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RESEARCH ARTICLE
Inhibitory effect of cow urine based plant extracts against Rhizoctonia solani causing sheath blight of rice
R.K.S. TIWARI* and KUNTAL DAS1
T.C.B. College of Agriculture and Research Station (I.G.K.V.), Bilaspur 1 Department of Botany, Government E.R.R. Science P College, Bilaspur .G.
ABSTRACT: Seventeen medicinal plant extracts prepared in cow urine, hot water and cold water were evaluated in vitro and in vivo against Rhizoctonia solani, the causal agent of sheath blight of rice. Irrespective of medicinal plants species, extracts prepared in cow urine were found significantly more efficacious in arresting the mycelial growth as well as in checking the disease severity compared with cold water and hot water extracts. In vitro, cow urine based extracts of P. corylifolia and S . suaveolens were significantly more effective whereas, in vivo, extracts of P. corylifolia, A. racemosus and C. forskohlii were highly efficacious and at par with hexaconazole. Moreover, cow urine extracts of C. gladiata, P. corylifolia and S. suaveolens were more consistent and showed identical results in vitro and in vivo. Key words: Cow urine, medicinal plant, Rhizoctonia solani, sheath blight
Sheath blight is a major disease of rice (Oryza sativa L.), prevails throughout temperate and tropical areas and being more prominent where rice is grown under intense, high fertility production systems. Chemical control is the only effective method for controlling the sheath blight disease however, most of the synthetic fungicides used now a days are non-biodegradable and pose problems to non-target organisms and their environment (Naidu and John, 1981; Nichols et al., 1993). In recent era, extracts from medicinal plants were widely reported to possess antimicrobial activity and disease controlling potential (Tripathi et al., 1985; Amuah, 1989; Shetly et al., 1989; Tiwari, et al., 2005; 2007). Successful determination of biologically active compound from medicinal plant is largely dependent on the type of solvent used in the extraction procedure. Properties of a good solvent in plant extractions include low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, preservative action and inability to cause the extract to complex or dissociate (Hughes, 2002). From the ancient period cows urine has been used singly or in combination with some other medicinal herbs against many diseases (Chauhan, 2004). Cow urine has a unique place in Ayurveda and has been described inSushrita Sumhita and Ashtanga Sangraha to be the most effective substance secretion of animal origin with innumerable therapeutic values. This kind of alternative treatment, termed as panchgavya therapy or cowpathy, are common in local healers and tribal peoples (Dharma, et al. 2005). An exhaustive reference of cows urine having curative properties in skin diseases, especially leprosy, is referred to in Caraka samhita (Sadhale, 2004). Furthermore, in the treatment of falling body parts, discharging lymphs and organism infested organs, use of cows urine (along with some other ingredients) has been recommended for
*Corresponding author: rkstiwari@rediffmail.com
bath, anointing and intake (Basham, 1998). The cow urine distillate has been patented as activity enhancer and availability facilitator for bioactive molecules including antiinfective and anti-cancer agents (Singh, 2001). The present study was undertaken to investigate the comparative impact of cow urine and aqueous based various medicinal plant extracts against mycelial growth of Rhizoctonia solani as well as their curative potential against sheath blight of rice.
2 Table 1. Characteristics details and uses of studied medicinal plants Plant Name Vernacular Name Ratti/ Gunja Kalmegh Satawar Sinduri Janglee sem Family Fabaceae Acanthaceae Liliaceae Bixaceae Fabaceae Celastraceae Asteraceae Habit Perennial, deciduous, twining shrub Branched herb Branched, spiny, shrub Shrub, evergreen tree Lage perennial shruby climber Climbing shrub Erect, aromatic herb
Therapeutic Use Antihistaminic, antiseptic, aphrodisiac, abortifacient, vermifuge Remedy for fever, laxative, acidity, syphilitic cachexia, antyphoid, antibiotic Plant is tonic for diuretic, galactogogue, dyspepsia and enhance milking of mother Use for leprosy, eczema, elephantiasis, for jaundice and snake bite Fruits are astringent, appetizer, digestive. Used in hyperdipsa, anorexia, ulcers. Used in headache, rheumatism, leprosy, paralysis Seeds are anthelmintic, diuretic and used in destroying pediculi Seeds have oil used as brain tonic. Roots are powerful cathartic and diuretic Used forheart and circulatory tonic, antispasmodic Bulbs are useful in rheumatic troubles while extract of leaves in earache Leaves are used in snake bite and rickets, improves milk deficiency in women Root part used in preparation of health tonic Diuretic, used in jaundice, rheumatism and disease of urino-genital tracts Antipyretic and used to relieve bodyache Used in leucoderma, seeds are astringent, stomachic, anthelmintic, diuretic Used for treatment of disease of spleen, piles, skin and muscular pain Root bark is diuretic, tonic and use to treat asthma and stomach diseases
Abrus precatorious L. Andrographis paniculata Burm. f. Asparagus racemosus Willd. Bixa orellana L . Canavalia gladiata (Jacq.) DC. Centratherum anthelminticum (L.) Kuntze. Clitoria ternatea L. Coleus forskohlii Briq. Crinum latifolium L. Cryptolepis buchananii Roen. & Schult.
Asclepiadaceae Glabrous twining shrub Boragainaceae Acanthaceae Bignoniaceae Papilionaceae Erect herb Gregarious, subshrub Medium size tree Erect annual herb
Cynoglossum lanceolatum Bhograj Forssk. Hygrophila auriculata (Schumach.) Heine. Millingtonia hortensis L.f. Psorelea corylifolia L. Plumbago zeylanica L. Stereospermum suaveolens (Roxb.) D.C.
Talmakhana Akashneem Bawchi Chitrak Garud
Preparation of the plant extract Crude extracts of the seventeen medicinal plant species were prepared for the present study (table 1). Fresh samples of plant parts were washed thrice under tap water to ward off the dirt and surface sterilized with Mercuric Chloride (0.01%) and rinsed with sterilized distilled water. Surface sterilized tubers were chopped finely in a blender before the extraction. For hot water extraction, the plant materials were plunged in required quantity of water (1:1 w/v) in a glass beaker and heated over a hot plate at 80-100C for 30-40 min. For the preparation of cow urine and cold water extract the plant materials were plunged in required quantity of respective solvents (1:1 w/v) in a glass beaker, covered and kept at room temperature for 7 days and overnight respectively. The pulp of the plant tissue along with the extraction solvent were then squeezed and filtered through 3 layers of muslin cloth. The filtrates were then subjected individually to low speed centrifugation (3000 rpm for 5 min) and the clear supernatant were collected. Plant extracts thus obtained was standard stock solution (100%) (Tiwari, et al., 2005).
Antifungal activity of crude plant extract (in vitro) To evaluate efficacy of plant extracts on the mycellial growth of Rhizoctonia solani (in vitro), poisoned food technique was employed (Grover and Moore, 1962). Stock solutions of crude plant extracts were mixed aseptically @ 5000 ppm and 10000 ppm in semisolid sterilized potato dextrose agar medium at a temperature of 40- 45C. Plants extract impregnated media were poured in sterilized petri dishes. Plates without extracts and with fungicide (Hexaconazole,100 ppm) were served as positive control and negative control respectively. Petr i dishes were inoculated with 7 mm diameter mycelial disc of 72 hrs old culture of R. solani and then incubated at 261C. Radial mycelial growth (mm) was recorded after 48 hrs of incubation. Pot experiment (in vivo) Rice cultivar Swarna was planted in small plastic pots containing autoclaved soil with single seed per pot. The pots were placed in greenhouse at a temperature of 30C day, 22C night and relative humidity of 70-85%. Inoculation of rice plant was done at maximum tillering stage. Inoculum
was prepared by mixing five days old culture of R. solani in sterilized pre moistened rice grains and incubated for seven days at 26C. Tillers were inoculated with rice grain culture placed in the leaf sheath at the collar, without wounding. Immediately after inoculation, plants were covered with a thin polythene bag and kept in greenhouse conditions. After the appearance of disease symptoms, three sprays with crude plant extract @ 5000 ppm were applied at an interval of ten days. Post treatment disease severity was recorded after third spray. Disease index percentage was calculated according to the following formula (Sharma, et al. 1990).
Table 2. Efficacy of different medicinal plant extracts in inhibition of mycellial growth of Rhizoctonia solani and sheath blight severity Botanical name Vernacular name Parts used Radial mycelial growth in different solvents (mm) Cow urine Cold water 18.0 28.8 34.5 26.3 20.5 39.6 27.9 35.6 49.3 35.8 40.5 36.5 39.8 40.5 40.0 38.3 35.5 90.0 0.0 34.6 Hot water 42.3 42.2 42.2 44.8 30.7 33.5 35.5 43.7 41.3 41.3 41.7 41.0 43.0 38.2 43.8 44.8 35.7 90.0 0.0 40.3 Mean 25.6 27.3 34.4 27.0 21.4 34.4 24.5 41.1 33.2 36.6 37.7 30.7 37.0 30.6 32.3 30.7 26.4 90.0 0.0 Cow urine 17.5 19.0 14.0 20.2 18.7 16.0 17.0 22.0 15.0 18.5 19.5 23.0 18.0 23.0 21.0 13.0 20.8 35.0 14.5 18.4 Disease severity in different solvents (%) Cold water 18.5 23.0 19.0 21.0 23.0 24.2 20.1 27.5 20.0 20.0 16.0 23.2 23.0 26.0 21.3 18.5 15.9 25.0 15.0 20.9 Hot water 22.5 24.0 19.0 21.6 16.2 25.9 23.6 18.0 18.0 28.0 18.0 22.0 28.5 23.5 22.5 27.0 20.2 35.0 14.2 21.7 Mean 20.0 22.0 17.0 21.0 19.0 22.0 20.0 23.0 18.0 22.0 18.0 23.0 23.0 24.0 22.0 20.0 19.0 32.0 15.0
Abrus precatorious Andrographis paniculata Asparagus racemosus Bixa orellana Canavalia gladiata Celastrus paniculatus Centratherum anthelminticum Clitoria ternatea Coleus forskohlii Crinum latifolium Cryptolepis buchananii Cynoglossum lanceolatum Hygrophila auriculata Millingtonia hortensis Plumbago zeylanica Psorelea corylifolia Stereospermum suaveolens
Positive Control (without extract) Negative Control (Hexaconazole) Mean C D at 5%
Ratti / Gunja Kalmegh Satawar Sinduri Jangali sem Malkangani Vanjeera Vishnukanta Pashanbhed Giloch Nagbel Bhograj Talmakhana Akashneem Chitrak Bawchi Garud -
Leaf Leaf Tuber Leaf Seed Leaf Seed Seed Leaf Bulb Leaf Leaf Leaf Leaf Root Seed Leaf -
16.3 11.0 26.7 10.0 13.2 30.0 10.0 44.0 14.0 32.7 30.8 14.5 28.3 13.3 13.0 8.8 8.0 90.0 0.0 18.8
Species (Factor A) - 1.250; Solvents (Factor B) 0.40 Species x Solvents (Interaction) 1.762 Species (Factor A) - 2.60, Solvent: Factor B) - 1.53 Species x Solvents (Interaction) 6.53
3.09 %
4.96 %
significant antimicrobial impact of cow urine and composted cow dung on the clinical pathogens and phytopathogenic fungi respectively. Earlier, Jarald et al. (2008) studied the antioxidant and antimicrobial activities of cow urine against clinical pathogens e.g. S. aureus, B. subtilis, K. pneumonia, E. coli, P. vulgaris, S. epidermitis and found that fresh cow urine is better inhibitory activity than its distillate.
In vivo, most of extracts prepared in cow urine, cold water and hot water were found significantly effective over control in controlling sheath blight disease however, cow urine based extracts of P. corylifolia, A. racemosus and C. forskohlii were highly efficacious and at par with hexaconazole followed by C. paniculatus, C. anthelminticum, A. precatorious, Crinum latifolium and C. gladiata (Table 3). The greater efficacy of extracts prepared in cow urine than the aqueous extract might be due to elution of different phytochemical groups having antimicrobial property in desired quantity (Singh, 2001). Irrespective of sheath blight severity, significant interaction between extracts and solvent was obtained. Mean data also indicated the overall greater efficacy of extracts of A. racemosus, C. buchananii, C. forskohlii, C. gladiata, S. suaveolens, P. corylifolia and A. precatorious for controlling sheath blight disease. Inhibitory effect of different plant extracts prepared in cow urine and cow dung suspension on Bipolaris sorokiniana was reported by Akhter et al. (2006). Jabin (2003) reported that cow urine has inhibitory activity against Fusarium semitectum and cow urine mixed with leaf extracts of C. procera, V. negundo and C. alata completely (100%) inhibited the mycelial growth of the pathogen. Cow urine has been proved to be inhibitory to the mycelial growth of F. oxysporum f. sp. cucumerinum, F. solani f. sp. cucurbitae and Sclerotinia sclerotiorum those cause diseases in cucumber (Basak and Lee, 2002).
In our study it was undoubtedly evident that medicinal plant extract prepared in cow urine showed better inhibitory impact than aqueous extract and worked as a bio enhancer for plant derived natural product. This study is novel due to the usage of cow urine as a natural buffer which circumvents the chemical extracting agents for phytochemicals. In conclusion, cow urine based plant extracts can be used for controlling sheath blight of rice and hence further on field studies are required to standardize cow urine base biorational fungicide.
REFERENCES
Akhter, N., Begum, M.F., Alam, S. and Alam, Md. S. (2006). Inhibitory effect of different plant extracts, cow dung and cow urine on conidial germination of Bipolaris sorokiniana. J. bioSci. 14: 47-92. Amuah, R.T. (1989). Fungitoxic effect of some West African plants. Annals of Applied Biology 115: 451-452. Basak, A.B. and Lee, M.W. (2002). In vitro inhibitory activity of cow urine and cow dung of Fusarium solani f. sp. cucurbitae. Mycobiology 30 : 51-54. Basham, A.L. (1998). Practices of medicine in ancient India in Asian Medical systems: A Comparative study. (Ed) by Charles leslie, Motilal Banarsidass, Delhi, pp: 22.