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African Journal of Microbiology Research Vol. 6(36), pp. 6572-6575, 20 September, 2012
Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR12.981
ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
Effects of essential oil extracted from Citrullus
colocynthis (CCT) seeds on growth of
phytopathogenic bacteria
Zahra Setayesh Mehr, Nima Sanadgol*
and LeylaVafadar Ghasemi
Department of Biology, Faculty of Science, Zabol University, P.O.Box 1568, Zabol, Iran.
Accepted 14 July, 2012
Citrullus colocynthis (CCT) is a non-hardy, herbaceous perennial vine, branched from the base.
Originally from Tropical Asia and Africa, it is now widely distributed in the Sistan phytogeographic
region of Iran. In a search of alternative ways to control plant disease, essential oil from seeds of CCT
was examined for antibacterial properties. The seeds are edible and have a high oil content with a large
proportion of linoleic acid (C18:2) which is important for human nutrition and an essential fatty acid
also contains only traces of linolenic acid (C18:3). Antibacterial activity of oil separated from the seeds
was tested against Xanthomonas campestris, Burkholderia cenocepacia, Pseudomonas syringae and
Agrobacterium tumefaciens. The agar disc diffusion method
was used to assess inhibitory effect by
measuring the inhibition zone against the test microorganisms. Antibacterial activity of the seeds oil
was confirmed for all bacterial, but with different ranges. This activity was observed to be dose-
independent. X. campestris was the most sensitive bacterium tested. A weak inhibitory effect was found
against Pseudomonas syringae. These results offer a scientific basis for the use of C. colocynthis seed
oil to prevent diseases caused by these bacteria.
Key words: Citrullus colocynthis, phytopathogens, agar disc diffusion.
INTRODUCTION
Bacterial pathogens and their control are a serious
problem in agriculture practice. Many of the currently
available antimicrobial agents for agriculture are highly
toxic, non-biodegradable, and cause extended environ-
mental pollution (Vyvyan, 2002). Diseases caused by
pathogens including bacteria and fungi significantly
contribute to the overall loss in crop yields worldwide
(Savary et al., 2006). Despite the existence of plant
defense mechanisms, a major difficulty encountered is
the lack of effective control agents against some severe
plant bacterial diseases. On the other hand, application of
chemical derivatives has effectively controlled the plants
from bacterial disease but this threatens to contaminate
the environment, hindering the management of diseases
in crops and agricultural products (Burhan, 2009). The
search for agents to cure infectious diseases began
*Corresponding author. E-mail: n.sanadgol@uoz.ac.ir,
sanadgol.n@gmail.com. Tel: +98-915-7444696. Fax: +98-542-
2240696
long before people were aware of the existence of
microbes. Herbal medicine represents one of the most
important fields of traditional medicine all over the world
(Hamil, 2003). Nowadays, medicinal plants receive
attention to research centers because of their special
importance in safety of communities (Mona, 2002).
The curative properties of medicinal plants are mainly
due to the presence of various complex chemical
substances of different composition which occur as
secondary metabolites (Karthikeyan, 2009). Plant based
natural constituents can be derived from any part of the
plant like bark, leaves, flowers, roots, fruits, seeds, etc
(Gordon and David, 2001). Medicinal and aromatic
plants form a large group of economically important
plants that provide the basic raw materials for indi-
genous pharmaceuticals, perfumery, flavor and cosmetic
industries. Citrullus colocynthis (Linn.) Schrad.,
(colocynth, wild-gourd or bitter-apple) is an important
medicinal plant belonging to the family Cucurbitaceae. It
is a well-recognized plant in the traditional medicine and
was used by people in rural areas as purgative, antidiabetic
and insecticide. Various oils are biocides against a broad
range of organisms such as bacteria, fungi, viruses,
protozoa, insects and plants (Dung et al., 2008;
Gurudeeban et al., 2010). Recent researches are showed
that essential oils of many plants possess antimicrobial
activities and maybe used for the treatment of
different diseases in the near future (Elaissi et al., 2011;
Erkan et al., 2012; Vairappan et al., 2012; Sivasothy et
al., 2012; Makhloufi et al., 2012; Abdelhady and Aly
,
2012). There is vast diversity among aromatic and
medicinal plants and different chemotypes of the same
species may grow in the same place and produce
different oils with different activity (Darokar et al., 1998).
The current work presents an evaluation of antibacterial
activity of essential oil from Iranian C. colocynthis and
their inhibitory effect against the growth of some
phytopathogenic bacteria.
MATERIALS AND METHODS
Oil isolation and extraction
Fresh fruits were collected from south-eastern of Iran during
2010/2011, especially Sistan region in large quantities. The seeds
were generally collected after fruit ripening, between September
and October. Dried seeds were powder and hydrodistilled for 5 h
using a Clevenger apparatus with a water-cooled oil receiver. The
oil was dried over anhydrous Na
2
SO
4
and preserved in a sealed vial
at 4°C in the dark until further analysis (yield 0.88%, w/w).
Test microorganisms
The test organisms used in this study (reference strains) were
Xanthomonas campestris pv. Campestris (ATCC33913),
Pseudomonas syringae pv. Syringae (B728a), Burkholderia
cenocepacia (HI2424) and Agrobacterium tumefaciens (str. C58).
The stock cultures were maintained in nutrient agar (NA) slant at
4°C and sub-cultured monthly. Working cultures were pre pared by
inoculating a loopful of each test microorganism in 3 ml of nutrient
broth (NB) from NA slants. Broths were incubated at 37°C for 12 h.
The suspension was diluted with sterile distilled water to obtain
approximately 10
6
CFU/ml.
Antibacterial testing
The seeds oil was tested for antibacterial activity by the disc agar
diffusion method (Murray et al., 199
5). Disk diffu
sion: 5 mm of sterile
disks were incorporated in 100 µl of plant extracts (5 mg/disk). The
disk (6 mm in diameter, Whatman No. 1) was completely saturated
with the extract and allowed to dry. Mueller Hinton (MH) agar plates
were swabbed with test bacteria and six extract disks with one of
the standard positive control disks (streptomycin) was placed on the
MH agar plate. Dimethyl sulfoxide (DMSO) was taken as the
negative control (10% DMSO did not show any antibacterial
activity). The plates were incubated at 37°C for 24 h and the
diameter of the inhibition zones were measured in mm.
Minimum inhibitory and minimum bactericidal concentrations
Micro-dilution susceptibility assay was performed using the NCCLS
and CLSI methods for the determination of minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC)
Mehr et al. 6573
(NCCLS, 1993; CLSI, 2009). Bacteria were cultured overnight at
30°C. The test samples of oil were dissolved in 5% DMSO.
Dilutions were prepared in a 96-well microtiter plates to get final
concentrations ranging from 0 to 4 µg/ml. Finally, 20 µl of inoculum
(10
6
– 10
7
CFU/ml) was inoculated onto the microplates and the
tests were performed in a volume of 200 µl. Plates were incubated
at 30°C for 24 h. The standard reference drug, ampici llin, was used
as a positive control for the tested plant pathogenic bacteria. The
lowest concentrations of tested samples, which did not show any
visual growth after macroscopic evaluation, were determined as
MICs, which were expressed in µg/ml. Using the results of the MIC
assay, the concentrations showing complete absence of visual
growth of bacteria were identified and 50 µl of each culture broth
was transferred onto the agar plates and incubated for the specified
time and temperature as mentioned above. The complete absence
of growth on the agar surface in the lowest concentration of sample
was defined as the MBC. Each assay in this experiment was
replicated three times.
Statistical analysis
The data obtained for antibacterial activity of essential oil and
various extracts were statistically analyzed and mean values were
calculated. A Student’s t test was computed for the statistical
significance of the results at p<0.05. All experiments were
performed at least, three times (unless indicated otherwise) and
were highly reproducible.
RESULTS AND DISCUSSION
The oil obtained from the seeds of CCT revealed
relatively potential antibacterial effects at the
concentrations utilized against all selected plant patho-
genic bacterial (Table 1). Xanthomonas campestris pv.
Campestris (ATCC33913) was found most sus-ceptible
pathogenic bacteria to the oil of CCT seeds. The
diameter of the inhibition zones of the oil against the
tested strains of Xanthomonas were in the range of 14-20
mm. On the other hand, standard streptomycin showed
both lower antibacterial (Xanthomonas) and comparable
(Burkholderia) effect as compared to the seeds oil
dependent of bacterial species (Table 1). The minimum
inhibitory concentration of the extracts varied between
35.0 - 81.86 µg/ml while the minimum bactericidal con-
centration was between 59.0-123.0 µg/ml (Table 1). As
shown in Table 1, the minimum concentrations of seeds
oil were found more susceptible to the tested plant
pathogenic bacteria of X. campestris pv. Campestris
(ATCC33913) as compared to the other bacteria. The
seeds oil had a detrimental effect on Xanthomonas. The
seeds oil displayed remarkable antibacterial activity
against tested strains such as X. campestris pv.
Campestris (ATCC33913), Burkholderia cenocepacia
(HI2424) and Agrobacterium tumefaciens (str. C58) with
MIC and MBC values of 35.26-70.1, 62.5-250 and 62.5-
250 µg/ml, respectively. On the other hand, the seeds oil
displayed better antibacterial effect against the tested
bacterial pathogens as MIC values as compared to
standard streptomycin (MIC: 300-500 µg/ml). However, in
some cases, the extracts had a higher antibacterial effect
6574 Afr. J. Microbiol. Res.
Table 1. Antibacterial activity, Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of seeds oil
of CCT against selected plant pathogenic bacterial.
Bacterial pathogen Seeds oil
a
Standard
b
IZ
c
MIC MBC IZ MIC
Xanthomonas campestris pv. Campestris (ATCC33913)
15.0±0.0
*
35.0 70.0 11.0±0.0 500
Pseudomonas syringae pv. Syringae (B728a)
8.0±0.0
*
65.2 65.0 13.0±0.0
*
350
Burkholderia cenocepacia (HI2424) 14.0±0.0 37.00 59.0 14.0±0.0 400
Agrobacterium tumefaciens (str. C58) 12.0±0.0
*
81.86 123.0 15.0±0.0
*
300
a
oil used at 1,000 µg/disc;
b
Standard: streptomycin (20 µg/ml, all values are in µg/ml);
*
P<0.05 significant,
c
Data are expressed as the
diameter of inhibition zones (IZ) in mm; Values are given as an average of triplicate experiments.
as compared to the standard antibiotic which might be
due to the presence of highly bioactive compounds in
seeds oil. The increased awareness of the environmental
problems
associated
with conventional non-biodegradable
agrochemicals has led to the search for non-conventional
chemicals of biological origin for the management of
post-harvest disease in fruits and vegetables (Abhay et
al., 2012). Bioactive compounds are naturally produced in
the plants and among of them essential oil are important
for the physiology of plants contributing properties confer
resistance against microorganisms, other organisms and
even antibacterial activities (Abhay et al., 2012; Cantore
et al., 2009; Kotan et al., 2010). The observed anti-
bacterial properties of CCT essential oil show its potential
for the practical use of the essential oil towards plant
pathogenic bacteria as a natural bactericide and it was
similar with previous studies (Marzouk et al., 2010). The
preliminary qualitative phytochemical screening of CCT
was reported in previous paper (Najafi et al., 2010;
Gurudeeban et al., 2010). Analysis of CCT fatty acid
methyl esters showed the presence of palmitic, stearic,
oleic, linoleic and linolenic acids in appreciable quantities
(Kulkarni et al., 2012). The obtained results suggest that
the use of CCT oil as antibacterial agent may be
judiciously applied to prevent the decay of fruits and
vegetables due to bacteria. The isolation and purification
of the phytochemical followed by a detailed study might
result in identification of lead compound and thus a
potential cure for the diseases caused by this bacteria.
REFERENCES
Abdelhady
MI, Aly HAH
(2012). Antioxidant and antimicrobial activities
of Callistemon comboynensis essential oil. Free Radic. Antioxidants
2(1):37-41.
Abhay K, Pooja SP, Palni UT, Tripathi NN (2012). In vitro antibacterial
activities of the essential oils of aromatic plants against Erwinia
herbicola (Lohnis) and Pseudomonas putida (Kris Hamilton). J. Serb.
Chem. Soc. 77(3):313-323.
Bauer AW, Kirby WMM, Sherris JC, Turck M (1966). Antibiotic
sensitivity testing by standardized single disk method. Am. J. Clin.
Pathol. 45:493-496.
Burhan M, Talib S, Ishfaq M, Ahmad S (2009). Effect of various
chemicals on citrus canker (Xanthomonas campestris pv. citri) in
nursery plants. J. Agric. Res. 47:465-468.
Cantore PL, Shanmugaiah V, Iacobellis NS (2009). Antibacterial Activity
of Essential Oil Components and Their Potential Use in Seed
Disinfection. J. Agric. Food Chem. 57(20):9454-9461.
CLSI (2009) Clinical and Laboratory Standards Institute (CLSI): Method
for dilution antimicrobial susceptibility tests for bacterial that grow
aerobically; approved standard - Eighth Edition. CLSI document M07-
A8, Wayne, PA, USA.
Cragg GM, Newman DJ, Snader KM (1997). Natural products in drug
discovery and development. J. Nat. Prod. 60: 52-60.
Darokar MP, Mathur A, Dwivedi S, Bhalla R, Khanuja SPS, Kumar S
(1998). Detection of antibacterial activity in the floral petals of some
higher plants. Curr. Sci. 75:187-189.
Dung NT, Kim JM, Kang SC (2008). Chemical composition, anti-
microbial and antioxidant activities of the essential oil and the ethanol
extract of Cleistocalyx operculatus (Roxb.) Merr and Perry buds.
Food Chem. Toxicol. 46:3632-3639.
Elaissi A, Hadj Salah K, Mabrouk S, Mohamed Larbi K, Chemli R, Skhiri
FH (2011). Antibacterial activity and chemical composition of 20
Eucalypt species’ essential oils. Food Chem. 129(4):1427-1434.
Erkan N, Tao Z, Rupasinghe HP, Uysal B, Oksal BS (2012).
Antibacterial activities of essential oils extracted from leaves of
Murraya koenigii by solvent-free microwave extraction and hydro-
distillation. Nat. Prod. Commun. 7(1):121-124.
Fluit AC, Wielders CLC, Verhoef J (2001). Epidemiology and
susceptibility of Staphylococcus aureus isolates from 25 university
hospitals participating in the Eur. SENTRY Study. J. Clin. Microbiol.
39:3727-3732.
Gordon MC, David JN (2001). Natural product drug discovery in the
next millennium. Pharm. Biol. 39:8-17.
Gurudeeban S, Rajamanickam E, Ramanathan T, Satyavani K (2010).
Antimicrobial activity of Citrullus colocynthis in gulf of manner. Int. J.
Curr. Res. 2:078-081.
Gurudeeban S, Satyavani K, Ramanathan T (2010). Bitter Apple: An
Overview of Chemical Composition and Biomedical Potentials. Asia
J. Plant Sci. 9(7):394-401.
Hamil FA, Apio S, Mubiru NK, Bukenya-Ziraba R, Mosango M, Maganyi
OW, Soejarto DD (2003). Traditional herbal drugs of southern
Uganda. J. Ethnopharma 84:57-78.
Harborne JH (1973). Phytochemical Methods. Chapman and Hill,
Tokyo, Japan.
Karthikeyan A, Shanthi V, Nagasathaya A (2009). Preliminary
phytochemical and antibacterial screening of crude extract of the leaf
of Adhatoda vasica. L. Int. J. Green Pharm. 3:78-80.
Kotan R, Cakir A, Dadasoglu F, Aydin T, Cakmakci R, Ozer H, Kordali
S, Mete E, Dikbas N (2010). Antibacterial activities of essential oils
and extracts of Turkish Achillea, Satureja and Thymus species
against plant pathogenic bacteria. J. Sci. Food Agric. 90(1):145-160.
Kulkarni AS, Khotpal RR, Karadbhajane VY, More VI (2012). Physico-
chemical Composition and lipid classes of Aegle marmelos(Bael)
and Citrullus colocynthis( Tumba) Seed Oils. J. Chem. Pharm. Res.
4(3):1486-1488.
Makhloufi A, Moussaoui A, Lazouni HA (2012). Antibacterial activities of
essential oil and crude extracts from Matricaria pubescens (Desf.)
growing wild in Bechar, South west of Algeria. J. Med. Plants Res.
6(16):3124-3128.
Marzouk B, Marzouk Z, Décor R, Mhadhebi L, Fenina N, Aouni M
(2010). Antibacterial and antifungal activities of several populations of
Tunisian Citrullus colocynthis Schrad. immature fruits and seeds. J.
Med. Mycol. 20(3):179-184.
Mona A, Abdel R, Gehan I, Samir AM (2012). Evaluation of antibacterial
properties and biochemical effects of monoterpenes on plant
pathogenic bacteria. Afr. J. Microbiol. Res. 6(15):3667-3672.
Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH (1995) ASM:
manual of clinical microbiology 6th edn. Washington.
Najafi SH, Sanadgol N, Sadeghi NB, Ashofteh BM, Sanadgol E (2010).
Phytochemical screening and antibacterial activity of Citrullus
colocynthis (Linn.) Schrad against Staphylococcus aureus. J. Med.
Plants Res. 4(22):2321-2325.
NCCLS (1993). National Committee for Clinical Laboratory Standards
(NCCLS), Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically. Approved Standard - NCCLS
document M7-A6, Wayne, PA, USA.
Mehr et al. 6575
Savary S, Teng PS, Willocquet L, Nutter FW (2006). Quantification and
modeling of crop losses: a review of purposes. Ann. Rev.
Phytopathol. 44:89-112.
Sivasothy Y, Awang K, Ibrahim H, Thong KL, Fitrah N, Koh XP, Tan LK
(2012). Chemical composition and antibacterial activities of essential
oils from Zingiber spectabile Griff. J. Essen. Oil Res. 24(3):305-313.
Vairappan CS, Nagappan T, Palaniveloo K (2012). Essential oil
composition, cytotoxic and antibacterial activities of five Etlingera
species from Borneo. Nat. Prod. Commun. 7(2):239-242.
