this study, we explored the antifungal effect
of citronella oil with Aspergillus niger ATCC 16404. The
antifungal activity of citronella oil on conidia of A. niger was
determined by poisoned food technique, broth dilution method,
and disc volatility method. Experimental results indicated that
the citronella oil has strong antifungal activity: 0.125 (v/v) and
0.25 % (v/v) citronella oil inhibited the growth of 5×105 spore/
ml conidia separately for 7 and 28 days while 0.5 % (v/v)
citronella oil could completely kill the conidia of 5×105
spore/ml. Moreover, the fungicidal kinetic curves revealed that
more than 90 % conidia (initial concentration is 5×105 spore/
ml) were killed in all the treatments with 0.125 to 2 % citronella
oil after 24 h. Furthermore, with increase of citronella oil
concentration and treatment time, the antifungal activity was
increased correspondingly. The 0.5 % (v/v) concentration of
citronella oil was a threshold to kill the conidia thoroughly.
The surviving conidia treated with 0.5 to 2 % citronella oil
decreased by an order of magnitude every day, and no fungus
survived after 10 days. With light microscope, scanning elec-
tron microscope, and transmission electron microscope, we
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-012-4460-y) contains supplementary material,
which is available to authorized users.
W.-R. Li : S.-S. Duan (*)
Research Center of Hydrobiology, Jinan University,
Guangzhou 510632, China
e-mail: tssduan@jnu.edu.cn
W.-R. Li : Q.-S. Shi : Y.-S. Ouyang (*) : Y.-B. Chen
Guangdong Provincial Key Laboratory of Microbial Culture
Collection and Application, State Key Laboratory of South China
Applied Microbiology (Ministry-Guangdong Province Jointly
Breeding Base), Guangdong Institute of Microbiology,
Guangzhou 510070, China
e-mail: ouyang6411@21cn.com
found that citronella oil could lead to irreversible alteration of
the hyphae and conidia. Based on our observation, we hypoth-
esized that the citronella oil destroyed the cell wall of the A.
niger hyphae, passed through the cell membrane, penetrated
into the cytoplasm, and acted on the main organelles. Subse-
quently, the hyphae was collapsed and squashed due to large
cytoplasm loss, and the organelles were severely destroyed.
Similarly, citronella oil could lead to the rupture of hard cell
wall and then act on the sporoplasm to kill the conidia. Never-
theless, the citronella oil provides a potential of being a safe and
environmentally friendly fungicide in the future.
Keywords Citronella oil . Antifungal effects . Act
mechanism . Aspergillus niger
Introduction
Molds, or mildewy fungi, are large filamentous fungi with more
developed myceliums but lacking large fruit bodies (Zhou
2003; Charles et al. 1947). They commonly grow and propagate
in moist climate, resulting in milden and rotting of industrial
and agricultural products such as food, feedstuff and leather, etc.
Mold contamination cannot only induce economic loss but also
threaten human health and environmental safety. In recent
years, the importance of the Aspergillus genus has increased
owing to its role in the etiology of systemic mycoses, allergic
reactions, and mycotoxin contamination of food (Tolouee et al.
2010). Aspergillus niger, a most important species of Aspergil-
lus genus, is nearly ubiquitous in our environment. Although A.
niger is also an important fermentation industry strain, produc-
ing many enzymes, such as amylase, cellulase, and pectinase
(Tolouee et al. 2010), it constantly cause food spoilage, indus-
trial and agricultural products moldy, and mycotoxin pollution
(ochratoxin A). Especially, A. niger could induced otomycosis
and pulmonary infections in immunocompromised persons
(Tolouee et al. 2010). While there are many ways to protect
7484
industrial and agricultural products from molding, such as
dehumidification, temperature control, and ultraviolet irradia-
tion etc., using mildew inhibitors is also a simple and easy
approach (Tolouee et al. 2010). With more strict environmental
regulations, many existing fungicides are prohibited, such as
dimethylfumarate (European Commission 2009, 2012). It is
urgent to research and develop natural, nontoxic, and environ-
mental friendly fungicides.
Essential oils are also called volatile or ethereal oils (Burt
2004). They are aromatic oily liquids obtained from some
aromatic plant materials by distillation and squeezing, such
as rosemary and sage from leaves, clove from flower buds,
garlic and onion from bulbs, asafetida from rhizomes, pep-
per from fruit, cardamom from seeds, and cinnamon from
barks (Shelef 1983; Ceylan and Fung 2004; Burt 2004). It
has been found that essential oils have both antibacterial and
antifungal activities (Arora and Kaur 1999; Inouye et al.
2001; Chuang et al. 2007; Bajpai et al. 2009; Al-Ja’fari et al.
2011; Tian et al. 2011), and fungi are more sensitive to
essential oils than bacteria (Shelef 1983; Ceylan and Fung
2004). In fact, essential oils have been widely used for
antibacterial, antifungal, antiviral, and cosmetic applications
since the middle ages (Bakkali et al. 2008). Nowadays,
essential oils are also applied in pharmaceutical, sanitary,
cosmetic, and agricultural and food industries (Bakkali et al.
2008). Currently, more than 300 essential oils are consid-
ered commercially important (Burt 2004). However, cur-
rently we know very little about the antifungal effect and
mechanism of essential oils. To study the antifungal effect of
essential oil, we screened the antifungal effects of some
essential oils on molds (the data have not been published),
and we found citronella oil extracted from Cymbopogon
nardus had very strong antifungal activity. Citronella is the
common name of the genus Cymbopogon plants, popularly
known by its characteristic insect-repellent feature (Beneti et
al. 2011). Citronella oil is composed mostly by monoter-
penes (±80 %), which mainly includes citronellal, citronel-
lol, and geraniol, although sesquiterpenes are also found
(Beneti et al. 2011). Citronella oil has been used tradition-
ally for antispasmodic, rubefacient, stimulant, insect repel-
lant, carminative, and diaphoretic (Man et al. 2012). It is
also widely used in the perfume industry and soap manu-
facturing (Man et al. 2012), and the citronella oil is known
for its antimicrobial activity (Billerbeck et al. 2001). One
research group in France screened 37 essential oils to find
the most antifungal ones, and they found that C. nardus and
Cymbopogon martinii oils were the most fungistatic species
(Delespaul et al. 2000). To figure out the antifungal effect
and mechanism of the citronella oil, in this paper, we select-
ed A. niger American Type Culture Collection (ATCC)
16404, the commonly contaminated mold, to investigate
the antifungal activity of citronella oil and attempt to illu-
minate its antifungal mechanisms.
Appl Microbiol Biotechnol (2013) 97:7483–7492
Materials and methods
Chemical reagents, organism, mediums, and cultivation
Citronella oil was purchased from Guangzhou Baihua Fla-
vours and Fragrances Company Ltd (Guangzhou, China). The
citronella oil is pure with a density of 0.866 g/ml. The strain of
A. niger ATCC 16404 was purchased from ATCC and con-
served in our laboratory. Potato dextrose agar (PDA) medium
(purchased from Guangzhou Huankai Microbial Sci. and
Tech. Co., Ltd, Guangzhou, China) contains the following
approximate formula (per liter): 300 g potatoes infusion,
20 g dextrose, 15 g agar, and 0.1 g chloramphenicol. Potato
dextrose water (PDB) medium (purchased from Qingdao
Hope Bio-Technology Co., Ltd., Qingdao, China) contains
the following (per liter): 6 g potato extract and 20 g dextrose.
The fungus was cultured on PDA slants for 7 days at 28 °C in
an incubator. Conidia suspension of A. niger was prepared by
gently scraping the culture surface using a sterile glass rod
after adding 3- to 5-ml 0.1 % aqueous solution of Tween 80.
After being filtered through gauze, the density of conidia
suspension was adjusted by hemacytometer. PDB medium
was used for a submerged culture of A. niger. The solution
of 0.5 % Tween 20 was used to dilute and to promote the
dispersibility of citronella oil. PDA- and PDB-modified media
were separately added 0.5 % Tween 20 in the PDA and PDB
to disperse the citronella oil better. All solvents and reagents
were of analytical grade.
GC-MS analyses of the citronella oil
The citronella oil was analyzed using a Thermo Finnigan
TraceGC ultra-gas chromatograph equipped with Finnigan
Trace DSQ mass spectrometer. DB 5-ms column (30 m
length×0.25 mm ID×0.25 μm particle diameter) was used.
Operating conditions were undertaken at oven temperature
programmed from 60 to 220 °C (retain for 15 min) at 10 °C/
min. The injection port temperature was from an initial 70 to
250 °C. Helium was used as the carrier gas at a flow rate of
1.0 ml/min. PTV split injection and the split ratio was 1:50.
Ionization was obtained by electron impact (source temperature
200 °C) and scan range was 30–400 m/z. Compounds were
tentatively identified by comparison of mass spectra of each
peak with those of authentic samples in the NIST MS library.
Antifungal activity measurements
The antifungal activity was measured by poisoned food tech-
nique, broth dilution method, and disc volatility method. First,
the poisoned food technique method (Sharma and Tripathi
2008) was as follows: PDA-modified medium (10 ml) includ-
ing different content of citronella oil was poured into sterilized
Petri dishes and the concentrations (v/v) of citronella oil were
Appl Microbiol Biotechnol (2013) 97:7483–7492
0 (as control), 0.0625, 0.125, 0.25, 0.5, and 1 %, respectively.
The spore suspension containing about 105 conidia was
streaked over the surface of PDA using a sterile glass stick
in order to get a uniform fungal growth on both control and
test plates. The plates were sealed by parafilm incubated at
28 °C in a static condition for 28 days. The lowest concen-
trations (MIC) of citronella oil showing no visible growth
until incubated for 7 and 28 days were determined separately.
Second, MIC and MFC determined by broth dilution method
(Li et al. 2010; Li et al. 2011) in test tubes was as follows:
different volume of PDB-modified medium, spore suspension
of A. niger, and 50 % dilutions of citronella oil by 0.5 %
Tween 20 were added separately to 5 ml cultures in six test
tubes, resulting in final concentrations (v/v) of citronella oil of
0 % (as control), 0.0625, 0.125, 0.25, 0.5, and 1 %, respec-
tively, and concentration of 105 spore/ml of A. niger. The
cultures were incubated in a water bath shaker at 28 °C with
shaking at 150 rpm. The germination of conidia and growth of
myceliums were observed by visual inspection and micro-
scope. The lowest concentrations (MIC) of citronella oil at
which no growth occurred by visual inspection until incubated
for 7 and 28 days were determined separately. After being
incubated for 28 days, cells from the tubes showing no growth
were subcultured on PDA plates to determine whether the
conidia were killed or inhibited. The lowest concentration at
which the conidia were killed thoroughly was regarded as
MFC. Third, the disc volatility method was as follows: PDA
(10 ml) was poured into sterilized Petri dishes of 90 mm
diameter and the spore suspension containing about 105 co-
nidia was streaked over the surface of PDA using a sterile
glass stick in order to get a uniform fungal growth. Five pairs
of filter paper discs of 5-mm diameter were distributed in the
cover of each Petri dish, and each pair of discs were soaked by
100 μl different dilutions of citronella oil. Two pieces of filter
paper discs were overlapped in each pair of discs. The differ-
ent dilutions of citronella oil were as follows: 0 (as control),
6.25, 12.5, 25, 50, and 100 %, respectively. The final concen-
tration of citronella oil was equal to that of poisoned food
technique. The plates sealed by parafilm were placed upside
down and incubated at 28 °C in a static condition for 28 days.
The lowest concentrations (MIC) of citronella oil showing no
visible growth until incubated for 7 and 28 days were deter-
mined. In all the three methods, two separate experiments
were carried out in triplicate.
Fungicidal kinetics of the citronella oil
To get the fungicidal kinetics of the citronella oil, different
volume of PDB-modified medium, spore suspension of A.
niger, and 50 % dilutions of citronella oil by 0.5 % Tween 20
were added separately to 10-ml cultures in six test tubes,
resulting in final concentrations (v/v) of citronella oil of 0 (as
control), 0.125, 0.25, 0.5, and 1 %, respectively, and
7485
concentration of 105 spore/ml of A. niger. The cultures were
incubated in a water baths shaker at 28 °C with shaking at
150 rpm. Samples were taken after incubating for 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, and 14 days, and the gradient dilutions
of the samples were cultured on PDA Petri dishes for 72 h at
28 °C. The fungal colonies were counted after the incubation
period, and the total number of conidia alive per milliliter was
calculated. The curves of fungicidal kinetics were made based
on the total number of conidia alive per milliliter. Two separate
experiments were carried out in triplicate.
Effect of citronella oil on the morphological structure
of hyphae and conidia of A. niger based on observation
by microscope, SEM and TEM
Experiment methods for antifungal effect of citronella
oil on hyphae of A. niger
Spore suspension of A. niger was inoculated to 4.8-ml PDB-
modified medium cultures in three test tubes, and the concen-
tration was 105 spore/ml. The cultures were incubated in a water
bath shaker at 28 °C with shaking at 150 rpm for 15 h. Then
0.2-ml dilutions of citronella oil of different concentrations were
added to the cultures separately resulting in final concentrations
(v/v) of citronella oil of 0 (as control), 0.5, and 2 %, respectively.
The cultures were further incubated under the same conditions
for 28 days. When further incubated for 0, 5, and 24 h, the
mycelium culture were sampled and centrifuged to discard the
supernatants. Then the mycelium samples of 0 and 24 h were
placed on glass slides and dyed in lactophenol cotton for optical
microscope observation (Leica DMLB), and the mycelium
samples of 0 (as control), 5, and 24 h were prepared for
scanning electron microscope observation (SEM; Hitachi S
3000N). The mycelium samples of 0 (as control) and 24 h were
also prepared as well as ultrathin sections of mycelium for
transmission electron microscope observation (TEM; Hitachi
H-7650). After further incubation for 28 days, samples were
taken and cultured on PDA Petri dishes to measure if the
myceliums were killed completely. Two separate experiments
were carried out in triplicate.
Experiment methods for observing the effect of citronella oil
on the morphological structure of conidia of A. niger
Spore suspension of A. niger was inoculated to 4.8-ml PDB-
modified medium cultures in three test tubes, and the concen-
tration was 105 spore/ml. Then 0.2-ml dilutions of citronella oil
of different concentrations were added to the cultures separately
resulting in final concentrations (v/v) of citronella oil of 0 (as
control), 0.5, and 2 %, respectively. The cultures were incubated
in a water bath shaker at 28 °C with shaking at 150 rpm for
28 days. Two separate experiments were carried out in tripli-
cate. When incubated for 2 days, the conidia were sampled and
7486
centrifuged to discard the supernatants. Then the conidia sam-
ples were prepared for microscope and SEM observation. The
methods were the same as described above.
Results
GC-MS analyses of citronella oil
The main constituents of citronella oil identified by GC-MS
are presented as follows: citronellal (37.51 %), geraniol
(22.57 %), citronellol (15.12 %), geraniol acetate (4.48 %),
2,6-octadien, 2,6-dimethyl- (4.06 %), dipentene (3.24 %),
cyclohexanemethanol, 4-ethenyl-à,à,4-trimethyl-3-(1- meth-
ylethenyl)-, [1R-(1à,3à,4á)]- (2.7 %), 6,7-dimethyl-
1,2,3,5,8,8a-hexahydronaphthalene (2.32 %), (−)-beta-ele-
mene (2.11 %), 1,6-cyclodecadiene, 1-methyl-5-methylene-
8-(1-methylethyl)-, [s-(E,E)]- (1.58 %), linalool (0.7 %), 1-
methylbicyclo[2.2.1]heptan-exo-2-ol (0.65 %), nopol
(0.59 %), and other constituents (2.37 %).
The antifungal activity of citronella oil on conidia of A. niger
The results of poisoned food technique experiment incubated
for 28 days were shown in Fig. 1. After incubation for 7 days,
the Petri dishes of the control and 0.0625 % citronella oil
group were full of myceliums and black conidia though the
fungus growth of 0.0625 % group had a 1 day delay. While
nothing grew in Petri dishes of 0.125, 0.25, 0.5, and 1 %
groups. Until incubation for 28 days, only tens of colonies
grew in the 0.125 % group, but no fungus grew in 0.25, 0.5,
and 1 % groups. So the MIC of citronella oil against A. niger
Fig. 1 The photos of antifungal
activity results incubated for A B
28 days measured by poisoned
food technique. a The control
with 0 % citronella oil; b
0.0625 % citronella oil group; c
0.125 % citronella oil group; d
0.25 % citronella oil group; e
0.5 % citronella oil group; and f
1 % citronella oil group
D E
Appl Microbiol Biotechnol (2013) 97:7483–7492
was 0.125 % (v/v) by poisoned food technique. The experi-
ment results of broth dilution method was as follows: after
incubated for 7 days, the control and 0.0625 % group showed
massive myceliums with many black conidia suspended in the
liquid surface though the 0.0625 % group had 1 day growth
delay compared with the control. While the 0.125, 0.25, 0.5,
and 1 % groups displayed no growth and germination by
visual inspection and microscope observation. So the MIC
of citronella oil against A. niger was 0.125 % (v/v) by the broth
dilution method. After incubation for 28 days, only the
0.125 % group showed growth while other groups did not
show any growth and germination by visual inspection and
microscope observation. When samples from the 0.25, 0.5,
and 1 % groups were subcultured on PDA plates for 7 days, no
fungus colonies were observed in 0.5 and 1 % groups. So the
MFC of citronella oil against 105 spore/ml of A. niger was
0.5 % (v/v) by broth dilution method. The experiment result of
disc volatility method was as follows (Fig. S-1 in the Elec-
tronic supplementary material (ESM)): after incubation for
7 days, the control and 0.0625 % group were full of hyphae
and black conidia of A. niger. While no fungus growth were
observed in 0.125, 0.25, 0.5, and 1 % groups. Therefore, the
MIC of volatility of citronella oil was 0.125 % (v/v). Fungus
began to grow in the 0.125 % group after incubation for more
than 14 days. But, after incubation for 28 days, no fungus
growth was found in the 0.25, 0.5, and 1 % groups.
Fungicidal kinetics of the citronella oil to conidia of A. niger
After incubation for 1 day, fungal growth and myceliums were
observed in control group by eyes, but no fungus growth was
observed in all the other groups treated with different
C
F
Appl Microbiol Biotechnol (2013) 97:7483–7492 7487

concentration of citronella oil. The total number of the surviving hardly grew. Moreover, cytoplasm in the hyphae was
conidia in every group was calculated based on the number of not uniform and was intermittent. Obviously, the hyphae
colonies that grew on the PDA Petri dishes. The initial concen- were damaged in different degrees.
tration of conidia alive in all the groups was about 5.0×105 The effects of citronella oil on the morphological
spore/ml. When treated with different concentration of citronel- structure of hyphae observed by SEM are shown in
la oil for 1 day, more than 90 % conidia were killed in all Fig. 3. The natural hyphae incubated for 15 h before
experimental groups (Fig. 2). After treated for 2 and 3 days, the being treated with citronella oil (Fig. 3a, b) had normal
quantity of conidia surviving in the 0.125 (v/v) and 0.25 % (v/v) and intact tubular hypha with smooth cell walls, and
groups had a little increase in a short term (Fig. 2). In the next the tubular hyphae were substantial and well rounded.
few days, the quantity of the surviving conidia decreased grad- The residual conidia from germinating were typical
ually and was less than 10/ml at last (Fig. 2). While the orbicular conidia covered with a warty surface. How-
surviving conidia in the 0.5 (v/v), 1 (v/v), and 2 % (v/v) declined ever, the hyphae treated with 0.5 % citronella oil for 5
rapidly and decreased by an order of magnitude every day, none (Fig. 3c) and 24 h (Fig. 3d) were observed with prom-
of the conidia was surviving after treatment for 10 days (Fig. 2). inent changes. The hyphae were wrinkled, collapsed,
and folded. The detachment of cytoplasm from the
The effect of citronella oil on the morphological structure hyphae cell wall was also observed from the photo-
of hyphae of A. niger graphs. Besides, the warty surface of the conidia was
damaged and wrecked. The hyphae treated with 2 %
The light microscope photographs of hyphae treated and citronella oil for 5 (Fig. 3e) and 24 h (Fig. 3f) had
untreated by citronella oil are shown in Fig. S-2 in the more changes and were destroyed more severely than
ESM. The hyphae of A. niger incubated for 15 h before that of 0.5 % group. Except for the more severe col-
being treated with citronella oil (Fig. S-2a in the ESM) lapse and folding, a lot of holes and damages were also
were intact and smooth tubular, and many warty surface found in the hyphae, and it seemed that the hyphae
of the conidia were still covering the hyphae base. The were empty and almost no cytoplasm was found. The
hyphae in control group further being incubated for residual conidia from germinating were also damaged
24 h (Fig. S-2b in the ESM) grew longer and more more seriously than that of those treated by 0.5 %
mature, and none residual warty surface of the conidia citronella oil.
were observed at the hyphae base. The hyphae were The effects of citronella oil on the internal structure of
also excellent without any damage. In contrast, the hyphae of A. niger by TEM are shown in Fig. 4. Figure 4a, b
hyphae treated by 0.5 (Fig. S-2c in the ESM) and shows the natural hyphae of A. niger subcultured for 24 h as the
2 % (Fig. S-2d in the ESM) citronella oil for 24 h control experiment. The control hyphae had healthy structure
did not grow, and many residual warty surfaces of the with clear cell nucleuses, vacuoles, and many mitochondria.
conidia were still observed at the hyphae base. Obvi- Their cell wall was uniform and was coated with an intact outer
ously, the hyphae treated with 0.5 and 2 % citronella oil fiber layer. The plasma membrane was integrated and closed to
the cell wall. The septum and main organelles such as the
mitochondria and the nucleus appeared normal. The cell meta-
Logarithmic quantities of survival conidia (lg)

bolic activities in the hyphae were very active, and the hyphae
6
0.125% displayed vigorous vitality. In contrast, the hyphae of A. niger
0.25%
5 treated with 0.5 (Fig. 4c, d) and 2 % (Fig. 4e, f) citronella oil for
0.5%
1% 24 h showed the outer fiber layer being damaged and falling
4 2% apart. Moreover, the plasma membrane was detached from the
cell wall resulting in the expanded periplasmic space, and the
3
plasma membrane was damaged in different levels and even
2 disappeared. The organelles such as the mitochondria were
damaged, broken, and wrecked, and so many vacuolations were
1 evident with vacuole fusion in the hyphae and some vacuola-
tions seemed coated with oils (Fig. 4e, f).
0
After the 0.5 and 2 % groups were cultured for 28 days, the
-1 samples were taken and cultured on PDA Petri dishes for
-2 0 2 4 6 8 10 12 14 16 7 days. No fungus grew out on PDA from the hyphae treated
Treatment time (days) by 2 % citronella oil, so the myceliums were killed completely
Fig. 2 The logarithmic fungicidal kinetic curves of citronella oil by 2 % citronella oil treated for 28 days. But the myceliums
against A. niger treated with 0.5 % citronella oil were not killed completely.
7488 Appl Microbiol Biotechnol (2013) 97:7483–7492

Fig. 3 The morphological

alteration of A. niger hyphae A B
observed by SEM. H hypha, C
residual conidia covered on the
hypha base. a, b The normal
morphology of A. niger hyphae
before being treated with
C
citronella oil. c, d The
morphology of A. niger hyphae C
treated with 0.5 % (v/v) H
citronella oil for 5 and 24 h H
separately. e, f The morphology
of A. niger hyphae treated with
2 % (v/v) citronella oil for 5 and
24 h separately
C D
C

C H
H

E F C
H

The effect of citronella oil on the conidia’s morphological (Fig. 5c), and some of conidia only leaved a part of hard
structure of A. niger cell wall with a warty surface (Fig.5d).

The conidia of A. niger ATCC 16404 were black spheroids

covered with a warty surface. When the conidia were cul-
tured for 1 day, the control samples grew out myceliums
while the 0.5 and 2 % group did not germinate. The obser-
vation by microscope (Fig. S-3 in the ESM) showed the
morphology of control conidia (Fig. S-3c in the ESM) and
the conidia treated with 0.5 % (Fig. S-3b in the ESM) and
2 % (Fig. S-3c in the ESM) citronella oil. Some damaged
and fragmentary conidia were shown in Fig. S-3b, c in the
ESM. Samples treated by 0.5 and 2 % citronella oil for
2 days were observed by SEM. The results (Fig. 5) showed
that the cell wall with a warty surface of some conidia in
0.5 % citronella oil was damaged. An obvious cleft was
observed in the center of the cell wall (Fig. 5b). While in the
2 % group, the cell wall with a warty surface of some
conidia were ruptured and the sporoplasm were exposed
Discussion
The inhibition of A. niger growth by essential oil has already
been previously reported (Tolouee et al. 2010; Sharma and
Tripathi 2008). They found that the growth inhibition of
essential oil from Matricaria chamomilla against A. niger
started at the lowest oil concentration of 15.62 μg/ml and
reached to a maximum of 92.5 % at the concentration of
1,000 μg/ml after cultured for 4 days on agar plates
(Tolouee et al. 2010), and 3.0 μg/ml Citrus sinensis oil
caused complete growth inhibition of A. niger on agar plates
(Sharma and Tripathi 2008). In our study, we studied the
antifungal effect of citronella oil against A. niger. Always,
citronella oil is popular as a mosquito repellent (Solomon et
al. 2012), but its high effective antifungal activity is rarely
Appl Microbiol Biotechnol (2013) 97:7483–7492 7489

Fig. 4 The internal

morphological alteration of A. A B FL
niger hyphae observed by
TEM. FL fiber layer, CW cell
wall, PM plasma membrane,
MIT mitochondria, V vacuoles,
N
N nucleus. a, b The internal MIT
morphology of control hyphae V
before being treated with
citronella oil. c, d The internal CW
morphology of A. niger hyphae
treated with 0.5 % citronella oil PM
for 24 h. e, f The internal
morphology of A. niger hyphae
treated with 2 % citronella oil
for 24 h
C D FL
V

PM

CW

E F
FL

CW

PM

known. The results indicated all the experimental groups
with different citronella oil concentration inhibited the
growth of A. niger conidia, and when the concentration
of citronella oil reached 0.125 % (v/v), the 105 spore/ml
conidia of A. niger could not grow until incubation for
7 days by poisoned food technique, broth dilution meth-
od, and disc volatility method. So the MICs of citronel-
la oil against A. niger were both 0.125 % (about
1,000 μg/ml) after being treated for 7 days by three
methods, and the MFC of citronella oil against 105
spore/ml A. niger was 0.5 % (v/v) after treatment for
28 days by broth dilution method. Therefore, both the
contacting antifungal activity and the volatile antifungal
activity of citronella oil are effective.
The different antifungal activity of different essential oils
may be due to their different constituents. The main con-
stituents of citronella oil are citronellal (37.51 %), geraniol
(22.57 %), and citronellol (15.12 %), which were similar to
the constituent analysis of Billerbeck et al. (2001) with
citronellal (42.0 %), geraniol (20.8 %), and citronellol
(14.5 %). Mahmoud reported that citronella oil components
geraniol and citronellol were effective in suppressing A.
flavus growth at 500 mg/l, and Viollon and Chaumont
reported that citral, geraniol, and citronellol showed the
highest antifungal activities among terpenoids (Billerbeck
et al. 2001). Sindhu et al. (2011) suggested that the fungi-
static and fungicidal effects of the essential oil may be due
to the effect of the individual constituent in the oil, and the
7490
Fig. 5 The morphological
alteration of A. niger conidia A B
observed by SEM. NC normal
conidia, Cleft obvious cleft in
the center of the conidia cell
wall, S sporoplasm of the
conidia, CW cell wall of the
conidia. a The normal
morphology of A. niger conidia NC
before being treated with
citronella oil. b The
morphology of A. niger conidia
treated with 0.5 % (v/
C D
v)citronella oil for 2 days. c, d
The morphology of A. niger
conidia treated with 2 % (v/v)
citronella oil for 2 days
CW
interactive effects of other minor components may also be
responsible for the antifungal effect.
Furthermore, as can be seen from the fungicidal kinetic
curves of citronella oil to A. niger (Fig. 2), the citronella oil
has strong antifungal activity. More than 90 % conidia were
killed in all the groups with 0.125 to 2 % citronella oil when
treated for only 1 day. Furthermore, with the increase of
citronella oil contents, the antifungal activity gradually
strengthened. And with the prolongation of treatment time,
the fungicidal rate was also improved. As showed in Fig. 2,
the antifungal kinetic curve of 0.125 % group was similar with
that of 0.25 % group. Both of them have a temporal upward
trend of survival rate of the conidia in treatment for 2 to 3 days.
Although most conidia were killed in the 0.125 and 0.25 %
groups after being treated for 1 day, the strong survivors may
temporally adapt to the new environment and some of them
germinate when treated for 2 to 3 days, resulting in the
improvement of survival rate. But with the continuous pres-
sure of citronella oil, the growth of the survival conidia was
restrained or gradually killed in the following days. When the
concentration of citronella oil arrived at 0.5 % (v/v), its anti-
fungal efficacy had been greatly strengthened, so the survival
conidia were decline rapidly every day; 0.5 % citronella oil
concentration was a threshold to kill the conidia thoroughly.
When the 5.0×105 spore/ml conidia of A. niger were treated
for about 10 days by 0.5 to 2 % citronella oil, none of conidia
survived. At the same time, the results showed that the fungi-
cidal kinetic curve was a good method to determine the
antifungal effect of fungicide.
Conidia of 105 spore/ml of A. niger were killed com-
pletely by 0.5 % citronella oil treated for 28 days while
hyphae grown out from 105 spore/ml conidia of A. niger
were not killed completely under the same condition. But it
Appl Microbiol Biotechnol (2013) 97:7483–7492
Cleft
Cleft
Cleft
S
CW
could not be concluded from this result that the conidia were
more sensitive than the hyphae to citronella oil because the
cell number of the hyphae grown out from conidia was more
than that of the conidia. A review (Ceylan and Fung 2004)
also reported that the effect of spices on spores might be
different than that on vegetative cells, but it did not conclude
which kind of cells was more sensitive.
The high effective antifungal activity of essential oils might
be related to their lipophilic characters which can penetrate the
plasma membrane (Nogueira et al. 2010; Rassoli and Owlia
2005). The hyphae morphology observed by light microscope
showed large cytoplasm loss caused by citronella oil. SEM
observation also revealed marked alterations in the morphology
of the hyphae, which appeared severely collapsed and squashed
due to the lack of cytoplasm. The result was consistent with that
of Rasooli et al. (2006) and Sharma and Tripathi (2006). They
found that the A. niger hyphae were squashed and severely
collapsed after C. sinensis essential oil application by SEM
observation (Sharma and Tripathi 2006). Yahyazadeh et al.
(2008) also found the same phenomenon in the Penicillium
digitatum hyphae treated with clove oil. Moreover, our results
of light microscope and SEM observation of hyphae suggested
that citronella oil penetrated across not only the cell wall, but
also the cell membrane. And the citronella oil changed the
cellular permeability and caused the leakage of cytoplasm by
interacting with the cytoplasm membrane of A. niger. Rassoli
and Owlia (2005) showed that the plasma membrane of A.
parasiticus treated with thyme essential oils dissociated from
the cell wall, invaginated, and associated with the formation of
lomasomes which were usually found in fungi treated with
imidazole components. Rammanee and Hongpattarakere
(2011) reported the plasma membranes of A. flavus hyphae
treated with acid lime essential oil became rough and irregular
Appl Microbiol Biotechnol (2013) 97:7483–7492
with continuous folding into cytoplasm and detached from the
fibrillar layer. Tolouee et al. (2010) also reported that the
essential oil of M. chamomilla changed the cell permeability
of A. niger van Tieghem. A change in cell permeability might
result in an imbalance in intracellular osmotic pressure, subse-
quent disruption of intracellular organelles, leakage of cyto-
plasm, and finally cell death (Tolouee et al. 2010). Indeed, our
TEM observation indicated that the citronella oil penetrated into
the cytoplasm and destroyed the organelles. Meanwhile, mas-
sive vacuolation of cytoplasm with vacuole fusion was ob-
served obviously as Rammanee and Hongpattarakere (2011)
found in A. flavus treated with acid lime essential oil. So, the
main target membranes of citronella oil included not only the
cytoplasm membrane but also the membrane of the organelles.
Razzaghi-Abyaneh et al. (2006) revealed morphological alter-
ations in Aspergillus parasiticus treated with a commercial
fungicide Akaci®plus which is composed of a mixture of two
different polymeric guanidine compounds. They found a
marked depletion of cytoplasmic contents of hyphae accompa-
nied with lysis and disruption of membranes of major organ-
elles such as nuclei, mitochondria, and endoplasmic reticulum.
In addition, the first target of citronella oil against the conidia
was the warty surface covering the cell wall. The center of the
conidia cell wall with warty surface displayed an obvious cleft,
and then the cell wall with warty surface was ruptured and the
sporoplasm was exposed. The tender sporoplasm was destroyed
by the citronella oil and cytolysis happened. Only some rup-
tured hard cell wall with a warty surface was left at last.
In conclusion, the citronella oil possesses has high strong
antifungal activity against A. niger. Just 0.125 (v/v) and
0.25 % (v/v) citronella oil inhibited the growth of 5×105
spore/ml conidia for 7 and 28 days while 0.5 % (v/v)
citronella oil could completely kill the conidia of 5×105
spore/ml. The observation by TEM and SEM indicated that
citronella oil could lead to irreversible alteration of the
hyphae and conidia. From the experimental results, it is
assumed that the citronella oil destroys the cell wall of the
A. niger hyphae, passed through the cell membrane, pene-
trated into the cytoplasm, and acted on the main organelles,
to kill the hyphae cells at last. Similarly, citronella oil could
lead to the rupture of hard cell wall and then act on the
sporoplasm to kill the conidia. So, except for being a pop-
ular mosquito repellent, the citronella oil is a promising
mold inhibitor which could be widely applied in food,
industry, agriculture, and household fungicide. In addition,
the citronella oil could be used for insect pest control in-
cluding granary and flying insects (Hiramatsu et al. 2008).
Acknowledgments The author gratefully acknowledges the support
by Guangdong Provincial Science and Technology Project
(2009B011000011, 2011B010400039), the National Natural Science
Fund of China (U1133003, 41176104, 20971028), and Guangdong
Natural Science Fund (10151007002000000).
7491
References
Al-Ja’fari AH, Vila R, Freixa B, Tomi F, Casanova J, Costa J, Canigueral S
(2011) Composition and antifungal activity of the essential oil from
the rhizome and roots of Ferula hermonis. Phytochem 72:1406–1413
Arora DS, Kaur J (1999) Antimicrobial activity of spices. Int J Anti-
microb Ag 12:257–262
Bajpai VK, Yoon JI, Kang SC (2009) Antifungal potential of essential
oil and various organic extracts of Nandina domestica Thunb.
against skin infectious fungal pathogens. Appl Microbiol Bio-
technol 83:1127–1133
Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008) Biological effects
of essential oils—a review. Food and Chem Toxicol 46:446–475
Beneti SC, Rosset E, Corazza ML, Frizzo CD, Luccio MD, Oliveira JV
(2011) Fractionation of citronella (Cymbopogon winterianus) es-
sential oil and concentrated orange oil phase by batch vacuum
distillation. J Food Eng 102:348–354
Billerbeck VG, Roques CG, Bessière J-M, Fonvieille J-L, Dargent R
(2001) Effects of Cymbopogon nardus (L.) W. Watson essential
oil on the growth and morphogenesis of Aspergillus niger. Can J
Microbiol 47:9–17
Burt S (2004) Essential oils: their antibacterial properties and potential
applications in foods—a review. Int J Food Microbiol 94:223–253
Ceylan E, Fung DYC (2004) Antimicrobial activity of spices. J Rapid
Meth Aut Mic 12:1–55
Charles S, Chester E, Henry T (1947) Molds, yeasts, and actinomy-
cetes. Soil Sci 63(5):418
Chuang PH, Lee CW, Chou JY, Murugan M, Shieh BJ, Chen HM
(2007) Anti-fungal activity of crude extracts and essential oil of
Moringa oleifera Lam. Bioresource Technol 98:232–236
Delespaul Q, Billerbeck VG, Roques CG, Michel G (2000) The anti-
fungal activity of essential oils as determined by different screen-
ing methods. J Essent Oil Res 12:256–266
European Commission (2009). 2009/251/EC: Commission decision of
17 March 2009 requiring member states to ensure that products
containing the biocide dimethylfumarate are not placed or made
available on the market. Official Journal of the European Union
European Commission (2012). 2012/48/EU: Commission implement-
ing decision of 26 January 2012 extending the validity of decision
2009/251/EC requiring member states to ensure that products
containing the biocide dimethylfumarate are not placed of made
available on the market. Official Journal of the European Union
Hiramatsu Y, Shida S, Miyazaki Y (2008) House dust mites and their
sensitivity to wood oils and volatiles. J Wood Sci 54:1–9
Inouye S, Takizawa T, Yamaguchi H (2001) Antibacterial activity of
essential oils and their major constituents against respiratory tract
pathogens by gaseous contact. J Antimicrob Chemoth 47:565–573
Li WR, Xie XB, Shi QS, Zeng HY, Ouyang YS, Chen YB (2010)
Antibacterial activity and mechanism of silver nanoparticles on
Escherichia coli. Appl Microbiol Biotechnol 85:1115–1122
Li WR, Xie XB, Shi QS, Duan SS, Ouyang YS, Chen YB (2011)
Antibacterial effect of silver nanoparticles on Staphylococcus
aureus. Biometals 24:135–141
Man HC, Hamzah MH, Jamaludin H, Abidin ZZ (2012) Preliminary
study: kinetics of oil extraction from citronella grass by ohmic
heated hydro distillation. APCBEE Procedia 3:124–128
Nogueira JHC, Gonçalez E, Galleti SR, Facanali R, Marques MOM,
Felício JD (2010) Ageratum conyzoides essential oil as aflatoxin
suppressor of Aspergillus flavus. Int J Food Microbiol 137:55–60
Rammanee K, Hongpattarakere T (2011) Effects of tropical Citrus
essential oils on growth, aflatoxin production, and ultrastructure
alterations of Aspergillus flavus and Aspergillus parasiticus. Food
Bioprocess Tech 4:1050–1059
Rasooli I, Rezaei MB, Allameh A (2006) Growth inhibition and
morphological alterations of Aspergillus niger by essential oils
7492
from Thymus eriocalyx and Thymus x-porlock. Food Control
17:359–364
Rassoli I, Owlia P (2005) Chemoprevention by thyme oils of Asper-
gillus parasiticus growth and aflatoxin production. Phytochemis-
try 66:2851–2856
Razzaghi-Abyaneh M, Shams-Ghhfarokhi M, Kawachi M, Eslamifar
A, Schmidt OJ, Schmidt A, Allameh A, Yoshinari T (2006)
Ultrastructural evidences of growth inhibitory effects of a novel
biocide, Akacid®plus, on an aflatoxigenic Aspergillus parasiticus.
Toxicon 48:1075–1082
Sharma N, Tripathi A (2006) Fungitoxicity of the essential oil of
Citrus sinensis on post-harvest pathogens. World J Microb
Biot 22:587–593
Sharma N, Tripathi A (2008) Effects of Citrus sinensis (L.) Osbeck
epicarp essential oil on growth and morphogenesis of Aspergillus
niger (L.) Van Tieghem. Microbiol Res 163:337–344
Shelef LA (1983) Antimicrobial effects of spices. J Food Safety 6:29–44
Sindhu S, Chemakam B, Leela NK, Bhai RS (2011) Chemoprevention
by essential oil of turmeric leaves (Curcuma longa L.) on the
Appl Microbiol Biotechnol (2013) 97:7483–7492
growth of Aspergillus flavus and aflatoxin production. Food
Chem Toxicol 49:1188–1192
Solomon B, Sahle FF, Gebre-Mariam T, Asres K, Neubert RHH (2012)
Microencapsulation of citronella oil for mosquito-repellent appli-
cation: formulation and in vitro permeation studies. Eur J Pharm
Biopharm 80:61–66
Tian J, Ban X, Zeng H, He J, Huang B, Wang Y (2011) Chemical
composition and antifungal activity of essential oil from Cicuta
virosa L. var. latisecta Celak. Int J Food Microbiol 145:464–470
Tolouee M, Alinezhad S, Saberi R, Eslamifar A, Zad SJ, Jaimand K,
Taeb J, Rezaee MB, Kawachi M, Shams-Ghahfarokhi M,
Razzaghi-Abyaneh M (2010) Effect of Matricaria chamomilla
L. flower essential oil on the growth and ultrastructure of Asper-
gillus niger van Tieghem. Int J Food Microbiol 139:127–133
Yahyazadeh M, Omidbaigi R, Zare R, Taheri H (2008) Effect of some
essential oils on mycelial growth of Penicillium digitatum Sacc.
World J Microbiol Biotechnol 24:1445–1450
Zhou DQ (2003). Filamentous fungi—mold. Textbook of microbiology.
Higher Education Press (pressed in Chinese), Beijing. pp. 53–54