Varaprasad Bobbarala et al.

/ Drug Invention Today 2009, 1(1),3-6

Available online through
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Research Article

Control of phytopathogenic fungi Colletotrichum graminicola using medicinal plant

methanolic extracts
1*
Varaprasad Bobbarala, 2Prasanthkumar Katikala, 1Vinila Duggirala, 4Somasekhar Penumajji
1For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam-17.
2A. U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-3.

4Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Habsiguda, Hyderabad, A.P.

Received on: 08-08-2009; Revised on: 18-09- 2009; Accepted on:14-10-2009

ABSTRACT
The antifungal activity of forty nine medicinal plants belonging to different families was tested in vitro on phytopathogenic fungus
Colletotrichum graminicola. In which methanolic extracts of forty two plants exhibited varying degrees of inhibition activity against C.
graminicola. The results revealed that extract of Terminalia chebula was highly effective in inhibiting the mycelial growth of C. graminicola
at 75µg/50µL. The following six plants Acalypha indica, Eichhornia crassipes, Gyanandropsis gyanandra, Suaeda maritime, Tephrosia
pumila and Tinospora cordifolia did not exhibit antifungal activity.

Keywords: Colletotrichum graminicola, Crude methanolic extracts, Antifungal activity, Potato Dextrose Agar (PDA), Well diffusion
assay.

INTRODUCTION

pathogen C. graminicola is a filamentous ascomycete that causes

Plants are the sources of natural pesticides that make excel-
lent leads for new pesticide development (1). Many of the plant mate-
rials used in traditional medicine are readily available in rural areas
and this has made traditional medicine relatively cheaper than mod-
ern medicine (2). The potential of higher plants as source for new
drugs is still largely unexplored. Among the estimated 250,000-500,000
plant species, only a small percentage has been investigated phy-
tochemically and the fraction submitted to biological or pharmaco-
logical screening is even smaller. Thus, any phytochemical investiga-
tion of a given plant will reveal only a very narrow spectrum of its
constituents. Historically pharmacological screening of compounds
of natural or synthetic origin has been the source of innumerable
therapeutic agents (3). Plant products still remain the principal source
of pharmaceutical agents used in traditional medicine (4, 5). The ef-
fects of plant extracts on bacteria have been studied by a very large
number of researchers in different parts of the world (6, 7). Much
work has been done on ethno medicinal plants in India (8, 9). Plant
*Corresponding author.
Dr. Varaprasad Bobbarala
Scientist In-Charge,
For U Biosciences/IMMA Labs,
A/4A, Park lane Residency, East point colony,
Visakhapatnam, A.P-530017, India.
Tel.: + 91-9949129539
E-mail:varaprasadphd@rediffmail.com
Drug Invention Today Vol.1.Issue 1.November 2009
anthracnose disease of maize. While the fungus can cause devastat-
ing foliar leaf blight and stalk rot diseases, little is known about its
ability to infect roots. Previously published reports suggest that C.
graminicola may infect maize roots and that root infections may con-
tribute to the colonization of aboveground plant tissues, leading to
disease (10).
MATERIALS AND METHODS
Plant material and extracts preparation:
The plant materials of forty nine plant species were col-
lected from different places in Visakhapatnam district, Andhrapradesh.
The collected plants were identified and authenticated by a botanist
Department of Botany, Andhra University. The selected parts of dif-
ferent medicinal plants were finely powdered and were extracted with
organic solvent methanol using soxhlet apparatus. The extracts ob-
tained were subsequently concentrated under reduced pressure to
get their corresponding residues. The extracts were screened for an-
tifungal activity. Methanolic extracts in different concentrations
(100mg/ml, 300mg/ml, and 500mg/ml) were used to get the final drug
concentration 5mg/well, 15mg/well, and 25mg/well respectively, con-
trol (DMSO) and standard (Bavistin 5µg/ml), were transferred to the
cupsofeachagarplate,incubatedatroomtemperature0(C) 28and
examined for inhibition zones after 36 hours of incubation.
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 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),3-6

0-20: Zone of inhibition in mm; A: 100mg/ml; B: 300mg/ml; C: 500mg/ml; Includes diameter of well (6 mm)

0-20: Zone of inhibition in mm; A: 100mg/ml; B: 300mg/ml; C: 500mg/ml; Includes diameter of well (6 mm)

The plant extracts were assayed for antifungal activity diffusion method 0.05ml of methanolic extracts of forty nine different
against the fungal strain Colletotrichum graminicola obtained from plant extracts were introduced serially after successful completion of
Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh. one plant. Incubation period of 24- 48hours at 280C was maintained
This fungus was grown on PDA plate at 28oC and maintained with for observation of antifungal activity of plant extracts. The antifungal
periodic sub-culturing at 4oC. activity was evaluated by measuring zones of inhibition of fungal
growth surrounding the plant extracts. The complete antifungal analy-
Antifungal activity: sis was carried out under strict aseptic conditions (Graph. 1a&b) and
the experiment was carried out in triplicates, the mean values reported
The methanolic extracts of forty nine different plant extracts here.
were screened for antifungal activity by agar well diffusion method
(11) with sterile cork borer of size 6.0mm. An aliquot (0.02ml) of C. Minimum inhibitory concentration (MIC) assays:
graminicola inoculum was introduced to molten PDA and poured in
to a petri dish by pour plate technique. After solidification, the appro- Based on the preliminary screening methanolic extracts were found to
priate wells were made on agar plate by using cork borer. In agar well

Drug Invention Today Vol.1.Issue 1.November 2009 3-6

 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),3-6
Graph.2 Minimum inhibitory concentration (MIC) (Volume per well: 50µl, Borer size used: 6mm, ‘x’ axis = Medicinal plants, ‘y’ axis =0-
200 = Extract concentration in mg/ml)
have potent antimicrobial activity and Minimum Inhibitory Concen-
trations (MIC) of the extracts was determined according to Elizabeth
(12). A final concentration of 0.5% (v/v) Tween-20 (Sigma) was used
to enhance crude extract solubility. A series of two fold dilution of
each extract, ranging from 0.2 to 100 mg/ml, was prepared. After ster-
ilization, the medium was inoculated with 3µl aliquots of culture con-
taining approximately 105 CFU/ml of each organism of 24 hours slant
culture in aseptic condition and transferred into sterile 6 inch diam-
eter petri dishes and allowed to set at room temperature for about 10
minutes and then kept in a refrigerator for 30 minutes. After the media
solidified a number 3-cup borer (6mm) diameter was properly steril-
ized by flaming and used to make three to five uniform cups/wells in
each petri dish. A drop of molten nutrient agar was used to seal the
base of each cup. Different plant crude extracts ranging from 0.2 to
100 mg/ml were added to the cups/wells of each petri dish and the
control plates without plant extract. Inhibition of organism growth in
the plates containing test crude extracts was judged by comparison
with growth in blank control plates. The MICs were determined as the
lowest concentration of extracts inhibiting visible growth of each
organism on the agar plate. Similarly the MICs of methanolic extracts
were determined against all other microorganisms. The results were
given in Graph -2.
RESULTS AND DISCUSSION
Antifungal activity of forty nine botanical extracts was as-
sayed and data on effect of plant extracts on the growth of C .
graminicola presented in graphs 1&2. The data revealed that signifi- REFERENCES
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All the extracts tested gave significant inhibition of mycelial growth
of C. graminicola over control. Among all the forty nine plant
methanolic extracts forty two plants showed activity among them
Drug Invention Today Vol.1.Issue 1.November 2009
nine plants Adenocelima allicia, Bridilia Montana, Cleome viscose,
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Source of support: Nil, Conflict of interest: None Declared

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