Antifungal effect of cow urine extracts of selected plants against

International Journal of Agriculture and Crop Sciences.
Available online at www.ijagcs.com
IJACS/2014/7-3/142-146
ISSN 2227-670X ©2014 IJACS Journal
Antifungal effect of cow urine extracts of selected
plants against Colletotrichum capsici isolated from
anthracnose of chilli
Prashith Kekuda T.R1, Vivek M.N1, Manasa M1, Yashoda Kambar1, Noor Nawaz A.S2,
Raghavendra H.L3*
1. P.G. Department of Studies and Research in Microbiology, Sahyadri Science College (Autonomous) campus,
Kuvempu University, Shivamogga-577203, Karnataka, India
2. Organic Farming Research Centre, University of Agricultural and Horticultural Sciences, Navile, Shivamogga577204, Karnataka, India
3. Faculty of Medical Sciences, Wollega University, Post Box No: 395, Nekemte, Ethiopia
*Corresponding author email: [email protected]
ABSTRACT: The present study was conducted to determine inhibitory effect of cow urine extract of four
plants namely Anacardium occidentale L., Pimenta dioica (Linn.) Merill., Alpinia galanga Willd. and
Anisomeles indica Linn. against Colletotrichum capsici isolated from anthracnose of chilli (Capsicum
annuum L.). Poisoned food technique was performed to investigate antifungal effect of cow urine extracts.
All extracts were found inhibitory against the fungus but to a varied extent. Leaf extract of A. galanga and
rhizome extract of P. dioica displayed marked inhibitory activity whereas leaf extract of P. dioica and bark
extract of A. occidentale showed less inhibitory efficacy. These cow urine based plant extract appears to
be promising and can be used to control anthracnose disease in chilli.
Key words: Cow urine extract, Anthracnose of chilli, Colletotrichum capsici, Antifungal activity, Poisoned
food technique
INTRODUCTION
Plants suffer from diseases caused by various kinds of pathogens such as bacteria, fungi, viruses,
nematodes, mycoplasma etc. Among these, fungi are considered as more aggressive. The plant diseases have
significant role in agriculture in terms of reduction of yield and economy. One of the most widely used strategies to
control plant diseases is the use of chemical agents. However, overuse and abuse of these chemical agents
resulted in certain hazardous effects. These chemicals suffer from drawbacks such as high cost, toxicity to nontarget organisms, residual problem and development of resistance in pathogens. This situation triggered interest in
searching alternates for disease control. Natural products, in particular from plants, can be the potential candidates
which can be used against phytopathogenic fungi. The use of these agents is risk-free when compared to synthetic
chemicals. The crude extracts, essential oils and purified compounds from plants have shown to possess marked
inhibitory activity against plant pathogenic fungi (Abou-Zeid et al., 2008; Yazdani et al., 2011; de Barros et al.,
2011; Bajpai and Kang, 2012; Rakesh et al., 2013b; Vivek et al., 2013). It has been experimentally shown that the
urine of cow has got agricultural importance in terms of control of insects and fungi. Cow urine is shown to control
root knot nematode in tomato (Abubakar et al., 2004) and melon aphids and pickle worms in watermelon cultivation
(Burubai and Eribo, 2012). Cow urine alone or in combination with plants is shown to inhibit a number of
phytopathogenic bacteria and fungi (Basak et al., 2002a; Basak et al., 2002b; Akhter et al., 2006; Murugan et al.,
2012; Rakesh et al., 2013a; Rakesh et al., 2013b; Kambar et al., 2013).
The species of the genus Capsicum (Solanaceae) are herbaceous, annual, dicotyledonous flowering plants
grown worldwide in tropical and subtropical regions. The term chilli refers to ripe fruit of the genus Capsicum. It is
extensively grown for consumption, nutritional and economy purposes. Chilli is used both as spice (ripe and dried
form) and as vegetable (green fruit). Among different countries, India is known to be the largest producer of chilli in
terms of international trade. Chilli is nutritionally rich as it contains steam-volatile oils, fatty oils, capsaicinoids,
carotenoids, vitamins, protein, fibre and mineral elements. Several factors are known to influence the production of
Intl J Agri Crop Sci. Vol., 7 (3), 142-146, 2014
chilli. The chilli cultivars exhibit susceptibility to diseases caused by fungi, bacteria and viruses which account for
considerable reduction in productivity. Among the diseases of chilli, anthracnose (pre-harvest and post-harvest) is
the most important disease resulting in drastic reduction in yield (up to 50%) and deterioration of the quality of fruit.
The typical symptoms of anthracnose include sunken necrotic tissues with concentric rings of acervuli on chilli fruit.
Anthracnose of chilli is reported to be caused by a number of Colletotrichum species viz., C. capsici, C. acutatum,
C. gloeosporioides, C. coccodes and C. dematium. Among the species of Colletotrichum, C. capsici is the most
important pathogen (Ushakiran et al., 2006; Anand et al., 2007; Ratanacherdchai et al., 2007; Than et al., 2008;
Kim et al., 2010; Narasimhan and Shivakumar, 2012; Susheela, 2012; Masoodi et al., 2013). The objective of the
present study was to determine inhibitory effect of cow urine extract of selected plants namely Anacardium
occidentale L., Pimenta dioica (Linn.) Merill., Alpinia galanga Willd. and Anisomeles indica Linn.
MATERIALS AND METHODS
PLANT MATERIALS USED
The details of plant selected, parts used and place of collection are given in Table 1.
Table 1. Plants used in this study
Plant name
A. occidentale
P. dioica
A. galanga
A. indica
Family
Anacardiaceae
Myrtaceae
Zingiberaceae
Lamiaceae
Part used
Leaf and bark
Leaf and bark
Leaf and rhizome
Leaf
Place of collection
Maragalale, Thirthahalli, Karnataka
Maragalale, Thirthahalli, Karnataka
Maragalale, Thirthahalli, Karnataka
Sahyadri Science College campus, Shivamogga
Cow urine
The urine was collected at early morning in a sterile container from an indigenous cow variety Malnad
gidda. After collection, the urine was filtered through Whatman No. 1 and used for the study (Rakesh et al., 2013b).
Preparation of cow urine extract of selected plants
10g of each powdered plant material was added to 100ml of cow urine in separate containers and left for
15 days and shaken regularly. The contents were filtered through sterile 4-fold muslin cloth followed by Whatman
no. 1. The filtrates were used to poison Potato dextrose agar (PDA) medium (Rakesh et al., 2013b).
Test fungus
The fungus C. capsici, isolated from anthracnose of chili in our previous study (Kambar et al., 2013), was
used as test fungus. The fungus was maintained on Potato dextrose agar (PDA) slants.
Antifungal activity of cow urine extract
In this study, we performed Poisoned food technique to determine the antifungal effect of cow urine
extracts of selected plants. Here, PDA media amended with cow urine extracts (5%) were sterilized by autoclaving,
poured into sterile petriplates and allowed to solidify. Fungal discs of 5mm diameter were cut using a sterile cork
borer from the periphery of 5 days old culture of C. capsici, the discs were transferred aseptically at the centre of
poisoned plates and the plates were incubated for 5 days at 28°C. The diameter of colonies in mutual
perpendicular directions was measured after incubation (Rakesh et al., 2013b). Antifungal activity of cow urine
extracts was recorded in terms of inhibition of mycelial growth (%) and was calculated using the formula:
Mycelial growth inhibition (%) = (C–T/ C) × 100 where ‘C’ is average colony diameter in control plates and ‘T’ is
average colony diameter in poisoned plates.
Statistical analysis
The experiment was performed in triplicates. The results are represented as Mean±Standard deviation
(SD).
RESULTS
The result of inhibition of C. capsici by cow urine extracts of selected plants is shown in Table 2, Figure 1
and Figure 2. All extracts were effective against the growth of the fungus. The diameter of the fungal colonies in
poisoned plates was lesser when compared to control plates and it indicates antifungal effect of cow urine extracts
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Intl J Agri Crop Sci. Vol., 7 (3), 142-146, 2014
of plants. High inhibitory activity (>60% inhibition) was shown by bark and leaf extract of P. dioica and A. galanga
respectively. Leaf extract of P. dioica caused least inhibition of test fungus.
Table 2. Colony diameter of C. capsici in control and poisoned plates
Treatment
Control
A. occidentale (leaf)
A. occidentale (bark)
P. dioica (leaf)
P. dioica (bark)
A. galanga (leaf)
A. galanga (rhizome)
A. indica (leaf)
Colony diameter in cm
3.1±0.0
2.1±0.1
2.7±0.1
2.8±0.1
1.2±0.0
1.2±0.0
2.4±0.2
2.1±0.1
Figure 1. Growth of C. capsici on control plates and plates poisoned with cow urine extracts of selected plants
Figure 2. Inhibition (%) of C. capsici by cow urine extracts of selected plates
DISCUSSION
Plants serve mankind as major source of food, fibre, fodder, medicines and other useful products. Various
parts of the plant such as roots, stem, leaves, fruits, flowers/inflorescence and seeds are utilized for different
requirements. Insect pests and a variety of microbes attack the plants at the various stages of their development
and results in productivity loss. When compared to all other plant parasites/pathogens, fungi cause the greatest
impact with regard to diseases and crop losses. The use of fungicides of synthetic origin is the most important
method of protecting the plants against the fungal infections. However, toxicity and non-biodegradability of
fungicides and the development of resistance in pathogenic fungi stimulated search for eco-friendly measures for
control of fungal diseases (Tapwal et al., 2011; de Barros et al., 2011; Vivek et al., 2013). Plant extracts, their
metabolites and plant based pesticides represents one of the best alternatives for fungal disease management. It
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has been found that plant extracts and their components possess inhibitory activity against C. capsici, the causal
agent of anthracnose of chilli. Nduagu et al. (2008) observed strong fungitoxicity in extracts of Azadirachta indica,
Vernonia amygdalina and Cochlospermum planchonii against C. capsici. Johnny et al. (2011) observed marked
inhibitory activity of Piper betle against mycelial growth of C. capsici. The solvent extracts and essential oil of
Magnolia liliflora were found to exhibit antifungal activity against C. capsici (Bajpai and Kang, 2012).
It has been found that cow urine possess antimicrobial activity against plant pathogenic microbes. It is
shown that cow urine possess inhibitory activity against Sclerotinia sclerotiorum (Basak et al., 2002a), Fusarium
solani f.sp. cucurbitae (Basak et al., 2002b), Bipolaris sorokiniana (Akhter et al., 2006), Xanthomonas oryzae pv.
oryzae (Venkatesh et al., 2011; Murugan et al., 2012), F. oxysporum f.sp. zingiberi (Rakesh et al., 2013a) and
Pythium aphanidermatum (Rakesh et al., 2013a), Colletotrichum capsici (Kambar et al., 2013). In the present
study, we found inhibitory activity of cow urine extract of four plants against C. capsici, causative agent of chilli
anthracnose. Marked inhibitory activity was observed in case of bark extract of P. dioica and leaf extract of A.
galanga. Leaf extracts of A. indica and A. occidentale showed moderate inhibitory activity. Studies have shown that
cow urine extracts of plants as well as plants in combination with cow urine possess inhibitory activity against
phytopathogens. Akhter et al. (2006) reported that the combination of extract of Calotropis procera and cow urine
exhibited 91% inhibition of conidial germination of Bipolaris sorokiniana, causative agent of leaf blight of wheat.
Tiwari and Das (2011) observed in vitro and in vivo inhibitory efficacy of cow urine extracts of some medicinal
plants against Rhizoctonia solani, causal agent of sheath blight of rice. Venkatesh et al. (2011) found inhibitory
effect of cow urine extract of a red algae Kappaphycus alvarezii against Xanthomonas oryzae pv. oryzae. Murugan
et al. (2012) showed the combined efficacy of cow urine and Pongamia pinnata seed against bacterial leaf blight of
paddy caused by Xanthomonas oryzae pv. oryzae. In another study, Rakesh et al. (2013b) observed inhibitory
activity of cow urine extract of some plants against F. oxysporum f.sp. zingiberi and P. aphanidermatum, causative
agents or rhizome rot of ginger.
CONCLUSION
The cow urine extracts of selected plants have shown inhibitory activity against mycelial growth of C.
capsici. These formulations can be employed in the field for the control of anthracnose disease in chilli. Further
field experiments are to be conducted to justify the results of the present study.
ACKNOWLEDGEMENTS
Authors are thankful to Dr. N. Mallikarjun, Associate Professor and Chairman, P.G. Department of Studies
and Research in Microbiology and Principal, Sahyadri Science College (Autonomous) for providing facilities and
moral support to conduct work.
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