Вы находитесь на странице: 1из 4

Varaprasad Bobbarala et al.

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


Available online through
www.ditonline.info
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- anthracnose disease of maize. While the fungus can cause devastat-
lent leads for new pesticide development (1). Many of the plant mate- ing foliar leaf blight and stalk rot diseases, little is known about its
rials used in traditional medicine are readily available in rural areas ability to infect roots. Previously published reports suggest that C.
and this has made traditional medicine relatively cheaper than mod- graminicola may infect maize roots and that root infections may con-
ern medicine (2). The potential of higher plants as source for new tribute to the colonization of aboveground plant tissues, leading to
drugs is still largely unexplored. Among the estimated 250,000-500,000 disease (10).
plant species, only a small percentage has been investigated phy-
tochemically and the fraction submitted to biological or pharmaco- MATERIALS AND METHODS
logical screening is even smaller. Thus, any phytochemical investiga-
tion of a given plant will reveal only a very narrow spectrum of its Plant material and extracts preparation:
constituents. Historically pharmacological screening of compounds
of natural or synthetic origin has been the source of innumerable The plant materials of forty nine plant species were col-
therapeutic agents (3). Plant products still remain the principal source lected from different places in Visakhapatnam district, Andhrapradesh.
of pharmaceutical agents used in traditional medicine (4, 5). The ef- The collected plants were identified and authenticated by a botanist
fects of plant extracts on bacteria have been studied by a very large Department of Botany, Andhra University. The selected parts of dif-
number of researchers in different parts of the world (6, 7). Much ferent medicinal plants were finely powdered and were extracted with
work has been done on ethno medicinal plants in India (8, 9). Plant 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-
*Corresponding author. tifungal activity. Methanolic extracts in different concentrations
Dr. Varaprasad Bobbarala
(100mg/ml, 300mg/ml, and 500mg/ml) were used to get the final drug
Scientist In-Charge,
For U Biosciences/IMMA Labs, concentration 5mg/well, 15mg/well, and 25mg/well respectively, con-
A/4A, Park lane Residency, East point colony, trol (DMSO) and standard (Bavistin 5µg/ml), were transferred to the
Visakhapatnam, A.P-530017, India. cupsofeachagarplate,incubatedatroomtemperature0(C) 28and
Tel.: + 91-9949129539
examined for inhibition zones after 36 hours of incubation.
E-mail:varaprasadphd@rediffmail.com
Drug Invention Today Vol.1.Issue 1.November 2009 3-6
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- nine plants Adenocelima allicia, Bridilia Montana, Cleome viscose,
trations (MIC) of the extracts was determined according to Elizabeth Heldigordia populipolia, Hyptis sueolences, Melia azedarach,
(12). A final concentration of 0.5% (v/v) Tween-20 (Sigma) was used Peltophorum pterophorus, Terminalia chebula and Withania
to enhance crude extract solubility. A series of two fold dilution of somnifera showed good activity, extract of plant Terminalia chebula
each extract, ranging from 0.2 to 100 mg/ml, was prepared. After ster- showed maximum activity even at lower concentrations. The follow-
ilization, the medium was inoculated with 3µl aliquots of culture con- ing six plants, viz, as Acalypha indica, Eichhornia crassipes,
taining approximately 105 CFU/ml of each organism of 24 hours slant Gyanandropsis gyanandra, Suaeda maritime, Tephrosia pumila and
culture in aseptic condition and transferred into sterile 6 inch diam- Tinospora cordifolia did not exhibited the antifungal activity against
eter petri dishes and allowed to set at room temperature for about 10 C. graminicola. This is interesting in that the traditional method of
minutes and then kept in a refrigerator for 30 minutes. After the media treating a fungal infection was by administering a decoction of the
solidified a number 3-cup borer (6mm) diameter was properly steril- plant or a part there of by boiling it in water, whereas according to our
ized by flaming and used to make three to five uniform cups/wells in results an organic solvent such as is better; hence this may be more
each petri dish. A drop of molten nutrient agar was used to seal the beneficial from the above results it can be concluded that plant ex-
base of each cup. Different plant crude extracts ranging from 0.2 to tracts have great potential as antimicrobial compounds against mi-
100 mg/ml were added to the cups/wells of each petri dish and the croorganisms and that they can be used in the treatment of infectious
control plates without plant extract. Inhibition of organism growth in diseases caused by resistant microorganism.
the plates containing test crude extracts was judged by comparison
with growth in blank control plates. The MICs were determined as the Therefore, this study suggests that methanolic extracts of
lowest concentration of extracts inhibiting visible growth of each screened plants would be helpful in treating diseases in plants caused
organism on the agar plate. Similarly the MICs of methanolic extracts by C. graminicola. In particular, the authors may recommend that the
were determined against all other microorganisms. The results were methanolic extract of Terminalia chebula to be used as potent bio-
given in Graph -2. cide to treat diseases in plants caused by C. graminicola as it showed
maximum activity even at lower concentrations. It was revealed in this
RESULTS AND DISCUSSION study, that increase in the antifungal activity of the extracts was en-
hanced by increase in the concentration of the extracts.
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
cant reduction in growth of C. graminicola was observed with ex- 1. Satish S, Raghavendra MP, Mohana DC, Raveesha KA, Antifungal
tracts of 42 plants. It was observed that extracts showed significant activity of a known medicinal plant Mimusops elengi L. against grain
differences in their efficacy to inhibit the growth of C. graminicola. moulds, Journal of Agricultural Technology, 2008, 4(1), 151-165.
2. Mann A, Banso A, Clifford LC, An antifungal property of crude plant
All the extracts tested gave significant inhibition of mycelial growth extracts from Anogeissus leiocarpus and Terminalia avicennioides.
of C. graminicola over control. Among all the forty nine plant Tanzania Journal of Health Research, 2008, 10(1): 34-38.
methanolic extracts forty two plants showed activity among them 3. Mahesh B, Satish S, Antimicrobial activity of some important me-

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


Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),3-6
dicinal plant against plant and human pathogens. World journal of and commercial plant extracts, Turk J Biol, 2003, 27, 157-162.
agricultural sciences, 2008, 4(S), 839-843. 8. Maheshwari JK, Singh KK, Saha S, Ethno botany of tribals of Mirzapur
4. Ibrahim MB, Anti-microbial effects of extract leaf stem and root bark District, Uttar pradesh, Economic Botany Information Service, NBRI,
of Anogeissus leiocarpus on Staphylococcus aureaus, Streptococcus Lucknow, 1986.
pyogenes, Escherichia coli and Proteus vulgaris, Journal of Pharma- 9. Negi KS, Tiwari JK, Gaur RD. Notes on ethno botany of five districts
ceutical and Development, 1997, 2, 20-30. of Garhwal Himalaya, Uttar pradesh, India. Ethnobotany, 1993, 5,
5. Ogundipe O, Akinbiyi O, Moody JO, Antibacterial activities of essen- 73-81.
tial ornamental plants. Nigeria Journal of natural Products and Medi- 10. Sukno SA, Garca VM, Shaw BD, Thon MR, Root infection and sys-
cine, 1998, 2, 46-47. temic colonization of maize by C. graminicola . Appl Environ Micro-
6. Reddy PS, Jamil K and Madhusudhan P. Antibacterial activity of iso- bial, 2008, 74(3), 823-832.
lates from Piper longum and Taxus baccata. Pharmaceutical Biol, 11. Perez C, Paul Mand Bezique P, Alta Biomedical group experiences,
2001, 39, 236-238. 1990, 15, 113.
7. Ateb DA, ErdoUrul OT, Antimicrobial activities of various medicinal 12. Elizabeth M, Adrien Szekely J, David W Warnock, Journal of Clinical
Microbiology 2001, 37(5), 1480-1483.

Source of support: Nil, Conflict of interest: None Declared

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