Indian Phytopath.

64 (3) : 0-0 (2011)

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.

MATERIALS AND METHODS

Isolation of pathogen Isolate of R. solani used for this study was collected from severely diseased rice plant from rice production fields identified in T.C.B. College of Agriculture and Research Station (IGKV), Bilaspur, Chhattaisgarh, India. Infected plant materials brought back from the field were washed, cut into 5 mm segments including the advancing margins of infection. The segments were surface disinfected in 0.5% Sodium Hypochloride solution for 5 min and rinsed in three changes of sterile water. The segments were separately dried in between sheets of sterile filter paper and plated (3 segments per plate) on fresh sterilized potato dextrose agar (PDA) medium, impregnated with Streptocycline (100 ppm), and incubated at 281C. Pure culture was obtained by sub-culturing three times and maintained on culture slants in the refrigerator until required. Collection of plant materials Fresh plant/plant parts were collected randomly from the medicinal plant nursery maintained at college campus, Bilaspur (Chhattisgarh). The plants and the plant parts used for the present study are documented in table 1.

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

Indian Phytopathology 64 (3) : 2-6 (2011)

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.

Celastrus paniculatusWilld. Malkangani

Vanjeera

Aparajita Pashanbhed Giloch Nagbel

Papilionaceae Lamiaceae Liliaceae

Twining shrub Perrenial herb Large herbs, bulbs globose

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

Plumbaginaceae Branched under shrub Vignoniaceae Deciduous tree

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

Indian Phytopathology 64 (3) : 2-6 (2011)

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).

RESULTS AND DISCUSSION

Data presented in table 2 indicated that in vitro, mycelial growth of R. solani differed significantly with respect to extract of medicinal plant species and solvents used and there was significant interaction between the extracts and solvents. All seventeen plant species were found significantly effective in inhibiting the growth of R. solani over control. However, extracts prepared in cow urine were highly significant and exhibited higher degree of anti fungal activity than the extracts prepared in cold water and hot water (Table 2, Fig. 1). Amongst cow urine plant extracts, S. suaveolens, P. corylifolia, B. orellana and C. anthelminticum were significantly more effective in inhibiting the mycelial growth closely followed by A. paniculata, M. hortensis, P. zeylanica and C. gladiata. Antifungal activity of above species against R. solani has earlier been reported by Tiwari, et al. (2007). The cow urine contains urea, uric acid, creatinine and enzymes which may act as buffer (pH~9) and aid in extracting the active principles from medicinal plant parts (Santhi et al., 2011). Moreover, Sathasivam et al., (2010) and Sharma et al. (2010) recently reported the

(n x r1) (n x r4) Disease severity index (%) = 100 N

where, n = number of infected tillers r1 - r4 = category number (category: 0=no infection, 1=25% infection, 2=26-50% infection, 3=51-75% infection and 4=76-100% infection) N = total number of examined tillers

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

Coefficient of variation (CV )

3.09 %

4.96 %

Indian Phytopathology 64 (3) : 2-6 (2011)

Chauhan, R.S. (2004). Panchagavya Therapy (Cow pathy). Current status and future directions. The Indian Cow 1: 3-7. Dharma, K., Rathore, R. and Chauhan, R.S. (2005). Panchagavya (Cow pathy): An Overview. International Journal of Cow Science 1: 26-29. Grover, R.K. and Moore, J.D. (1962). Toximetric studies of fungicides against brown rot organisms, Sclerotia fructicola and S. laxa. Phytopathol . 52: 876-880. Hughes, I. (2002). Isoprenoid compounds and phenolic plant Constituents, Elsevier, New York, N.Y. Science in Africa Magazine 9(56). Jabin, M. (2003). In vitro inhibitory activity of cow urine, cow dung and some medicinal plant extracts against Fusarium semitectum Berk & Rav. of Chilli. M Sc Thesis, Department of Botany, Chittagong University, Bangladesh.107. Jarald, E., Edwin, S., Tiwari, V., Garg, R. and Toppo, E. (2008). Antioxidant and antimicrobial activities of cow urine. Global Journal of Pharmacology 2: 20-22. Naidu, A.D. and John, V.T. (1981). In vitro inhibition of rice fungal pathogens by extract from higher plants. International Rice Research News Letter 6: 12-15. Nichols, A.E., Birch, M.R., Walter and Lindda, E. (1993). Plant products to control plant parasitic nematodes. Pesticides Sci. 39: 141-145. Sadhale, N., (2004). Vishvallabba Agri History Bulletin 5. Asian Agri-History Foundation, Secunderabad 500009. pp: 134 Sathasivam, A., Muthuselvam, M. and Rajendran, R. (2010). Antimicrobial activities of cow urine distillate against some clinical pathogens. Global Journal of Pharmacology 4 : 41-44. Shanthi, S., Elamathy, Panneerselvam and S., Radha, A. (2011). Antixanthomonas activity of Pongamia pinnata linn leavescow urine extract-a natural cost effective ecofriendly remedy to bacterial leaf blight of paddy (BLB). Journal of Pharmacy Research. 4: 650-652. Sharma, N. R., Teng, P.S. and Olivares, F. M., (1990). Comparison of assessment methods for rice sheath blight disease. Philippine Phytopath. 26: 20-24. Sharma, R., Sharma, A., and Agarwal, A.K. (2010). Impact of cow urine and composted cow dung on the incidence of seed borne Phytopathogenic fungi. International Journal of Plant Science, 5: 579-581. Shetly, S.A., Prakash H.S. and Shetly, H.B. (1989). Efficacy of certain plant extracts against seed-borne infection of Trichoconiella padwickii of paddy (Oryza sativa). Canadian J. Botany 67: 1956-1958. Singh, S. (2001). Cow urine has anti Leshmania donovani effect in vitro. International J. Cow Sci. 1: 72-73. Tiwari, R.K.S., Chandravanshi, S.S. and Ojha, B.M. (2005). Efficacy of extracts of medicinal plant species on growth of Sclerotium rolfsii root rot in tomato. J. Mycol. Pl. pathol. 34: 461-464. Tiwari, R.K.S., Singh, A., Das, K. and Sinha, A. (2007). Efficacy of extracts of medicinal plants against Rhizoctonia solani. Annals Pl. Prot. Sci. 15: 460-539. Tripathi, R.D., Banerji, R., Sharma, V.R., Balasu, B., and Nigam, S.K. (1985). Toxicity of essential oils from a new strain of Ocimum gratissimum (Clocimum) against betelvine pathogenic fungi. Agric. Biol. Chem. 49: 2277-2282. Received for publication: April 7, 2011 Accepted for publication: July 20, 2011

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.