ARTICLE IN PRESS

Microbiological Research 163 (2008) 337344

www.elsevier.de/micres

Effects of Citrus sinensis (L.) Osbeck epicarp

essential oil on growth and morphogenesis of
Aspergillus niger (L.) Van Tieghem
Neeta Sharma, Abhishek Tripathi
Mycology and Plant Pathology Division, Department of Botany, University of Lucknow, Lucknow 226007, India
Accepted 15 June 2006

KEYWORDS
Aspergillus niger;
Citrus sinensis;
Essential oil;
GC/MS analysis;
Scanning electron
microscopy

Summary
Essential oils from different plant parts are known for their antimicrobial activity but
the antifungal effects of essential oil from Citrus sinensis (L.) Osbeck epicarp on
growth and morphogenesis of Aspergillus niger has not been observed so far. The
mycelial growth was inhibited at 2.5 and 3.0 mg/ml of oil in Potato Dextrose Broth
and Agar medium, respectively. These concentrations were fungicidal under the test
conditions. The fungitoxicity of oil did not change even at exposure to 100 1C and
autoclaving. The main changes observed under light and scanning electron
microscopy after oil treatment were loss of cytoplasm in fungal hyphae, and
budding of hyphal tip. The hyphal wall and its diameter became markedly thinner,
distorted and resulted in cell wall disruption. The attened and empty hyphal tips
bifurcated into bud like structures. GC-MS studies of the oil revealed the presence of
10 chemical constituents. Limonene has been found to be major component (84.2%).
& 2006 Elsevier GmbH. All rights reserved.

Introduction
The protection of crops, stored food grains and pest
control in the public health sector continues to place
heavy reliance upon the use of chemicals (Sharma,
2000). The history of pesticide development has been
instructive to us in terms of benets derived as well as
Corresponding author. Tel./fax: +91 522 2308594.

E-mail address: dr_neeta_sharma2003@yahoo.com

(N. Sharma).

the hazards, which accompany indiscriminate use of

these poisons. A 1986 National Academy of Sciences
(NAS) report on pesticides residues on food indicated
that fungicides pose more of a carcinogenic risk than
insecticides and herbicides together besides developing resistance towards pathogens (Research Council,
Board of Agriculture, 1987).
This negative consumer perception of chemical
preservatives drives attention towards natural
alternatives. Particular interest focused on the
potential application of plant essential oils and

0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2006.06.009

ARTICLE IN PRESS
338
extracts from plants have recently been of great
interest. Their possible use as natural additives
emerged from a growing tendency to replace
synthetic antimicrobial agents with natural ones.
Phyto-compounds are expected to be far more
advantageous than synthetic pesticides for sheer
magnitude of complexity, diversity and novelty of
chemicals, reactions and phenomenon (Sharma,
1998) as they are bio-degradable in nature, nonpollutant and posses no residual or phytotoxic
properties (Badei et al., 1996; Bishop and Thornton, 1997; Tewari, 1990).
Aspergillus spp., are capable of growing upon a
wide range of organic substrates, and often cause
deterioration of stored food material (Barrios et
al., 1997; Misra and Dubey 1994; Paster et al 1990).
There are also reports of Aspergillus niger induced
spoilage of mangoes (Prakash and Raoof, 1989),
grapes (Sharma and Vir, 1986), and tomatoes (Sinha
and Saxena, 1987). Adebajo and Popoola (2003)
reported that A. niger damages kolanuts during
storage. Biodeterioration of the African star apple
(Chrysophylum albidum) in storage was found to be
due to A. niger (Amusa et al., 2003). Thus, the
presence and growth of this fungus in food and feed
threatens human and animal health.
Very few publications have documented the
antimicrobial activity of Citrus sinensis oil against
different microbial species (Shukla et al., 2000;
Singh et al., 1993). Karapinar (1985) reported the
effects of citrus oils and some spices on growth and
aatoxin production by Aspergillus parasiticus.
Fungicidal activity of citrus oil was also reported
against the causal agent of anthracnose disease in
tropical fruits (Ernestina et al., 2003). Control of A.
niger has been evaluated previously by Paster et al.
(1990, 1995) using oregano oil and by de Billerbeck
et al. (2001) using Cymbopogon nardus oil. These
ndings thus, indicate the possibility of exploiting
Citrus sinensis essential oil as an effective inhibitor
of this biodegrading and storage-contaminating
fungus. In this study the essential oil from epicarp
of Citrus sinensis was used to study the effect on
morphological changes of A. niger in order to
investigate its mode of action. No studies have
been carried so far with regard to precise target of
Citrus sinensis essential oil on A. niger.

Materials and methods

Extraction of essential oil
Fresh epicarp of Citrus sinensis (L.) Osbeck
(Musambi) was collected from various juice shops

N. Sharma, A. Tripathi
of Lucknow, India during the months of May
2004October 2004. The essential oil was extracted
from collected material by hydro-distillation for 5 h
using Clevenger-type apparatus (Guenther, 1948). A
clear, light yellow coloured, oily layer was obtained
on the top of the aqueous distillate which was
separated from the latter and dried with anhydrous
sodium sulphate. The extracted essential oil was
kept in air-tight sealed glass vials, covered with
aluminum foil at 4 oC until further analysis.

GC-MS analysis of essential oil

The GC-MS of essential oil was analysed on a
Shimadzu QP-2000 instrument at 70 eV and 250 1C.
GC Column: ULBON HR-1 equivalent to OV-1, fused
silica capillary 0.25 mm  50 M with lm thickness
0.25 mm. The GC-MS was operated under the
following conditions 60-5-5-250 meaning that the
initial temperature was 60 1C for 5 min and then
heated at the rate of 5 1C/min to 250 1C. Carrier gas
(helium) ow was 2 ml/min.
The identication of component was based on
comparison of their mass spectra fragmentation
patterns with those of Mass Spectrometry Data
Centre, the Royal Society of Chemistry. UK (Eight
Peak Index of Mass Spectra, 3rd Ed. 1983) and those
reported in the literature (Adams, 1995).

Fungal species
Strain of A. niger (L.) Van Tieghem strain MPPLU
10 from the collection of Mycology and Plant
Pathology Division, Botany Department, University
of Lucknow was used. The fungus was maintained
on Potato Dextrose Agar (PDA) at 28 1C.

Antifungal activity measurements

Poisoned food technique
The fungi-toxicity of the oil was evaluated
against the test fungi by the poisoned food
technique of Grover and Moore (1962). PDA
(20 ml) was poured into sterilized Petri dishes and
measured amount of oil was added to give desired
concentrations of 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0,
2.5, 3.0 and 3.5 mg oil/ml. In medium 0.05% Tween80 was also added for even distribution of the oil in
the medium. For control sets, the medium was
supplemented with the same amount of distilled
water instead of oil and 0.05% Tween-80. Plates
were incubated at 2871 1C. The growth of the test
fungi were recorded for seven days and percent
inhibition was computed after comparison with the
control.

ARTICLE IN PRESS
Effects of Citrus sinensis (L.) Osbeck epicarp
Fungitoxicity was expressed in terms of percentage of mycelia growth inhibition and calculated as
per formula of Pandey et al. (1982).
Percentage of mycelial
dc  dt
 100,
growth inhibition
dc
where dc average diameter of fungal colony in
control and dt average diameter of fungal colony
in treatment.
Determination of mycelial weight
To determine the effect of essential oil on the dry
weight of the test fungus different concentrations of
oil in potato dextrose broth (PDB) medium were
prepared in Erlenmeyer ask and inoculated with
107 spores/ml of A. niger. Spore population was
counted using haemocytometer. In the corresponding
control equal amount of distilled water was added.
After 15 days dry weight of mycelium was determined.
Flasks containing mycelia were ltered through Whatman lter no. 1 and then washed with distilled water.
The mycelia were allowed to dry at 60 1C for 6 h and
then at 40 1C over night. The lter paper containing
dry mycelia were weighed. Percent growth inhibition
on the basis of dry weight was calculated as
Control weight  Sample weight=Control weight
 100.

Spore germination assay

Nine concentrations of oil (0.1, 0.3, 0.5, 0.7, 1.0,
1.5, 2.0, 2.5, 3.0 and 3.5 mg oil/ml) were tested for
spore germination of the test fungi. Fungal spores
obtained from 10-day-old cultures of the fungi were
taken and placed on glass slides in triplicate. Slides
containing the spores were incubated in a moist
chamber at 2572 1C for 24 h. Each slide was then
xed in lacto-phenol-cotton blue and observed
under the microscope for spore germination. About
200 spores were counted and the number of spores
germinated was scored using haemocytometer to
calculate the percentage of spore germination
(Surender et al., 1987).
Nature of toxicity
The fungitoxicity (fungistatic/fungicidal) of the
essential oil was tested by using the technique of
Thomson (1989).
Effect of temperature on toxicity of oil
Experiments were performed to determine the
thermostable nature of the oil. Different glass vials
containing 3 ml oil each were subjected to different

339
temperature treatments for 3 h in incubators
already adjusted to 40, 60, 80 and 100 1C.
Antifungal activity of oil was also tested after
autoclaving it at 121 1C for 15 min. The fungitoxicity of the treated oil from each set was tested
against the test fungi at minimum inhibitory
concentration (MIC) of 3.0 mg oil/ml on agar medium by the usual poisoned food technique.
Light microscopy
A sample of mycelium was taken from the
periphery of the colony grown on PDA with 0, 0.5,
1.0 and 2.0 mg/ml treatments of oil after 4 days of
incubation. The samples were xed in lactophenol-cotton blue and examined under the microscope (Nikon ECLIPSE E200, Japan) at 400  to
examine structural abnormalities. Samples from
control plates without oil were also stained and
observed. Photographs were taken with the help of
computer attached Samsung COLOR CAMERA SAC410PA.
Scanning electron microscopy
Four day old fungal cultures on PDA treated with
0, 0.5, 1.0 and 2.0 mg/ml concentrations of oil were
used for all scanning electron microscopic (SEM)
observations. 5  10 mm segments were cut from
cultures growing on potato dextrose plates and
promptly placed in vials containing 3% glutaraldehyde in 0.05 M phosphate buffer (pH 6.8) at 4 1C.
Samples were kept in this solution for 48 h for
xation and then washed with distilled water three
times for 20 min each. Following which they were
dehydrated in an ethanol series (30%, 50%, 70%, and
95%), for 20 min in each alcohol dilution and nally
with absolute ethanol for 45 min. Samples were
then critical point dried in liquid carbon dioxide.
Fungal segments were placed in desiccators until
further use. Following drying, samples prepared
were mounted on standard 1/2 in Cambridge SEM
stubs using double-stick adhesive tabs and coated
with gold-palladium electroplating (60 s, 1.8 mA,
2.4 kV) in a Polaron SEM Coating System sputter
coater. All samples were viewed in a Cambridge
LEO S-430 SEM operating at 15 kV at various levels
of magnication (300  to 1.5 k  ).

Results
Citrus sinensis essential oil and its
components
Essential oils were extracted from epicarp (waste
product) of C. sinensis by hydro-distillation yielded

ARTICLE IN PRESS
340

N. Sharma, A. Tripathi

1.8% essential oil. GC-MS analysis of the oil led to

identication of 10 components. The main components of the Citrus sinensis epicarp essential oil
studied and their percentages are presented in
Table 1, the major constituents were Limonene
(84.2%), Linalol (4.4) and Myrcene (4.1).

Inhibitory effect of citrus oil on test fungus

Results of inhibitory activity of the essential oil in
agar medium are presented in Table 2. At 3.0 mg/ml
concentration fungal development was completely
inhibited after 7 days of incubation. This concentration was found to be fungicidal. Radial growth
and biomass of A. niger was signicantly reduced in
response to various concentrations of citrus oil
ranging from 0.1 to 3.0 mg/ml. At 0.7 mg/ml, growth
was reduced to about half that of the control
(Table 2). It was found that the oil was fungicidal
against A. niger at 3.0 mg oil/ml concentration
which was also the MIC for the test fungi.
Determination of mycelial weight was done and it
Table 1. Components of Citrus sinensis (L.) Osbeck
epicarp essential oil identied by GC-MS
Peak no.

Components

Percentage in
total oil

1
2
3
4
5
6
7
8
9
10

a-pinene
b-pinene
Myrcene
Limonene
Linalol
Citral
a-Terpineol
Terpinolene
Citronellal
Geraniol

0.9
0.6
4.1
84.2
4.4
0.5
0.8
1.3
1.9
1.3

was found that insignicant growth occurred at

2.5 mg/ml concentration. Percent inhibition on the
basis of radial growth and dry weight was calculated and it was found that the oil was more
effective in liquid medium than solid medium
(Table 2). The oil exhibited 100% inhibition of spore
germination at 1.5 mg/ml concentration, while at
0.3 mg/ml concentration only 50% inhibition of
spore germination was observed (Table 2).

Effect of temperature on antifungal activity

of oil
The thermostability of the oil was tested and it
was found that at temperature ranging from 40 to
100 1C and even after autoclaving (121 1C for
15 min) the oil, its activity was not altered.

Light microscopy
The observations of A. niger examined under
light microscope at 400  magnication, after
treatment with different concentrations (0.5, 1.0
and 2.0 mg/ml) of Citrus sinensis epicarp oil are
presented in Fig. 1. In addition to inhibited growth,
mycelial colonies grown in the presence of essential
oil seemed to consistently exhibit distinct morphological changes when compared to control. These
variations included lack of sporulation, visible loss
of pigmentation and aberrant development of
conidiophores. In microscopic examination of untreated mycelium hyphae had homogenous, clear
cytoplasm and profuse conidiation on conidial
heads which were large and radiate, sterigmata
bearing conidia were clearly visible (Figs. 1a and b)
while treatment with 0.5 mg/ml concentration
clearly showed reduction in conidial heads, poorly
developed sterigmata with less or absence of

Table 2. Effect of different concentrations of Citrus sinensis epicarp oil on per cent radial growth inhibition (after 7
days), dry weight (after 15 days) and inhibition of spore germination of Aspergillus niger at 2871 1C
Conc. of oil (mg/ml)

Radial growth inhibition (%)

Dry weight (mg7SEa)

Spore germination (%)

Control
0.1
0.3
0.5
0.7
1.0
1.5
2.0
2.5
3.0
3.5

61571.6
52770.9
48871.3
32670.7
12370.3
6871.1
5170.5
2670.1
0.070.0
0.070.0
0.070.0

100.0
74.28
46.19
21.00
10.20
4.50
0.00
0.00
0.00
0.00
0.00

5.9
14.11
31.41
47.05
63.05
78.82
87.05
92.94
100
100

()
(14.33)
(20.65)
(46.99)
(80.00)
(88.94)
(91.70)
(95.77)
(100.00)
(100.00)
(100.00)

All data shown are averages and standard errors from three determinations of experiment.
a
Figure in parenthesis indicates per cent growth inhibition on the basis of dry weight.

ARTICLE IN PRESS
Effects of Citrus sinensis (L.) Osbeck epicarp

341

Figure 1. Microphotographs of Aspergillus niger mycelium grown on PDA with or without Citrus sinensis essential oil
during 4 days of incubation at 2871 1C. (a) A. niger control mycelium, structure is homogenous, Bar 10 mm. (b) Control
conidial head of A. niger, large and radiate, clear development of vesicle on conidiophore, primary and secondary
sterigmata bearing conidia clearly visible, Bar 10 mm. (c) Conidial head modications induced by 0.5 mg/ml of Citrus
sinensis essential oil, clear absence of conidia, conidiophore distorted, Bar 10 mm. (d) The apical hyphal tip become
distorted, budding of hyphal tip and unusual structures clearly visible, some anomalies such as little swellings along the
hypha and apex bifurcations are visible, modications seen at 0.5 mg/ml treatment of essential oil, Bar 10 mm. (e)(h)
Treatment with essential oil at 1.0 mg/ml, showing hyphae with greater anomalous structures, budded apical tip, with
cytoplasmic granulations, hyphae showing clear separation of cytoplasm from cell wall Bar 10 mm. (i)(l) Effect at
2.0 mg/ml of essential oil, showing clear decrease in cytoplasmic content and note the cytoplasmic retraction and
empty hyphae. Bar 10 mm.

conidiation and conidiophores becoming distorted

(Fig. 1c). The alterations in hyphal structures
started with budding of hyphal tip, anomalous
structures such as swelling, localized along the
hyphae and at their extremities with the apexes

showing an irregular growth in dimension with

irregular shape, and the formation of anomalous
apex bifurcations were also visible at this concentration (Fig. 1d). At 1.0 mg/ml concentration of oil
there was clear decrease in cytoplasmic content,

ARTICLE IN PRESS
342
with clear separation of cytoplasm from cell wall in
hyphae and bifurcated apical tip without cytoplasm
were observed (Figs. 1eh). At higher concentration of 2.0 mg/ml clear retraction of cytoplasm from
hyphae and ultimately hyphae without cytoplasm
were found (Figs. 1il).

Scanning electron microscopy

The effect of Citrus sinensis essential oil on the
morphology of A. niger examined by SEM are shown

N. Sharma, A. Tripathi
in Fig. 2. The A. niger mycelium grown on PDA
medium as control showed the characteristic
morphology with lengthened, regular, homogenous
hyphae of constant diameter with smooth external
surface and with rounded apex. After 4 days of
incubation in the treatments (0.5, 1.0, and 2.0 mg/
ml) fungal mycelium showed alterations in the
morphology of the hyphae. Treatment with essential oil at 0.5 mg/ml clearly showed distorted
mycelium, squashed and attened conidiophore
bearing damaged conidial head (Fig. 2c). At 1.0 mg/
ml of concentration highly budded hyphal tip and

Figure 2. Scanning electron micrographs of Aspergillus niger mycelium grown on PDA with or without Citrus sinensis
essential oil during 4 days of incubation at 2871 1C. (a) Control, magnication of a single hyphal tip, Bar 10 mm. (b)
Control, mycelium showing conidial heads on conidiophore, Bar 30 mm. (c) Treatment with essential oil at 0.5 mg/ml,
showing distorted mycelium, squashed and attened conidiphore bearing damaged conidial head, Bar 30 mm. (d) and (e)
Effect of 1.0 mg/ml essential oil, showing highly budded apical tip of hyphae, the attened hyphae clearly visible, note
the presence of punctured hyphae Bar 10 mm. (f) Treatment at 2.0 mg of oil/ml, causing cell wall disruption, squashed
and degrading hyphae clearly visible, Bar 10 mm.

ARTICLE IN PRESS
Effects of Citrus sinensis (L.) Osbeck epicarp
attened empty hyphae with the presence of
undulations along the hyphal borders were
clearly visible. On the hyphae some punctured
portions were also found (Figs. 2d and e). On
increasing the concentration up to 2.0 mg/ml
complete cell wall disruption and squashed hyphae
were found (Fig. 2f).

Discussion
Citrus sinensis essential oil caused complete
growth inhibition of A. niger at 3.0 mg/ml on agar
plates. This concentration was found to be lethal
under the test conditions. The oil showed fungistatic activity at 1.5 mg/ml with about 79% growth
inhibition after 7 days of incubation and a delay of
conidiation compared to control. The essential oil
signicantly reduced the growth of A. niger in a
dosage response manner. After calculating the
percent inhibition with respect to radial growth
(PDA) and dry weight (PDB) basis we found that the
oil was more effective in liquid medium.
Earlier fungitoxic investigations on the essential
oil of Citrus sinensis were done by Singh et al.
(1993). Caccioni et al. (1998) tested the antifungal
efcacies of citrus oils against Penicillium digitatum and Penicillium italicum and found P. digitatum was more sensitive to the inhibitory action of
the oils. Shukla et al. (2000) also reported Citrus
sinensis as a potent source of natural pesticide.
Cymbopogon nardus oil was used previously to
investigate the growth, conidiation and ultrastructure of A. niger (de Billerbeck et al., 2001). Our
observations can be related to the results presented by Cymbopogon nardus oil, but we found
that Citrus sinensis essential oil caused bifurcation
of apical hyphae and profuse budding in vegetative
hyphae leading to complete loss of cytoplasm from
the hyphae. A comparison of the results of the
present study with those of de Billerbeck et al.
(2001) clearly show that Citrus sinensis is more
effective than Cymbopogon nardus oil against
A. niger.
The observation by SEM was earlier done against
pathogens as Rhizoctonia solani, Fusarium solani,
Pythium ultimum and Colletotrichum lindemuthianum using treatment of Thymus vulgaris, Lavandula
and Mentha piperata essential oils. Thyme oil was
more effective than that of lavender or mint
(Zambonelli et al., 1996). A some what similar
result of surface modications in SEM study was
shown by de Billerbeck et al. (2001) using Cymbopogon nardus essential oil against A. niger and by
Mares et al. (2004) using Tagetes patula extract on

343
Pythium ultimum. But in our results, with Citrus
sinensis oil, completely squashed and severely
collapsed hyphae were recorded, which results in
attening and ultimately death of hyphae. The
hyphae showed lack of cytoplasm, damage and loss
of integrity and rigidity of the cell wall. These
observations indicate that the mode of antifungal
activity of essential oil of Citrus sinensis is a result
of attack of oil on the cell wall and retraction of
cytoplasm in the hyphae and ultimately death of
the mycelium. Such modications induced by
essential oil may be related to the interference of
essential oil components with enzymatic reactions
of wall synthesis, which affects fungal morphogenesis and growth. Zambonelli et al. (1996) reported
fungal growth inhibition associated with degeneration of fungal hyphae after treatment with Thymus
vulgaris essential oil. Our observations are in
agreement with the above reports.
The present study also conrmed the antifungal
activity of citrus oil already described in literature,
but the mode of action and morphological alterations were not studied yet. Based on the present
study, it could be concluded that essential oil from
Citrus sinensis possess fungitoxic activities inhibiting the growth of A. niger, leading to irreversible
deleterious morphological alterations and thus
it is worth exploiting for the biomanagement of
A. niger.

Acknowledgements
Abhishek Tripathi, gratefully acknowledge the
University Grant Commission (UGC), New Delhi,
India for the nancial support in the form of Junior
Research Fellowship (JRF). Special thanks to staff
of Electron Microscopy Unit, Birbal Sahni Institute
of Palaeobotany, Lucknow for their help.

References
Adams, R.P., 1995. Identication of Essential Oil Components by Gas Chromatography/Mass Spectroscopy.
Allured Publishing Corporation: Carol Stream, IL.
Adebajo, L.O., Popoola, O.J., 2003. Mycoora and
mycotoxins in kolanuts during storage. Afri. J.
Biotech. 2 (10), 365368.
Amusa, N.A., Ashaye, O.A., Oladapo, M.O., 2003.
Biodeterioration of the African star apple (Chrysophylum albidum) in storage and the effect on its food
value. Afr. J. Biotech. 2 (3), 5659.
Badei, A.Z.M., El-Akel, A.T.M., Morsi, H.H., Baruah, P.,
Sharma, R.K., Singh, R.S., Ghosh, A., 1996. Fungicidal
activity of some naturally occurring essential oils

ARTICLE IN PRESS
344
against Fusarium moniliforme. J. Essen. Oil Res. 8,
411412.
Barrios, M.J., Medina, L.M., Cordba, M.G., Jordano, R.,
1997. Atoxin-producing strains of Aspergillus avus
isolated from cheese. J. Food Prot. 60, 192194.
Bishop, C.D., Thornton, I.B., 1997. Evaluation of the
antifungal activity of the essential oil of Monarda
citriodora var. citriodora and Melaleuca alternifolia
on post harvest pathogens. J. Essen. Oil Res. 9, 7782.
Caccioni, D.R.L., Guizzardi, M., Biondi, D.M., Renda, A.,
Ruberto, G., 1998. Relationship between volatile
components of citrus fruit essential oils and antimicrobial action of Penicillium digitatum and Penicillium italicum. Int. J. Food Micro. 43 (1&2), 7379.
de Billerbeck, V.G., Roques, C.G., Bessiere, 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. Micro. 47, 917.
` .M., Maria, M.H., Socorro, V.,
Ernestina, A., Miguel, A
Eduardo, P., Irasema, V., 2003. Fungicidal potential of
methoxylated avones from citrus for in vitro control
of Colletotrichum gloeosporioides, causal agent of
anthracnose disease in tropical fruits. Pest Manage.
Sci. 59 (11), 12451249.
Grover, R.K., Moore, J.D., 1962. Toxicometric studies of
fungicides against brown rot organisms Sclerotinia
fructicola and S. laxa. Phytopath. 52, 876880.
Guenther, E., 1948. The essential oils, Vol. 1, p. 774.
Karapinar, M., 1985. The effects of citrus oils and some
spices on growth and aatoxin production by Aspergillus parasiticus NRRL 2999. Int. J. Food Micro. 2 (4),
239245.
Mares, D., Tosi, B., Poli, F., Andreotti, E., Romagnoli, C.,
2004. Antifungal activity of Tagetus patula extracts on
some phytopathogenic fungi: ultrastructural evidence
on Pythium ultimum. Microbiol. Res. 159, 295304.
Misra, A.K., Dubey, N.K., 1994. Evaluation of some
essential oils for their toxicity against fungi causing
deterioration of stored food commodities. Appl.
Environ. Micro. 60, 10011005.
Pandey, D.K., Tripathi, N.N., Tripathi, R.D., Dixit, S.N.,
1982. Fungitoxic and phytotoxic properties of the
essential oil of H. suaveolens. Zeit. Pazenkran.
Panzensch. 89, 344349.
Paster, N., Juven, B.J., Shaaya, E., Menasherov, M.,
Nitzan, R., Weisslowicz, H., Ravid, U., 1990. Inhibitory
effect of Oregano and Thyme essential oils on molds
and food borne bacteria. Lett. Appl. Micro. 11, 3337.
Paster, N., Menasherov, M., Ravid, U., Juven, B., 1995.
Antifungal activity of oregano and thyme essential oils

N. Sharma, A. Tripathi
applied as fumigants against fungi attacking stored
grain. J. Food Prot. 58, 8185.
Prakash, O., Raoof, M.A., 1989. Control of mango fruit
decay with post harvest application of various
chemicals against black rot, stem end rot and
anthracnose disease. Int. J. Trop. Plant Dis. 6, 99106.
Research Council, Board of Agriculture, 1987. Regulating
Pesticides in FoodThe Delaney Paradox. National
Acadimy Press, Washington, DC.
Sharma, N., 1998. Control of post-harvest diseases with
natural plant products. In: Sharma, N., Alam, M.M.
(Eds.), Postharvest Diseases of Horticultural Perishables. International Book Distrubuting Company,
Lucknow, 226004 (India).
Sharma, N., (2000). Preservation of dried fruits and nuts
from biodeterioration by natural plant volatiles. In:
Proceedings of International Conference Controlled
Atmosphere and Fumigation in Stored Products.
Executive Printing Services, Clovis, CA, USA,
pp. 195208.
Sharma, R.C., Vir, D., 1986. Post harvest diseases of
grapes and studies on their control with benzimidazole
derivatives and other fungicides. Pesticides (Bombay)
20, 1415.
Shukla, A.C., Shahi, S.K., Dixit, A., 2000. Epicarp of
Citrus sinensis: a potential source of natural pesticides. Ind. Phytopath. 53 (4), 468471.
Singh, G., Upadhyay, R.K., Narayanan, C.S., Padmkumari,
K.P., Rao, G.P., 1993. Chemical and fungitoxic
investigations on the essential oil of Citrus
sinensis (L.). Pers. J. Plant Disease Prot. 100 (1),
6974.
Sinha, P., Saxena, S.K., 1987. Effect of treating tomatoes
with leaf extract of Lantana camara on development
of fruit rot caused by A. niger in presence of
Drosophila busckii. Ind. J. Exp. Biol. 25, 143144.
Surender, P., Janalah, C., Reddy, V.K., Reddy, S.M., 1987.
Antifungal activity of secretions of scent glands
from Heteropteram bugs. Ind. J. Exp. Biol. 25,
233234.
Tewari, S.N., 1990. Toxic effect of few botanicals on
three fungal pathogens of rice. In: Chari, M.S.,
Ramprasad, G. (Eds.), Proceedings of Symposium
Botanical Pesticides in IPM, Rajmundry, India, pp.
397403.
Thomson, D.P., 1989. Fungitoxic activity of essential oil
components on food storage fungi. Mycologia 81 (1),
151153.
Zambonelli, A., Zechini dAulerio, A., Bianchi, A.,
Albasini, A., 1996. Effects of essential oil on phytopathogenic fungi. Phytopath 144, 491494.