Sunilbabu Koppula et al.

/ Journal of Pharmacy Research 2010, 3(9),2334-2336

Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
Inhibition of plant pathogenic fungi by ethnobotanically selected plant extracts
Sunilbabu Koppula1 , K. Ammani1 , Varaprasad Bobbarala2* and PV.Bramhachari 3
1
Department of Botany and Microbiology, Acharya Nagarjuna University, A.P, India
2
Vivimed Labs Ltd., Veeranag Towers,Habsiguda,Hyderabad, AP.
3
Department of Biotechnology, Krishna University, A.P, India
Received on: 15-04-2010; Revised on: 18-05-2010; Accepted on:13-07-2010

ABSTRACT
The inhibitory effects of two extracting solvents methanol and chloroform on the antibacterial and antioxidant activity of extracts of selected medicinal plants
viz, as Butea monosperma, Hildegardia populifolia, Elephantopus scaber, Morus alba and Plumbago zeylanica arjuna were screening against four plant
pathogens Rhizoctonia solani Kuhn, Bipolaris spp., Ustilago maydis, Alternaria alternate. Methanol extracts exhibited a higher degree of antifungal activity as
compared with chloroform extracts. The methanolic extract of Hildegardia populifolia showed highest activity compared to other plant extracts. All the tested
plant extracts showed moderate to potent antioxidant activity and/or free radical scavenging activity. The antifungal and antioxidant activity of Hildegardia
populifolia has not been reported before. More detailed studies on chemical composition of the plant extracts are essential to characterize them as potent
biological antioxidants.

Key words: Antioxidant, Antimicrobial, Medicinal plants, Hildegardia populifolia, Rhizoctonia solani Kuhn, inhibitory effects.

INTRODUCTION

The schematic search of higher plants for antifungal activity has shown that
some plant extracts have the ability to retard fungal growth or completely
inhibit the fungus. Interest in more effective antifungal agents has grown with
the increasing incidents of fungal resistance especially in several agrochemical
fungicides1. Medicinal plants represent a rich source of antimicrobial agents2.
Several plant materials used in traditional medicine are readily available in
rustic areas at fairly cheaper rates than modern medicine 3. In general Plants
produce many secondary metabolites which comprise an important source of
microbiocides and many pharmaceutical drug molecules. The plant products
still remain the principal source of pharmaceutical agents used in traditional
medicine4,5. The effects of plant extracts on bacteria have been studied by
several researchers in different parts of the world 6. However, much work has
been done on ethnomedicinal plants in India7,8. Despite the fact that, atten-
tion in a large number of traditional natural products has greatly increased9.
Plants are the sources of natural microbiocides that make an excellent lead for
the development novel biomolecules10,11,12,13.
the leaves of various sorghum species. Smut diseases are caused by the patho-
genic plant fungus Ustilago maydis on various plants such as maize ( Zea mays)
and teosinte (Euchlena mexicana). Plethora of reports has been documented
on Alternaria alternata to cause leaf spot and other diseases on over 380 host
species. It is an opportunistic pathogen on numerous hosts causing leaf spots,
rots and blights on many plant parts. All these fungal pathogens used in the
present study have a worldwide distribution. These diseases occur most often
when the plants are subjected to hot, dry weather or when unfavorable envi-
ronmental conditions prevail. The main objective of this study was to inves-
tigate the inhibitory effects of chloroform and methanol extracts from five
medicinal plant species against plant pathogens Rhizoctonia solani, Bipolaris
spp, Ustilago maydis, Alternaria alternate and to evaluate the potential appli-
cation of medicinal plant based treatments to control diseases caused by the
above plant pathogens. The present research has been conducted to study the
medicinal properties like antifungal and antioxidant properties of some lo-
cally available medicinal plants against plant pathogens.

Natural products offer an untold diversity of chemical structures. These natu-
ral compounds often serve as lead molecules whose activities can be enhanced
by manipulation through combinations with chemicals and by synthetic chem-
istry. An important source of natural products is plants which are rich in a wide
variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and
flavonoids14. In order to promote the use of medicinal plants as potential
sources of antimicrobial compounds, it is pertinent to thoroughly investigate
their composition and activity and thus validate their use 15 . Some
phytochemicals produced by plants have antimicrobial activity allowing these
plants to be studied and used for the development of new antimicrobial drugs16.
The effectiveness of phytochemicals in the treatment of various diseases may
lie in their antioxidant effects17. Antioxidants are important in the prevention
of human diseases. Antioxidant compounds may function as free radical scav-
engers, complexing agents for pro-oxidant metals, reducing agents and
quenchers of singlet oxygen formation 18,19,20. Antioxidants are often used in
oils and fatty foods to retard their autoxidation; therefore, the importance of
search for natural antioxidants has greatly increased in the recent years21.
Damping off, caused by Rhizoctonia solani is a prevalent plant pathogenic
fungus with a wide host range and causes death of seedlings in agriculture. It is
also responsible for wire stem, a disease of cabbage, cauliflower and related
plants. Target leaf spot disease is caused by Bipolaris spp, and mostly affects
*Corresponding author.
Dr. Varaprasad Bobbarala
Plot No. 92, Phase 1, IDA, Cherlapally
Hyderabad, A.P 500051 India
Tel.: + 91-9949129539
E-mail:phytodrugs@gmail.com Journal of Pharmacy Research Vol.3.Issue 9.September 2010
MATERIALS AND METHODS
Plant material and extracts preparation:
The plant materials of five plant species such as Butea monosperma (Lam.)
Taub. (Fabaceae) , Hildegardia populifolia (Roxb.) (Sterculiaceae),
Elephantopus scaber L. (Asteraceae), Morus alba L. (Moraceae) and Plum-
bago zeylanica L (Plumbaginaceae) were collected from different places in
Guntur district, Andhrapradesh (Table-4). The selected parts of different me-
dicinal plants were cut into small pieces and shade dried at room temperature
for fifteen days, finely powdered plant materials were successively extracted
with organic solvent chloroform and methanol basing on order of polarity
using soxhlet apparatus. The different extracts obtained were subsequently
concentrated under reduced pressure to get their resultant residues. Chloro-
form and methanolic extracts in different concentrations (100mg/ml, 300mg/
ml, and 500mg/ ml) to get the final drug concentration 5mg/well, 15mg/well,
and 25mg/ well respectively, control (DMSO) and standard (Bavistin 5µg/ml),
were transferred to the cups of each agar plate, incubated at room temperature
(28ºC) and observed for inhibition zones after 36 hours of incubation. The
extracts were screened for antifungal activity.
Microbial cultures and growth conditions:
The plant extracts were assayed for antifungal activity against the fungal
strains Rhizoctonia solani Kuhn. (4633), Bipolaris spp. (2105), Ustilago
maydis (1474) and Alternaria alternate (149) obtained from Microbial Type
Culture Collection and Gene Bank (MTCC), Chandigarh, India. This fungus
was grown on Potato Dextrose Agar plate at 28 oC and maintained with peri-
odic sub-culturing at 4oC.
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Table.1. Ethnobotanical information of the investigated medicinal plants
S.No. Botanical Name Family Parts used Uses / Ailments treated

1 Butea monosperma (Lam.) Taub. Fabaceae Bark, flowers and leaves Leprosy, anthelmintic and tumerous haemorrhoids
2 Elephantopus scaber L. Asteraceae Roots and leaves Dysuria and diarrhea.
3 Hildegardia populifolia (Roxb.) Sterculiaceae Stem bark Dog bite, Malaria.
4 Morus alba L Moraceae Bark and fruit asthma, bronchitis, cold and constipation
5 Plumbago zeylanica L. Plumbaginaceae Roots Ulcers, piles, skin diseases and influenza.

Figure.1. Antifungal activity of B. monosperma against plant pathogens Figure.4. Antifungal activity of Plumbago zeylanica against plant pathogens

Figure.5. Antifungal activity of Plumbago zeylanica against plant pathogens

Figure.2. Antifungal activity of Hildegardia populifolia against plant

pathogens

Fig.6. Total Antioxidant activity (DPPH) of some medicinal plant ex-

tracts (chloroform and methanol)

Figure.3. Antifungal activity of Morus alba against plant pathogens

1, 1-Diphenyl-2-picrylhydrazyl (DPPH) assay:

Antifungal activity:
The chloroform and methanolic extracts of five different plant extracts were
screened for antifungal activity by agar well diffusion method with sterile cork
borer of size 6.0mm22. The cultures of 48hours old grown on potato dextrose
agar (PDA) were used for inoculation of fungal strain on PDA plates. An aliquot
(20 ul) of inoculum was introduced to molten PDA and poured into a petri dish
by pour plate technique. After solidification, the appropriate wells were made on
agar plate by using cork borer. In agar well diffusion method 50 ul of chloroform
and methanolic extracts of five different plant extracts were introduced serially
after successful completion of one plant analysis. Incubation period of 24-48
hours at 280C was maintained for observation of antifungal activity of plant
extracts. The antifungal activity was evaluated by measuring zones of inhibition
of fungal growth adjacent to the plant extracts. The antifungal analysis was
carried out under strict aseptic conditions.
The scavenging activity of five plant extracts on DPPH was determined using
the method described by 23Choi et al. This method depends on the reduction of
purple DPPH to a yellow colored diphenyl picrylhydrazine. The determination
of the disappearance of free radicals was done using spectrophotometer24. The
remaining DPPH which showed maximum absorption at 518 nm was measured.
Each plant extract sample’s stock solution (1.0 mg/mL) was diluted to final
concentrations of (0.5) mg/mL, in ethanol. One mL of a 0.3 mM DPPH
ethanol solution was added to 2.5 mL of sample solution of different concentra-
tions. One mL DPPH solution plus 2.5 mL of ethanol was used as a negative
control. The blank for this solution is ethanol. As DPPH is sensitive to light, it
is exposed to the minimum possible light. These solutions were allowed to react
at room temperature for 30 minutes. The absorbance values were measured at
518 nm and converted into the percentage antioxidant activity using the fol-
lowing equation:
Scavenging capacity (%) = 100 – [(absorbance of sample – absorbance of blank)
× 100/absorbance of control]. The tests were done in triplicate.

Journal of Pharmacy Research Vol.3.Issue 9.September 2010 2334-2336

 Sunilbabu Koppula et al. / Journal of Pharmacy Research 2010, 3(9),2334-2336
RESULTS AND DISCUSSION
The ethno botanical information of the screened plants is presented in (Table.1).
Antifungal activity of five selected medicinal plant extracts was assayed against
plant pathogens Rhizoctonia solani, Bipolaris spp, Ustilago maydis, Alternaria
alternate as presented in (Table 1). The data revealed that all the crude extracts
have significant antifungal activities against most of the fungi, but the activity
of inhibition varied for the fungi with respect to the type of plant extract. The
chloroform and methanolic extracts of five medicinal plants viz, as Butea
monosperma, Hildegardia populifolia, Elephantopus scaber, Morus alba and
Plumbago zeylanica were used for the study. Methanol extracts exhibited a
higher degree of antifungal activity as compared with chloroform extracts (Fig
1-5). The methonolic extract of Hildegardia populifolia showed highest activ-
ity compared to other plant extracts. The growth of Rhizoctonia solani was
inhibited by all the plant extracts where as Ustilago maydis showed considerably
less inhibitory activity.
In this study methanol extract of bark of Butea monosperma showed antibacte-
rial activity against Rhizoctonia solani and Alternaria alternate (Fig.1). 25Gaurav
et al also evaluated the extracts of Butea monosperma Gum against human
pathogen Candida albicans. Hildegardia populifolia and Morus alba produced
the largest zones of inhibition against Rhizoctonia solani and Bipolaris spp.
26
Sehajpal et al reported good antifungal activity in solvent extract of Morus
alba against Rhizoctonia solani. In general, among the tested phytopathogens,
all the fungi were found to be sensitive. The most sensitive fungi was Rhizocto-
nia solani, which was inhibited by methanol and chloroform extracts of 5
plants, however the chloroform extracts of Hildegardia populifolia and Morus
alba inhibited Rhizoctonia solani and Bipolaris spp. (Fig 2 and 3). On the other
hand, no inhibition was observed in the Plumbago zeylanica against Ustilago
maydis (Fig 4). The plant pathogen Alternaria alternate exhibited only slight
susceptibility all plant extracts. (Fig 1-4). The control plate representing DMSO
did not exhibit inhibition on the tested fungi where as standard antifungal drug
Bavistin showed antifungal activity even at 5µg/well.
We believe that methanolic extracts of Hildegardia populifolia could be used as an
effective biocide to treat various plant diseases caused by fungal pathogens, as it
showed maximum antifungal activity even at lower concentrations. These observa-
tions suggest that, increase in the antifungal activity of the plant extracts was im-
proved by increase in the concentrations. Extensive bioprocess optimization studies
should be undertaken for the methanolic extract of Hildegardia populifolia as a strong
antifungal agent against various plant diseases.The antifungal, antibacterial and anti-
oxidant activity of Hildegardia populifolia has not been reported before. The results
of the present study suggest that tested plant materials have moderate to potent
antioxidant activity and/or free radical scavenging activity. Copious studies on plant
phytochemicals possessing antioxidant properties may reduce the risk of disease
spectrum. All the plant extracts showed antioxidant activity of 63-82% and among
which H. populifolia showed potent antioxidant property of 82.3 % (Fig. 6). How-
ever, we haven’t explored the components in the plant extracts. More detailed studies
on chemical composition of the plant extracts are essential to characterize them as
biological antioxidants which are beyond the scope of this study. Literature survey
indicated that antioxidant activity and radical scavenging activity of some potent
plant materials used in the study27,28,29,30 .
In conclusion, the results obtained from this study indicated that chemical
constituents from these plant species may be developed as potential biofungicides
in agriculture. Further phytochemical studies are required to determine the types
of compounds responsible for the antifungal effects of these species. The results
also indicate that scientific studies carried out on medicinal plants having tradi-
tional claims of effectiveness might warrant prolific results. Purification and
identification of the active components from some of these plants are in
progress.
Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.3.Issue 9.September 2010
ACKNOWLEDGEMENTS:
The authors acknowledge T. Pullaiah for assisting us with the identification of
plant materials. This work was supported by grants from the Rajiv Gandhi
National fellowship as Senior Research Fellowship, UGC Grants, India.
REFERENCES
1. Comitini F, Ciani M, Influence of fungicide treatments on the occurrence of yeast flora
associated with wine grapes Annals of Microbiol., 2008, 58: 3, 489-493.
2. Mahesh B, Satish S. Antimicrobial activity of some important medicinal plant against plant
and human pathogens. World J. Agri. Sci., 2008, 4 (S), 839-843.
3. Mann A, Banso A, Clifford LC, An antifungal property of crude plant extracts from
Anogeissus leiocarpus and Terminalia avicennioides. Tanzania J. Health Res. 2008., 10 (1)
34-38.
4. Ibrahim MB, Antimicrobial effects of extract leaf, stem and root bark of Anogeissus
leiocarpus on Staphylococcus aureaus, Streptococcus pyogenes, Escherichia coli a n d
Proteus vulgaris. J. Pharma. Devpt., 1997, 2, 20-30.
5. Ogundipe O, Akinbiyi O, Moody JO, Antibacterial activities of essential ornamental plants.
Nigeria J. Nat. Products & Medicine., 1998, 2, 46-47.
6. Ateb DA and ErdoUrul OT, Antimicrobial activities of various medicinal and commercial
plant extracts. Turk. J. Biol., 2003, 27, 157-162.
7. Maheshwari JK, Singh KK, Saha S, Ethnobotany of tribals of Mirzapur District, Uttar
pradesh, Econ. Bot. Inform Service, NBRI, Lucknow., 1986.
8. Negi KS, Tiwari JK, Gaur RD, Notes on ethno botany of five districts of Garhwal Himalaya,
Uttarpradesh, India. Ethno Botany., 1993, 5,73-81.
9. Taylor RSL, Manandhar NP, Hudson JB, Antiviral activities of Nepalese medicinal plants.
J Ethnopharmacol., 1996, 52,157-163.
10. Gangadevi V, Yogeswari S, Kamalraj S, Rani G, Muthumary J, The antibacterial activity
of Acalyphaindica L. Indian J. Sci. Technol., 2008, 1 (6), 1-5. Domain site: http://
www.indjst.org.
11. Brindha V, Saravanan A, Manimekalai R, Drug designing for ring finger protein 110 in-
volved in adenocarcinoma (human breast cancer) using casuarinin extracted from
Terminalia arjuna. Indian J. Sci. Technol., 2009, 2 (2), 22-26. Domain site: http://
www.indjst.org.
12. Jagadish L, Anand Kumar VK, Kaviyarasan V, Effect of Triphala on dental bio-film.
Indian J. Sci. Technol., 2009, 2 (1), 30-33. Domain site: http://www.indjst.org.
13. Vetrivel Rajan A, Shanmugavalli N, Greety Sunitha C, Umashankar V, Hepatoprotective
effects of Cassia tora on CCl4 induced liver damage in albino rats. Indian J. Sci. Technol.,
2009, 2 (3), 41-44. Domain site: http://www.indjst.org.
14. Houghton P, The Role of Plants in Traditional Medicine and Current Therapy. The J.
Alternat Complem Medicine 1995., 1:131-143.
15. Nair R, Chanda S, Activity of some medicinal plants against certain pathogenic bacterial
strains. Indian J. Pharmacol., 2006, 38: 142-144.
16. Houghton P, The Role of Plants in Traditional Medicine and Current Therapy. The J.
Alternat Complem Medicine 1995., 1:131-143.
17. Baker D, Mocek U, Garr C, Natural products vs. combinatorials: a case study. Biodiversity:
new leads for pharmaceutical and agrochemical industries The Royal Society of Chem-
istry, Cambridge: United Kingdom Wrigley SK, Hayes MA, Thomas R, Chrystal EJT,
Nicholson N 2000, 66-72.
18. Bell EA, The possible significance of secondary compounds in plant. In: Secondary Plant
Products; Bell, E.A., Charlwood, B.V., Eds.; Springer-Verlag: New York, NY, USA., 1980,
pp. 11–21.
19. Constable F, Gamborg OL, Kurz WGW, Steek W, Production of secondary metabolites in
plant cell cultures. Planta Med., 1974, 25, 158–165.
20. Rice-Evans CA, Miller NJ, Paganaga G, Antioxidant properties of phenolic compounds.
Trends Plant Sci., 1997, 2, 152–159.
21. Zollman C, Vickers A, Complementary medicine and the patient. Br. Med. J., 1999, 319,
1486–1494.
22. Perez C, Paul M, Bezique P, An Antibiotic assay by the agar well diffusion method. Alta
Biomed. Group Experiences., 1990, 15: 113.
23. Choi CW, Kim SC, Hwang SS, Choi BK, Ahn HJ, Lee MY, Park SH, Kim SK, Antioxidant
activity and free radical scavenging capacity between Korean medicinal plants and fla-
vonoids by assay-guided comparison. Plant Sci., 2002, 163: 1161–1168.
24. Abdel-Hameed ESS, Total phenolic contents and free radical scavenging activity of cer-
tain Egyptian Ficus species leaf samples. Food Chem., 2008, 1133–1138.
25. Gaurav SS, Gulkari VD, Duragkar NJ, Patil AT, Antimicrobial activity of Butea monosperma
Lam. Gum Iran J. Pharmac Therapeutics., 2008,7:21-24.
26. Sehajpal A, Arora S, Kaur P, Evaluation of plant extracts against Rhizoctonia solani caus-
ing sheath blight of rice. The J. Plant Protec Sciences., 2009, 1(1) : 25-30.
27. Yam MF, Basir R, Asmawi MZ, Mariam A, Rosidah, Akowuah GA, Antioxidant and
Hepatoprotective Activities of Elephantopus tomentosus, Ethanol Extract. Pharmaceuti-
cal Biol,, 1744-5116, 46:2008, 46: 3 , 199-206.
28. Nikkhah E, Khayami M, Heidari R, In vitro antioxidant activity of berry (Morus alba
var.nigra) Int J. Plant Production., 2009, 3 (4).
29. Shivraj HN, Khobragade CN, Antioxidant activity and flavonoid derivatives of Plumbago
zeylanica. J. Nat. Products., 2010, 3130-133.
30. Lavhale MS, Mishra SH, Evaluation of free radical scavenging activity of Butea
monosperma. Indian J. Exp. Biology., 2007 45: 376-384.
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