ANTIFUNGAL ACTIVITIES OF SEED OIL OF NEEM (Azadirachta indica A. Juss.)

Abstract

Abstract
This study was carried out to investigate the effects of Neem (Azadirachta indica A. Juss.) seed oil on four fungi, namely: Fusarium sp. Rhizopus sp. Curvularia sp. and Aspergillus sp. which are pathogenic in nature. The crude extract of neem seed was obtained using petroleum ether. The extract inhibited the growth of all the fungi tested. The extent to which the extract inhibited the growth of the fungi was observed to be different for each of the fungi. Growth inhibition was highest in Curvularia sp. (which did not grow beyond the initial point of its radial growth before introduction of the extract), while the lowest effect was observed in Rhizopus sp.

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G.J.B.A.H.S.,Vol.3(1):106-109 (January – March,2014) ISSN: 2319 – 5584
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ANTIFUNGAL ACTIVITIES OF SEED OIL OF NEEM (Azadirachta indica A. Juss.)
1ADEPOJU Adeyinka Olufemi; 2OGUNKUNLE Adepoju Tunde Joseph & 3FEMI-ADEPOJU Abiola Grace
1&2Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso, Nigeria.
3Department of Biosciences and Biotechnology, Kwara State University, Malete, Kwara State, Nigeria.
Abstract
This study was carried out to investigate the effects of Neem (Azadirachta indica A. Juss.) seed oil on four fungi,
namely: Fusarium sp. Rhizopus sp. Curvularia sp. and Aspergillus sp. which are pathogenic in nature. The crude extract
of neem seed was obtained using petroleum ether. The extract inhibited the growth of all the fungi tested. The extent to
which the extract inhibited the growth of the fungi was observed to be different for each of the fungi. Growth inhibition
was highest in Curvularia sp. (which did not grow beyond the initial point of its radial growth before introduction of the
extract), while the lowest effect was observed in Rhizopus sp.
Keywords: Azadirachta indica, Antifungal, Neem seed oil, Curvularia sp.
1.0 Introduction
Fungi are the major cause of plant diseases and are responsible for large scale harvest failures in crops like maize
and other cereals all over the world (Suleiman and Omafe, 2013). The fungi genera typically found in stored grains are
Aspergillus, Penicillium, Fusarium and some xerophytic species, several of them with capabilities of producing toxins
(Castellari et al., 2010). Seed fungi especially species of Aspergillus, Diplodia, Penicillium, Fusarium, Trichoderma and
a number of phycomycetes affect the seed of all forest species. Previous studies by Pacin et al. (2009) identified
Aspergillus and Fusarium as mycotoxigenic species in stored grains. So also, their mycotoxins and fumonisins were
reported in different concentrations by Moreno et al. (2009) in stored grains.
Chemical control of fungal pathogens has been of help in the increase of crop yield. However, usage of these
chemical products is being discouraged due to the resultant environmental pollution which leaves toxic residues in soil,
water and food. Some chemicals are also harmful to non-target organisms and this leads to ecological imbalance and
development of fungicidal resistant strains. All these limitations call for an alternative plant disease management strategy
such as biological control (Gardener and Fravel, 2002; Lokesha and Benagi, 2007). Biological control method is
preferred because it is selective with no side effects, and is relatively cheap. Moreover, resistance to biological control is
rare and biological control agents are self-propagating and self-perpetuating.
Neem (Azadirachta indica) is a widely prevalent tree, mainly cultivated in India subcontinent (Karl, 1997). Various
parts of the tree have been used as traditional Ayurvedic medicine in India (Brahmachari, 2004). Neem oil in particular
was widely used as a traditional medicine in India, Sri Lanka, Burma, Thailand, Malaysia and Indonesia and already has
more than 2000 years history. Neem oil was often administered orally, for deworming and constipation, and is applied
topically to relieve rheumatism, ulcer, itching and cure chronic skin diseases (Aggarwal and Dhawan, 1995). There is
evidence that neem oil has acaricidal, antibacterial, antifungal, antimalarial, antiparasitic, anti-inflammatory as well as
immunomodulatory properties in different animal species (Mulla and Su, 1999; Biswas et al., 2002; Brahmachari, 2004;
Gossé et al., 2005; Du et al., 2007, 2008, 2009; Xu et al., 2010; Zhang et al., 2010). Due to its efficacy, biodegradability
and minimum side effects, azadirachtin, a tetranortriterpenoid obtained from neem seeds, has emerged as a natural
biopesticide (Locke, 1995; Martinez, 2002).
The objective of this study was to evaluate the efficacy of neem seed oil against selected pathogenic fungi species
that constitute major threats to agricultural products in Nigeria.
2.0 Materials and Methods
2.1 Media Preparation and Culture
Potato Dextrose Agar (PDA) combined with Chloramphenicol was used for fungi isolation and enumeration. Plates
were incubated at room temperature for 3–5 days. For identification purpose, the microscopic and macroscopic features
of the hypha mass, morphology of cells and spores, nature of the fruiting bodies, among other criteria were used
according to the method of Uzma and Shahida (2007).
2.2 Preparation of Neem Seed Oil
Seed oil extraction was carried out using the method of Eksteen et al. (2001) with slight modifications. Seeds from
neem trees were obtained from the campus of Kwara State College of Education, Ilorin, Nigeria. The shells were
separated from the kernel and were sun-dried. The shells were blended, air-dried and later oven-dried, in order to remove
every moisture trace that might remain. The powder of the neem seed kernel (250g) obtained was soaked in one litre of
petroleum ether and placed on a shaker for about 72hours. Using a muslin cloth, the mixture was filtered and the cake
was kept. The filtrate obtained was made to undergo distillation to separate the oil obtained from the neem seed powder
from the solvent.

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2.3 Tests for Sterility of Extract
About 1ml of the oil obtained was inoculated onto 4 sterile petri dishes. About 9ml of agar medium were then
poured into the plates and mixed and then allowed to set. Subsequent incubation showed no growth on the mixture thus
confirming the sterility of the extract, the environment and the apparatus used.
2.4 Determination of Effectiveness of Neeem Seed Oil on the Test Organisms
The neem seed oil was used directly in its pure form without varying its concentration. The use of 100%
concentration of the test oil was informed by the outcome of a preliminary survey of its efficacy at different
concentrations which led to the exclusion of lower concentrations on account of their ineffectiveness. Application of
extracts against the test organisms was carried out using the pour plate and the cork-boring methods, both of which were
undertaken according to the method of Suleiman and Omafe (2013). Two control experiments were also set up using
distilled water on the one hand and petroleum ether on the other hand in place of the test oil.
2.5 Determination of Percentage Inhibition
The diameter of radial growth of each test organism on the control plates was measured at regular intervals of 24
hours for 5 days and the mean was calculated and designated as X. The radial growth on each experimental plate was
also measured and the mean calculated and designated as Y. The percentage inhibition of each organism by the test oil
was calculated using the conventional formula as follows:
(X-Y) x 100%
X
[X= mean radial growth on control plates. Y= mean radial growth on experimental plates]
3.0 Results
3.1 The Effects of the Neem Seed Oil on the Test Fungi
From the results of the experiment, neem seed oil can be observed to have completely inhibited the growth of
Curvularia sp. and substantially retarded the growth of Aspergillus and Fusarium species. However, the treatment had no
notable effect on Rhizopus stolonifer (Tables 1 and 2).
Table I: 5-Day radial growth of some fungi species following treatment with neem seed oil
Organisms
DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
MEAN
(mm)
Aspergillus sp
2.05
3.00
3.00
3.05
3.10
2.84
Curvularia sp
1.50
1.50
1.50
1.50
1.50
1.50
Fusarium sp
2.05
2.60
3.60
3.80
3.85
3.18
Rhizopus sp
6.00
8.70
11.00
13.50
16.00
11.00
Table 2: 5-Day radial growth of some fungi species in the control set up*
Organisms
DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
MEAN
(mm)
Aspergillus sp
2.50
3.00
3.50
3.90
4.90
3.56
Curvularia sp
1.80
2.10
2.40
2.70
3.05
2.41
Fusarium sp
2.50
3.00
3.40
3.90
4.40
3.44
Rhizopus sp
6.00
8.70
11.40
14.10
15.80
11.20
*Measurements indicate observations in both distilled water and petroleum ether.
A comparison of the results in Tables 1 and 2 shows that neem seed oil contains anti-fungal properties which made
it possible for it to successfully retard the growth of the four test organisms used in this experiment. However, the
effectiveness of the oil varied across the organisms as indicated by their % inhibition (Table 3). Table 3 shows that the
lowest percentage inhibition was recorded in both Rhizopus and Fusarium species i.e.1.79 and 7.56 respectively. In
contrary, the percentage inhibition observed in Aspergillus and Curvularia sp were substantial, being 20.22 and 37.76
respectively. Thus, the neem seed oil had the highest inhibitory activity on the growth of Curvularia sp and the lowest on
Rhizopus sp.
Table 3: Comparison of the mean radial growths of organisms in the treatment and control experiments
Organisms
Mean radial growth (mm)
Percentage
inhibition (%)
Treatment
Control
Difference
Aspergillus sp
2.84
3.56
0.72
20.22
Curvularia sp
1.50
2.41
0.91
37.76
Fusarium sp
3.18
3.44
0.26
7.56
Rhizopus sp
11.00
11.20
0.20
1.79
4.0 Discussion

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The results of this study show that petroleum ether extract of neem seed oil had antimicrobial properties against
three of the four fungi species studied, Rhizopus sp being the only exception. Many workers have reported the use of
plant extracts in the control of fungal diseases (e.g. Dubey et al., 2009; Satish et al., 2008). Many phytofungicides have
been obtained from a number of plant extracts. These include “Fitoekols-IF” from Pinus sylvestris and Picea abies
greens extract, “Fitosativum” from Allium sativum extract, “Fitocapsicum” from Capsicum annuum extract,
“Fitokrisanthemium” from Chrisanthemum sp. leaf extract, “Fitoarmoracium” from Armoracia rusticana root and leaf
extract, “Fitotabacum” from Nicotiana tabacum and N. rustica extracts, “Fitopelargonium” from Pelargonium sp. leaf
extract and “Fitosinepium”-from white mustard (Sinapis alba) plant and seed extract (Zarins et al., 2009). Citrus fruits
have also been acknowledged by Munoz and Marcos (2006) to possess a variety of phytofungicides that help to inhibit
fungal growth and development.
Afzal et al. (2010) reported Allium sativum to have a wide antifungal spectrum that effected 60-82% inhibition in
the growth of seed borne Aspergillus and Penicillium fungi. This was attributed to phytochemical properties of garlic
plant, allicin which could decompose into several effective antimicrobial compounds such as diallyl sulphide, diallyl
disulphide, diallyl trisulphide, allyl methyl trisulphide, dithiins and ajoene (Salim 2011; Tagoe, 2011).
According to Mulla and Su (1999) and Biswas et al.(2002), neem oil, extracted from the seeds of Azadirachta
indica, has versatile medicinal properties, including antifertility, antifungal, antibacterial, immunostimulant, antipyretic
and acaricidal activities. Chloroform extracts and petroleum ether extracts of neem oil have also been found to exhibit
potent acaricidal activity against Sarcopte scabiei var. cuniculi larvae (Du et al., 2008, 2009). Neem extract was also
found by Da-Costa et al. (2010) to have inhibited the fungal growth (i.e. mycelia dry weight, diameter of colony and
growth rate) of Aspergillus flavus on solid media at concentrations from 0.5 to 5.0% v/v, although it significantly
increased sporulation in the same conditions. Bhutta et al. (2001) tested 32 different seed diffusates against Aspergillus
alternata and Fusarium solani and found that the diffusates from Corriander sativum and Memoranda charata exhibited
inhibitory effects at 0.5% and 1% concentrations. Eksteen et al. (2001) also tested 11 plant extracts against different
pathogenic fungi including F. oxysporum and Rhizopus solani by the agar dilution method and obtained encouraging
results comparable inhibitory effects on mycelial growth with reference to those obtained using Carbendazim and
Difenconazole. Similar observations were recorded against Alternaria solani by using Allium cepa extract (Khallial,
2001).
Locke (1995), Martinez (2002) and Da-Costa et al. (2010) all reported that due to the antifungal efficacy of neem
seed extract, its biodegradability and minimum side effects, azadirachtin, a tetranortriterpenoid obtained from the seed
has emerged as a natural biopesticide. In addition, the percentage inhibition against the tested fungi were found to
increase at different rates by increasing the concentration of neem leaf and seed extract with the result that neem seed
organic extracts had higher inhibition percentage than that of neem leaf organic extracts.
5.0 Conclusion
From the results of this study, it can be concluded that the antifungal effects of neem seed extract was highest
against Curvularia sp. (37.76% inhibition) followed by that of Aspergillus sp. (20.22% inhibition) and Fusarium sp.
(7.56% inhibition). The extract had no significant inhibitory effect on Rhizopus sp.
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