J. Env. Bio-Sci., 2015: Vol.

29 (2):571-573
(571) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

ANTIFUNGAL ACTIVITIES AND CHEMICAL CHARACTRIZATION OF NEEM LEAF

EXTRACTS ON THE GROWTH OF ALTERNARIA SPECIES - IN VITRO CULTURE MEDIUM
Naina Srivastava
Department of Botany, D.A.V. (P.G.) College, Dehradun.

Received: 18-07-2015 Accepted: 24-10-2015

Azadirachta indica (Neem) leaf extract was taken to test its antifungal activity against three Alternaria spp. Ethanolic and Methanolic
extracts in different concentrations (25%, 50%, 75% and 100%) was prepared and tested against test organisms using disc
diffusion method. The crude aqueous and alcoholic leaf extracts of neem was more effective in inhibitions of growth of the fungi
Alternaria alternata comparison to inhibitory effects on Alternaria solani growth in the artificial culture medium. Leaf extracts of
neem which are cheap and environmentally safe are promising for protecting crop species against the fungal infestation and
leading towards improvement of the crop in terms of yield and productivity. The assessment of fungitoxicity was carried out by
food poison technique using four different extracts and their activity was recorded as radial growth and percentage inhibition.
Ethanol and acetone extracts showed inhibition of growth of fungus; while methanol extract showed 50% inhibition and aqueous
extract did not produce any inhibition of fungus. Findings from present study confirmed that ethanol and acetone extract of
Azadirachta indica can be used as biofungicide to control this phytopathogenic fungus. Thus, the study ascertains the value of
neem plant, which could be of considerable interest to the development of new antifungals.

Neem is very common tree in India. It is a large evergreen
dense tree growing some 10 to 15 meter tall with a girth of
about 2-3 meter. The leaves of this are divided into numerous
leaflets, each resembling a full grown leaf. The Neem tree has
played an important role in Ayurvedic medicines and agriculture
since time immemorial. The earliest documentation of Neem
mentioned the fruit, seeds, oil, leaves, roots and bark for their
medicinal properties. Fungal diseases of crop plants have
always been one of the major constraints in successful crop
production which causes severe yield loss every year.
Injudicious use of synthetic fungicides for controlling plant
diseases have given rise to negative effects on human and
animal health and agro-ecosystem.
The Meliaceae specially neem contains at least 35 biologically
active principles of which nimbin and azadirachtin are the
most active insecticidal ingredients and are present
predominantly in the seeds, leaves and other parts of the neem
tree. A. indica, a common medicinal plant that could be
exploited as the source of a potent biocide that has immense
fungicidal effect against several fungal pathogens including
Aspergillus and Rhizopus1. Similarly, aqueous extract of neem
extract have potential to inhibit powdery mildew and ability to
induce systemic resistance in pea and balsam 2. Azadirachta
indica is commonly used for the treatment of Diabetes and
show the potential role of anti diabetic activity3. Azadirachta
indica leaves possessed good anti fungal activity, confirming
NAAS Rating (2016)-4.20
the great potential of bioactive compounds and is useful for
rationalizing the use of this plant in primary health care4. Almost
every part of the tree has been in use since ancient times to
treat a number of human ailments and also as a household
pesticide5.
MATERIAL AND METHODS
1. Collection of raw materials and preparation of extracts:
Mature leaves were collected separately from Azadirachta
indica plants. For antifungal and secondary metabolite studies,
fresh leaves were collected. Collected fresh leaves of A. indica
were washed thoroughly in tap water and sterile distilled water,
air-dried at 27°C, weighted and ground in a sterile mortar. The
pastes was added to 100ml of sterile distilled water in 250 ml
beaker, stirred vigorously and allow to stand for 1 hour and
then filtered through four folds of sterile cheese cloth to obtain
water extract.
2. Determination of antimicrobial activity: Potato dextrose
agar (PDA) slants at 40C. For evaluation of in vitro antifungal
activity of the biocide (plant extract of Azadirachta indica) the
phyto-extracts were added to Potato Dextrose Agar (PDA)
medium in different concentrations (0.1%;0.5% and 1%) in
separate sterilized petri plates. Each plate was inoculated
with a mycelial disc taken from 7-day-old culture raised on
PDA. The inoculated plates were incubated at 30 ±1°C and
the diameter of colony of the pathogen was measured in each

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 ANTIFUNGAL ACTIVITIES AND CHEMICAL CHARACTRIZATION (572)

case for successive 7 days. investigate the bioactivity, mechanism of action,

pharmacokinetics and toxicity of compounds isolated from
3. Determination of Minimum inhibitory concentration
neem plant.
(MIC)- In vitro tests: Results of the present investigation shows
that the growth fungus Alternaria spp.was inhibited with the RESULTS AND DISCUSSION
crude aqueous and alcoholic extract of different aged leaves
Antifungal activity of neem extract was determined against
of Azadirachla indica. From the result it is evident that the
plant pathogenic fungi viz . Alternaria spp. The neem extract
inhibition of growth of both the fungus was more pronounced
0.15%, 0.25% and 0.35% concentration tested by agar
with ethanolic leaf extracts as compared to aqueous leaf
diffusion plate method caused significantly reduction in the
extracts. Significant inhibition of growth of Alternaria spp.
growth of above fungi. The rate of growth reduction was directly
observed in the artificial culture media containing older leaf
proportional to the concentration of tested material in the
extracts of Azadirachta indica. The concentration of aqueous
medium. Result showed that neem extract significantly
and alcoholic leaf extracts of different aged leaves it was
inhibited the growth of all tested fungi. 0.15% concentration of
observed that higher concentrations leaf extracts were more
neem extract possesses remarkable antifungal activity toward
effective on growth inhibition of Alternaria spp. and it was also
some fungi compared to control only two fungal species viz.,
noted that from early period of incubation inhibition of growth
Alternaria alternata and Alternaria brassicae had strong
occurred. Percentage inhibition of fungi growth by the leaf
antifungal properties towards neem extract at same
extracts was calculated using the formula:
concentration.
% FG = Dc-Dr/Dc x100
The result showed that natural neem extract suppress the
Where: %FG = % inhibition of fungi growth
mycelial growth of Alternaria alternata and A. brassicae. At
Dc = diameter of control
0.25% concentration, neem extract showed greater
Dr = diameter of test
suppression in the growth of Alternaria spp. However, the least
The MIC was interpreted as the lowest concentration of the
inhibitory effect was found on Alternaria solani compared to all
extract that did not show any visible growth when compared
fungi and gave only 4.15% mycelial growth6. They used plants
to control tubes. Although crude extracts from various parts of
extract against pathogenic species of genus Alternaria viz.,
neem plant have medicinal applications from time immemorial,
A. alternata, A. citri and A. tenuissima. The results of this
very little work has been done on the biological activity and
study clearly reflect that neem has inherent ability to induce
plausible medicinal applications of isolated compounds. Hence
toxic effect on mycelial growth and proliferation of these fungi.
drug development programmes could be undertaken to

Table-1.Comparative view showing the effect of Neem extract on different fungal species.

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 (573) SRIVASTAVA

Workers observed that 0.25% dose of neem extract had more
inhibitory effect in Alternaria brassicae compare to A. solani7.
They also proved that plant extract had antifungal effect on A.
flavus, A. glaucus, Candida albicans, C. tropicalis,
Trichophyton mentagrophytes and T. rubrun. Plant extact
0.35% concentration was most significntly effective towards
all tested fungi. At this concentration, the inhibition in mycelial
growth was 24.29% against A. brassicae, 9.26% for A.
alternata and only 6.24% for A. solani. It was also examined
vitro study suggested a potential inhibitory effect of neem leaf
extracts against growth and viability of Alternaria alternata, A.
brassicae and A. solani. Plant may be further investigated for
their anti -fungal properties under in vivo conditions.
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