African Crop Science Conference Proceedings Vol. 8. pp.

1755-1758
Printed in El-Minia, Egypt
ISSN 1023-070X/2007$ 4.00
2007, African Crop Science Society
Phytochemical Composition and I nVitroAntifungal Activity Screening of
Extracts from Citrus Plants against FusariumOxysporumof Okra Plant
(Hibiscusesculentus)
D. E. OKWU
1
, A. N. AWURUM
2
& J. I. OKORONKWO
1
1
. Department of Chemistry,Michael Okpara University of Agriculture Umudike, P.M.B 7267 Umuahia Abia State,
Nigeria, [email protected]
2
. Department of Plant Health Management ,Michael Okpara University of Agriculture, Umudike
P.M.B 7267 Umuahia Abia State, Nigeria
Abstract: Phytochemical studies of five varieties of citrus species, sweet orange (Citrus sinensis), tangerine (Citrus
reticulata), lemon (Citrus limonum), lime (Citrus aurantifolia) and grape (Citrus grandis) revealed the presence of
bioactive compounds comprising alkaloids (0.22-1.60%), saponin (0.30-0.98%), flavonoids (0.30-0.89), phenols (0.02-
0.64%) and tannins (0.23-1.45%). The growth of Fusarium oxysporum which causes damping-off diseases of okra
(Hibiscus esculentus) was inhibited in vitro by the extracts of citrus species. The extracts from the peels of C. sinensis,
C. aurentifolia and C. reticulata showed 83.55%, 71.10% and 68.14% inhibition activity, respectively. An analysis of
chemical composition showed that the most active constituents have relatively high alkaloids and phenolic constituents.
The fungitoxicity of the extracts from the peels of C. sinensis was the same as that of benomyl, a synthetic fungicide.
Keywords: Antifungal properties, Citrus, Synthetic fungicide, inhibition, phenolic compounds
Introduction
Numerous natural products of plant origin are pesticidal
and have the potentials to control fungal diseases of crops.
Considerable effort has been directed and devoted to
screening plants in order to develop new natural
fungicides as alternatives to existing synthetic fungicides,
which are associated with problems such as phytotoxicity,
vertebrate toxicity, pest resistance and resurgence,
widespread environmental hazards and high cost (Okwu
2003). This effort has resulted in the use of extracts from
citrus plants as botanical fungicides.
Effects of oil extracts from various citrus varieties on
the in vitro mycelia growth of Phaeoramularia angolensis
were evaluated and the result showed positive inhibition
of P. angolensis (Dangmo et al., 2002). Research has
shown (Sexena and Kidiavai 1997) that extracts of
medicinal plants such as citrus tree, neem tree, pyrethrum
and some herbs contain toxic substances and so have
potential for use in the development of natural pest
control products.
It is generally assumed that the active constituents
contributing to these antifungal properties are the
phytochemicals (Okwu 2004, 2005). Phytochemicals
constitute one of the most numerous and widely
distributed groups of substances in the plant kingdom.
Woody plants and herbs synthesize and accumulate in
their cells a great variety of phytochemicals including low
molecular phenolics (hydroxybenzoic and
hydroxycinamic acids, acetophenone, flavonoids,
stilbenes, and lignans) as well as oligo- or polymeric
forms (hydrolysable and condensed tannins and lignins)
(Close and McArthur 2002; Okwu 2004; Okwu and
Omodamiro 2005).
Flavonoids belong to a group of polyphenolic
compounds found in fruits and vegetables (Waladkhani
and Clemens 2001).The family includes monomeric
flavanols, flavanones, anthocyanidins, flavones and
flavonols (Waladkhani and Clemens 2001). In addition to
their free-radical scavenging activity (Kandaswni and
Middleton 1994) flavonoids have multiple biological
functions: antibacterial, antifungal and antiviral effects as
well as being inhibitors of phospholipase A2,
cycloxygenase and lipoxygenase (Middleton and
Kandaswani 1992).
Inhibition of germination and viability of sclerotia of
M. phaseolina by the essential oils of Citrus medica and
Ocimum canum was due to volatile and non-volatile
substances such as citral, limonenes and dipentene found
in C. medica and citral, citronellal, linalol, methyl
cinnamats, -camphor and traces of phenols and acetic
acid available in O. canum (Huang and Chung 2003).
Phenolic compounds possessing a C3 side chain at a
lower level of oxidation and containing no oxygen are
classified as essential oils. These compounds were often
considered as antifungal agents (Huang and Chung 2003),
inhibiting the growth of microorganisms.
Okra (Hibiscus esculentus) is cultivated as a vegetable
and its fruit pods are consumed as vegetables. It
originated in tropical Africa and has now been widely
spread throughout the tropics (Purseglove 1979;
Thompson and Kelly 1987). Okra plants are attacked by a
number of seed and soil diseases caused by different
fungi. Rots, blights and wilts are caused by Fusarium
oxysporum. The fungus is reported (Thompson and Kelly
1987) to reproduce the disease due to its presence in the
soil and seed as well as on infected plants. Naturally-
infected pods appear brown to black. The affected plants
show a dark brown to black discoloration from the base of
D. E. OKWU et al.,
1756
the stems. F. oxysporum caused damage, including flower
and pod abortion, pod and seed rot, shrunken pods, pod
and leaf necrosis, discoloration, reduced germination and
reduction in plant vigor in cowpea (Awurum et al., 2005).
In order to maximize yield in okra production, the plants
should be healthy and disease-free.
Citrus fruits are well endowed with a variety of
phytofungicides that are necessary to inhibit fungal
growth and development. In this report, the antifungal
properties of the extracts from the peels and leaves of
citrus tree (C. aurantifolis) against F. oxysporum was
evaluated in vitro.The present study was undertaken to
evaluate the phytochemical composition of citrus peels
and leaves and consequently to employ the extracts as a
low cost fungicide for peasant farmers.
Materials and Methods
The experiment was carried out in the Department of
Chemistry and Plant Health Management laboratories,
Michael Okpara University of Agriculture, Umudike,
Nigeria.
Source of materials
The fruits and leaves of sweet orange (Citrus
sinensis), lime (C. aurantifolia), grape (C. grandis),
tangerine (C. reticulata) and lemon (C. limonum) were
harvested from the National Root Crops Research
Institute (NRCRI) orchard, Umudike, Nigeria. The citrus
species were identified by Mr. John Ibe, the manager of
the Forestry Department, NRCRI. An infected okra plant
(Hibiscus esculentus) was collected from the research
farm of Michael Okpara University of Agriculture,
Umudike, Nigeria.
Preparation of plant extracts
The epicarps of the five citrus species were peeled off.
The leaves and peels were air dried on the laboratory
bench for 10 days and then ground into a uniform powder
using a Thomas Wiley mill machine (model Ed-5, USA).
The powdered materials (650 g powder for each sample)
were stored in air tight bottles for chemical analysis.
Extraction
Each of the powdered plant materials (100 g) was
packed into a Soxhlet apparatus (2L) and extracted
exhaustively with 500 ml of diethyl ether (60-80C) for 6
hours. The ether was evaporated using a water bath and
then left overnight at laboratory temperature for
evaporation of the remaining ether. The test solution of
each extract was prepared by dissolving 10 g of crude
plant extract separately in 100 ml sterile distilled water in
a 250 ml Erlenmeyer flask in a water bath at 80C for 2 h.
Extracts were subsequently filtered through four folds of
cheese cloth.
Isolation of the inoculum
The petiole leaf and stem of the infected plant were
cut in bits using a sharp blade and then placed in a Petri
dish and were disinfected with 70% alcohol and finally
rinsed with three changes of sterile water, after which the
tissues were placed in a Petri dish containing a moist filter
paper at room temperature of 27C. After a week, a
pronounced whitish growth was observed on the surface
of the tissues. The organism was sub-cultured twice to get
a pure culture, which was finally examined using a
compound microscope and the identity of the organism
confirmed to be F. oxysporum with the aid of an
identification manual by Barnett and Huntter (1972).
I nvitroexperiment
Each of the Petri dishes contained potato dextrose
agar (PDA) and a 10% concentration of 5 ml plant extract
were mixed together and allowed to solidify. Dishes were
inoculated with the fungus by cutting a 4 mm-diameter
disc from a pure culture of F. oxysporum growing on the
PDA using a cork borer. This was done for each of the
extracts as well as for two controls: a plate containing
benomyl and another without extract. The cultures were
incubated at 27C in an inoculation chamber for 9 days.
Radial growth of the fungus for each treatment was
measured at the 9
th
day of inoculation using a ruler and
the percentage inhibition was calculated using the formula
of Amadioha (2003), as shown below:
(DC DT) X 100
% Growth inhibition =
DT
where DC = colony diameter of control and DT = colony
diameter of treated plates.
Phytochemical analysis
Alkaloids and phenols were determined according to
the method of Harborne (1973) while tannins were
determined using the method of Van Burden and
Robinson (1981). Saponin was determined using the
method of Obadoni and Ochuko (2001). Flavonoids were
determined according to the method of Boham and
Kocipai (1994).
Statistical analysis
All measurements were replicated three times and
standard deviations determined. T Student t-test at P<
0.05 was applied to assess the difference between the
means (Steel and Torrie 1980).
Results and Discussion
The phytochemical contents of the citrus peels and leaves
are shown in Table 1. The alkaloid content of the peels of
C. sinensis was very high (1.60%), followed by C.
limonum which contained 1.35% of alkaloids while the
peels of C. aurantifolia contained 1.0% of alkaloids. The
content of alkaloids was low in the leaves compared to the
peels.
1757
Table 1 Phytochemical composition of leaves and peels citrus fruits (%).
Alkaloids Phenols Flavonoids Tannins Saponins Species
Leaf Peel Leaf Peel Leaf Peel Leaf Peel Leaf Peel
C.
reticulata
0.22
0.11
a
1.20
0.20
a
0.02
0.10
a
0.23
0.10
a
0.03
0.02
a
0.07
0.10
a
0.23
0.10
a
0.45
0.20
a
0.83
0.10
a
0.56
0.02
a
C.
aurantifolia
0.44
0.03
a
1.00
0.20
a
0.05
0.20
a
0.47
0.22
a
0.07
0.11
a
0.52
0.02
a
1.45
0.22
a
0.63
0.20
a
0.66
0.10
a
0.98
0.12
a
C. limonum 0.54
0.20
a
1.35
0.11
a
0.25
0.22
a
0.64
0.11
a
0.64
0.14
a
0.48
0.20
a
1.31
0.02
a
0.59
0.10
a
0.34
0.22
a
0.58
0.22
a
C. sinensis 0.39
0.05
a
1.60
0.03
a
0.02
0.11
a
0.40
0.20
a
0.62
0.33
a
0.34
0.12
a
0.54
0.11
a
0.92
0.20
a
0.99
0.10
a
0.72
0.30
a
C. grandis 0.28
0.11
a
1.20
0.22
a
0.10
0.02
a
0.56
0.10
a
0.89
0.20
a
0.15
0.11
a
0.76
0.20
a
0.50
0.02
a
0.86
0.20
a
0.30
0.10
a
Data are means standard deviation of triplicate determination on a dry weight basis. Means followed by the same
superscript in each row are not significant (P<0.05).
This observation was also made in the results of the
phenolic content where the values of phenol in the peels
are higher than those in the leaves. The reason may be
that during fruiting, more alkaloids and phenolic
constituents are produced in the peels in order to protect
and preserve the seeds from microbial attack (Okwu and
Emenike 2006). The mechanism of inhibitory action of
these alkaloids and phenolic compounds on micro-
organisms may be due to impairment of a variety of
enzyme systems, including those involved in energy
production, interference with the integrity of the cell
membranes and structural component synthesis (Huang
and Chung 2003). The antimicrobial activities of phenols
are further evidenced by their active role in plant disease
resistance and prevention. (Matern and Kneusel 1988;
Russel and Chopra 1990).Moreover, phenolic compounds
from plant extracts act as antimicrobial agents (Okwu
2005). Phenolic compounds are also considered to be
bacteriostatic and fungistatic. These compounds caused
swelling of hyphal tips, plasma seeping around hyphae,
leaking of plasma, cell wall distortion, abnormal
branching or fusion of hyphae and consequently
wrinkling of hyphae surface (Huang and Chung 2003).
Flavonoids are another phytochemical found in citrus
samples. The results revealed the presence of 0.03% and
0.07% (w/v) flavonoids in the peels and leaves,
respectively of C. reticulata, 0.52% and 0.07% for C.
aurantifolia while C. limonum leaves contained 0.64% of
flavonoids. Tannins content were found more in C.
aurentifolia leaf (1.45%) and C. limonum leaf contained
(1.31%) of tannins, followed by C sinensis peels which
contained 0.92% of tannins. The presence of tannins may
be a reason for the bitter and astringent taste of unripe
citrus fruits particularly lime, lemons and grapes.
Saponins were found to be available at 0.99% in C.
sinensis leaf ,C.aurantifolia peel contained 0.98% of
saponins and C.grandis leaf contained 0.86% while
C.limonum leaf contained 0.34% of saponins. The
presence of saponins may also be responsible for bitter
taste and antifungal properties these plants exhibits
particularly lemons, lime and grapes.Table 2 showed the
inhibitory effects of 10% concentration peel and leaf
extracts of citrus and benomyl on in vitro growth of
Fusarium oxysporum. C.sinensis peel extract has
significant (P< 0.05) inhibiting effect. Both C. sinensis
and synthetic fungicide (benomyl) have 83.55% and
83.64% inhibition respectively.
Table 2 Inhibitory effects of 10% concentration of leaf
and peel extracts of citrus and benomyl on in vitro growth
of Fusarium oxysporum.
Treatment % Inhibition
Benomyl 83.64 0.10
Control 0.00
Citrus samples Leaf extract Peel extract
C. reticulata 44.25 0.05
b
68.14 0.17
b
C. aurantifolia 44.25 0.05
b
71.10 0.09
b
C. limonum 27.75 0.13
a
48.48 0.07
b
C. sinensis 26.12 0.13
a
83.55 0.13
b
C. grandis 24.56 0.15
a
42.15 0.13
a
Data are means standard deviation of triplicate
determinations. Values with superscript that are the same
are not significantly different at P<0.05.
This could be linked to the fact that C. sinensis has
enormous deposit of alkaloids and phenolic content.
Better inhibition was obtained with the peels than on the
leaf extracts. This could also be attributed to the fact that
more phytochemicals were deposited on the peel
particularly alkaloids and phenols. Plants store these
antifungal, antibacterial and antiviral chemicals on the
peels to protect and preserve the seeds from microbial
attack. This agreed with the findings of Okwu and
Emenike (2006) who reported that phytochemicals are
reserved in plants to protect the plant against the attack
and inversion of micro-organisms. The extracts and
synthetic fungicide (benomyl) inhibited the growth of the
organism, Fusarium oxysporum. This is in agreement
with the work of Amadioha and Obi (1998). In the in vitro
experiment, all the extracts were highly effective in the
inhibition of the organism. Extracts of citrus plants
contain antifungal compounds that can be used as
alternative to synthetic fungicides, including fumigants
and contact pesticides. The prospect of using citrus plant
extracts for development of natural fungicides is
appealing and acceptable. This is because citrus peels and
leaves are readily available, environmentally safe, and
less risky for developing resistance in pests, less
hazardous to non target organisms and pest resurgence,
less adverse effect on plant growth, less harmful to seed
viability and quality and above all less expensive (Prakash
and Rao 1997). Based on these findings, citrus plant
extracts are viable and can be possible alternative to
synthetic pesticides for control of fungal diseases.
D. E. OKWU et al.,
1758
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