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Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) ANTIFUNGAL AND ANTIBACTERIAL
POTENTIALITY OF SIX ESSENTIAL
OILS EXTRACTED FROM PLANT
Department of Studies in Botany and Microbiology Maharanis Science College for Women, Palace Road CMR Institute of Management Studies(Autonomous) C.A. #2, 3rd ‘C’ Cross, 6th ‘A’ Main, HRBR layout, 2nd Block 3. DR.RAVEESHA.K.A. M.Sc., M.Phil., Ph.D Department of Studies in Botany, Manasagangotri ABSTRACT: In vitro evaluation of six essential oils viz., Allium sativum, Capsicum annum Cassia fistula,
Coriandrum sativum, Cuminum cyminum and Curcuma longa were tested against ten seed borne fungi
of paddy viz., Pyricularia oryzae, Bipolaris oryzae, Alternaria alternata, Tricoconis padwickii,
Drechslera tetramera, Drechslera halodes, Curvularia lunata, Fusarium moniliforme, F. oxysprorum and
F. solani and five human pathogenic bacteria viz., Escherichia coli, Pseudomonas aeruginosa, Salmonella
typhi ,Vibrio cholera and Streptococcus pneumonia at 500,1000,1500 and 2000 ppm concentration.
Among the six essential oils, A. sativum recorded a complete inhibition in eight fungi compared to
control. A. sativum is followed by C. sativum , C. longa and C. cyminum . No significant activity was
observed in C. annum and C. fistula against all the test fungi. In antibacterial assay, A. sativum recorded
a maximum inhibition of all test bacteria in the range of 10.9 to36.9 mm, followed by C. longa (5.6 -25.6
mm), C. cyminum (10.9-30.2 mm). Least inhibition was observed in Capsicum annum and no
antibacterial activity was observed in C. fistula.

Key words: Antifungal, Antibacterial, Essential oils, Antibiotics, pesticides.

INTRODUCTION

Fungal diseases of plants are primarily controlled by the application of fungicides and bacterial diseases of human is controlled by synthetic antibiotics. It is not an eco-friendly approach to use synthetic fungicides as many of them are reported to have carcinogenic, teratogenic, oncogenic and genotoxic properties Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) (Dalvi and Whittiker, 1995; Hussain et al., 2011 ). Many of these fungicides are biohazardous and adversely affect the components of ecosystem. Further, the cost of these fungicides and antibiotics is comparatively high and their constant application results in development of resistance in the pathogens against these fungicides and antibiotics (Nigam et al., 1994). Thus use of ecofriendly alternative approaches for the management of plant diseases and human diseases is suggested (Mishra and Tewari, 1990). A good number of plant derived natural products and its oils are reported to be antifungal and antibacterial in nature (Chattopadhyay et al., 2004; Bhalodia et al., 2011). Green plants and their products have proved their fruitfulness in providing less phytotoxic, more systemic, easily biodegradable and host metabolism stimulatory fungicides (Dubey and and Tripathi, 1987). Plant products have been proved toxic for large number of fungal and bacterial pathogens (Gulluce et al., 2004). Essential oils have been used by many workers for controlling fungi, bacteria, viruses and insect pests (Singh, et al., 2001). The essential oils are known for possessing antifungal and antibacterial properties (Tepe et al., 2004; Stojanovic et al., 2006). The antimicrobial properties of essential oils invariably depend on the chemical nature of the constituents present in them (Nidiry, 1998). Essential oils, being lipophilic in nature can easily penetrate deeper through living tissue unbarred by the selective permeability of the cell membrane, hence they are of interest in the management of fungal and bacterial diseases. It has been reported that about 60% of the essential oils posses antifungal and about 35% antibacterial properties (Gangrade et al., 1991). Information on the use of essential oils against fungal pathogens of paddy in
general and human bacterial pathogens in particular is lacking. The non toxic, non-pollutive and biodegradable
nature of these essential oils prompted to evaluate some volatile natural products of higher plants against fungal
and bacterial pathogens. Hence in the present investigation, the antifungal and antibacterial properties of six
essential oils were evaluated against ten important seed borne fungal pathogens of paddy known to cause
significant loss in yield and quality of paddy and five human pathogenic bacteria.
MATERIALS AND METHODS


TEST PLANT

Six plant materials viz., Allium sativum (Alliaceae) (Berry), Capsicum annum (Solanaceae) (Fruit), Cassia fistula (Fabaceae) (Leaves), Coriandrum sativum (Apiaceae) (Seed), Cuminum cyminum (Apiaceae)
(Seed) and Curcuma longa(Zingiberaceae)Rhizome collected form market, Mysore.

ESSENTIAL OIL ISOLATION

All the test plant materials (100 g) was air dried and each plant material was introduced into conical flask with 100 ml of Alcohol and plugged with cotton wool. It was hydrodistilled for 3 hours using a Clevenger
type apparatus with a small quantity of n-Hexane (0.3 ml) which was dried over anhydrous sodium sulfate and
kept in a sealed vial at 4°C until analysis and tests (Osho and Adetunji, 2010).

TEST FUNGI

Important and frequently occurring seed borne pathogens of paddy with relative percent occurrence of more than 45 were selected. The selected pathogens were Pyricularia oryzae, Bipolaris oryzae, Alternaria
alternata, Tricoconis padwickii, Drechslera tetramera, Drechslera halodes, Curvularia lunata, Fusarium
moniliforme, F. oxysprorum
and F. solani. These fungi were isolated from the infected seeds of paddy by
Standard Blotter Method (ISTA, 2003) and the pure culture was maintained on CDA agar slants which served as
test fungi.

TEST BACTERIA

Four Gram negative human bacteria viz., Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Vibrio cholera and one Gram positive bacteria Streptococcus pneumonia were obtained from Kumar
hospital, Bangalore. All Gram negative bacteria were grown in Mac Conkey agar medium and Gram positive
bacteria in blood agar medium and maintained at 4oC until further use.
ANTIFUNGAL ACTIVITY

The evaluation of antifungal effects of essential oils on the mycelial growth of test pathogens was studied by poisoned food technique (Hadizadeh et al., 2009). The CDA containing 500, 1000, 1500 and 2000ppm concentrations of the each test oils were prepared. The CDA medium without any oil but with same quantity of absolute alcohol served as control. The test oils were added to CDA medium before sterilization. The oil amended medium was poured into sterile petriplates (9 cms diameter). Twenty ml of the medium was poured to each petriplate. The mycelial agar discs (5mm diameter) of the test pathogen were obtained from the Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) margin of the seven-day-old culture of the test fungi and the disc was inoculated to the centre of petriplates. The inoculated petriplates were incubated for seven days at 22±2o C and the colony diameter was measured in millimeters. The percent mycelial growth inhibition (P) if any with respect to the control was computed from the formula. P=C-T X100/C. Where C is the colony diameter of the control and T is the colony diameter of the treated ones. Three replicates were maintained for each treatment and repeated three times (Pinto et al., 1998; Sarala et al., 2002). ANTIBACTERIAL ACTIVITY:
Preparation of standard culture inoculums of test organism: Three or four isolated colonies of all the test
Gram negative bacterial species were inoculated in the 2 ml Mac Conkey broth and Gram positive bacteria to
nutrient broth and incubated at 37oC for 24 hours till the growth in the broth was equivalent with Mac-Farland
standard(0.5%) as recommended by WHO.
Agar cup diffusion method: Agar cup diffusion method described by Joshi et al., 2009 was employed. An
overnight culture of E. coli, P. aeruginosa, S. typhi, V. cholera and S. pneumonia was standardized to contain
approx.107cfu/ml and inoculated into 20 ml of Mac Conkey broth. The culture medium was allowed to set.
Thereafter, all the inoculum was swabbed over the surface of the Mac Conkey agar medium for Gram negative
bacteria and blood agar medium for Gram positive bacteria plate using sterile cotton swab. Using a sterile cork
borer of 5 mm diameter, five wells were made in solidified sterile Mac Conkey agar medium and blood agar
medium plate (one in the centre and four wells at the corner). The agar plugs were removed with a flamed and
cooled wire loop. Then 10,20,30,40 and 50µl of all the test oil samples were placed in the wells made in
inoculated plates. The treatment also includes 50 µl of absolute alcohol served as control. All the plates were
incubated for 24hours at 37oC and zone of inhibition if any around the well were measured in millimeter (mm).
For each treatment ten replicates were maintained. The same procedure were followed for standard antibiotics
Gentamicin (25mg), Tetracycline (25mg) and Streptomycin (25mg) to compare the efficacy of plant extract
against test organisms.
RESULT
ANTIFUNGAL ACTIVITY

Among the six medicinal plants tested, A. sativum recorded a complete inhibition of eight fungal pathogens among ten tested. At 2000ppm concentration P.oryzae, A.alternata, D.tetramera, F.oxysporum and F.solani were completely inhibited. T. padwickii was completely inhibited at 1000ppm concentration and C. lunata and F.moniliforme were inhibited at 1500ppm concentration tested. Significant activity was also observed in 500ppm concentration in all the pathogens tested(Table 2). In C. sativum at 2000ppm concentration, B.oryzae, A.alternata, D.halodes and F.oxysporum were completely inhibited. P.oryzae, T. padwickii and F.moniliforme were completely inhibited at 1000ppm concentration of the extract. D.tetramera and F.solani were showed maximum activity of 84.75% and 84.31% at 2000ppm concentration tested (Table 3). In C. longa at 2000ppm concentration tested, P.oryzae, D.tetramera, D.halodes, C. lunata recorded 80.83, 83.88, 88.33 and 85.49% inhibition respectively and B.oryzae, A.alternata, T. padwickii, F.moniliforme, F.oxysporum and F.solani recorded 76.86, 76.44, 75.47, 60.60, 73.33 and 63.67% inhibition respectively. D.halodes and T. padwickii showed 78.88 and 71.69% inhibition at 500ppm concentration. Rest of the pathogens showed a significant activity against all the fungi at 500ppm concentration (Table 3). In C. cyminum, C. lunata recorded 100% inhibition at 2000ppm concentration, T. padwickii at 500ppm concentration . P.oryzae, B.oryzae, A.alternata, D.tetramera, D.halodes, F.moniliforme, F.oxysporum and F.solani showed 82.16, 87.44, 91.66, 74.16, 82.41, 80.85, 80.51 and 80.44% inhibition respectively. At 500ppm concentration, all the nine fungi recorded significant activity in the range of 29 -80% inhibition (Table 2). Least inhibition of 5-38% was observed in C. annum tested against all the ten test fungi (Table 2). In C. fistula, no significant activity was observed against all the fungi tested. Compared to synthetic fungicides
Thiram and Bavistin at 2000ppm concentration, Thiram showed 87- 100% inhibition against all the test fungi
and in Bavistin, all the fungi was completely inhibited at 2000ppm concentration tested.


Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) ANTIBACTERIAL ACTIVITY
Among the six plants tested against five bacterium at 500,1000,1500 and 2000ppm concentration, A. sativum recorded a maximum antibacterial activity against all the bacterium tested. Compared to standard antibiotics Gentamycin, Tetracycline and Streptomycin at 2000ppm concentration. S. typhi showed 36.9mm inhibition and E. coli (33.8mm), P. aeruginosa (32.4 mm), S. pneumonia (26.7 mm) and V. cholera recorded 22.5mm inhibition at 2000ppm concentration. At 500ppm concentration least inhibition was observed in V. cholera(10.9mm) followed by P. aeruginosa(15.6 mm) and S. pneumonia(16.3 mm). Compared to standard antibiotics Gentamycin, Tetracycline and Streptomycin, it recorded 27-36mm inhibition against all the bacterium (Table 4). A. sativum is followed by C. sativum and at 2000ppm concentration, V. cholera recorded 32.2mm inhibition followed by S. typhi(27.6mm), S. pneumonia(25.7mm), P. aeruginosa (21.8mm) and E.coli (19.0mm). At 500ppm concentration the inhibition zone is in between the range of 9.0mm to 15.8mm in all the test fungi(Table 5). In C. longa, S. typhi recorded 10.2, 15.2, 19.0 and 25.6mm inhibition respectively at 500, 1000, 1500 and 2000ppm concentration, least inhibition was observed in E.coli which recorded 5.6, 12.3, 15.8 and 19.1mm inhibition at 500, 1000, 1500 and 2000ppm concentration tested. Standard antibiotics Gentamycin, Tetracyclin and streptomycin, it recorded 27 – 36mm inhibition against all the test bacterium (Table 5). In C. cyminum, S. pneumonia recorded 30.2mm inhibition at 2000ppm concentration followed by E.coli (29.0mm), S.typhi(29.0mm), V. cholera (25.0mm) and P. aeruginosa (24.6mm) respectively. At 500ppm
concentration tested, the inhibition zone is in between 10.9- 18.9mm (Table 4). Least inhibition was observed
in C. annum at all the concentration tested and no antibacterial activity was observed in C. fistula against all
test bacteria (Table 4 and Table 5).

DISCUSSION

The use of many synthetic fungicides and antibiotics has been cautioned due to their pollutive effects, non-biodegradability and residual toxicities. Most of these fungicides and antibiotics have become a popular target of conservationists and are treated to be one of the most vital man-made pollutants (Athukoralage, 2001). During recent years, many essential oils have been found as potent antimicrobial agents (Tripathi et al., 2004). Since such antifungal and antibacterial essential oils have penetration action, these may especially be used to control seed-borne fungal pathogens and human pathogenic bacteria. The volatility, ephemeral nature and biodegradability of these compounds of angiosperms will be especially advantageous if they are developed as pesticides and antibiotics. This finding of the present study reveals that this essential oils may constitute an ideal fungi toxicant for the protection of seeds from infestation by fungi and ideal natural antibiotics. Since these plants are widely cultivated, its essential oil will be an indigenous and renewable source in plant protection in place of synthetic chemicals and as a potent antibiotics in in vitro condition. During recent years, the antimicrobial principles of higher plants variously referred to as plant antibiotics, pseudo antibody, phytocide and their possible use in plant disease management particularly seed borne fungi and many pathogenic bacteria has been advocated often and again (Mishra et al., 2003). The resurgence of interest in natural therapies and increasing consumer demand for effective, safe, natural products means that quantitative data on plant oils are required (Hammer et al., 1999). Various publications have documented the antimicrobial activity of essential oils and plant extracts including Clove, Bay, Basil, Angelica, Calamus, Citronella, Lime, Palmorosa etc (Singh and Pandey, 1998). Thus the oil constitutes an ideal, indigenous and effective fumigant preservative for protection of paddy in particular and other food commodities in general. CONCLUSION: this study confirms that many essential oils and plant extracts possess in vitro antifungal and
antibacterial activity. However, if plant oils and extracts are to be used for food preservation or medicinal
purposes, issues of safety and toxicity need to be addressed. The reports shows that the maximum activity was
observed in 1500 to 2000ppm concentration. These finding suggest that the investigated essential oils have
strong fungi toxicity against important seed borne pathogens of paddy and strong antibacterial activity against
Gram Positive and Gram negative human pathogenic bacteria . Thus these oils can be exploited as alternative
source of natural fungicide for the management of seed borne pathogens of paddy and bactericides for the
management of human bacteria. Further study is necessary to identify the bioactive principle responsible for
antifungal and antibacterial activity.

ACKNOWLEDGEMENT
: The authors are thankful to the Department of Studies in Botany and Department
of Studies in Microbiology, University of Mysore, Mysore and Department of studies in Botany and
Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) Microbiology, Maharanis science college for women, Palace road, Bangalore, and CMR Institute of
Management Studies(Autonomous), PG Department of Biosciences, Bangalore for providing facilities and
financial support.

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Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) Table 1: Essential oils evaluated for antifungal activity against important seed borne fungal pathogens of paddy. Plant species
Common name
Extract type
Plant part
Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) Table 2: Antifungal activity of essential oils of Allium sativum, Capsicum annum and Cuminum cyminum Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) • Values are means of three replicates, ± standard error, Th: Thiram, Ba: Bavistin • Analysis of Variance (ANOVA); d.f =9, 20; P < 0.001 Table 3: Antifungal activity of essential oils of Cassia fistula, Coriandrum sativum and Curcuma longa Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) • Values are means of three replicates ± standard error, Analysis of Variance (ANOVA); d.f =9, 20; P < 0.001, a to h means with different letters are significantly Table 4: Antibacterial activity of essential oils of Allium sativum, Capsicum annum and Cuminum cyminum • Values are means of three replicates ± standard error • Analysis of Variance (ANOVA); d.f =9, 20; P < 0.001 • a to h means with different letters are significantly different from each other. • G.M: Gentamycin, T.C: Tetracycline, S.M: Streptomycin Dr.Lalitha.V et al. / International Journal of Engineering Science and Technology (IJEST) Table 5: Antibacterial activity of essential oils of Cassia fistula, Coriandrum sativum and Curcuma longa • Values are means of three replicates ± standard error • Analysis of Variance (ANOVA); d.f =9, 20; P < 0.001 • a to h means with different letters are significantly different from each other. • G.M: Gentamycin, T.C: Tetracycline, S.M: Streptomycin

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