International Journal of Phytopharmacology
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64 Parashurama TR et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 64-71. e- ISSN 0976 - 3651 Print ISSN 2229 - 7480 International Journal of Biological & Pharmaceutical Research Journal homepage: www.ijbpr.com IJBPR CERCOSPORA LEAF SPOT DISEASE IN CENTELLA ASIATICA AND ITS EFFECT ON PHARMACEUTICAL COMPONENTS Thaygarthi Ramappa Parashurama, Parinitha Mahishi, G Eswarappa Mallikarjunaswamy, Shivanna Manchanahally Byrappa* Department of studies in Applied Botany, Kuvempu University, Jnana Sahyadri, Shankaraghatta-577451, Shimoga District, Karnataka, India. ABSTRACT Centella asiatica is an important medicinal herb. Studies were conducted to determine the casual organism of leaf spot disease in Centella asiatica in Bhadra Wildlife Sanctuary during 2006-09. The foliar disease incidence and severity and distribution pattern of disease in eight forest regions of the sanctuary were determined. The effect of foliar infection on secondary metabolite content was also determined. The present study indicated that Cercospora centellae is the major leaf spot disease causing pathogen in the study regions of the sanctuary. The foliar disease incidence and severity was high in Madla forest region during September-October. The disease is homogeneously distributed in the study area. The secondary metabolites- alkaloids and steroids in diseased foliages decreased with increase in foliar infection due to C. centellae, while phenols and flavonoids increased. The study suggested that foliar infection of C. asiatica by C. centellae caused significant decrease in therapeutically important alkaloids and steroids. Key Words: Centella asiatica, Cercospora centellae, disease severity, alkaloids, steroids. INTRODUCTION modulatory properties (Plohmann et al., 1994; Coldren et Centella asiatica (L). Urban (Apiaceae) is an al., 2003). The plant is diuretic and alterative and is used in important traditional medicinal herb (Devkota and Jha, the treatment of leprosy (Wolfram, 1965; Kartnig, 1988), 2008) native to India, China, Nepal, Indonesia, Sri Lanka, asthma, bronchitis, dropsy, elephantiasis, gastric catarrh, Australia, Madagascar and Southern and Central Africa. kidney trouble, leucorrhoea, skin disease and urethritis Previous work on C. asiatica has led to the isolation of (Kakkar, 1998). It has been shown with antibacterial, anti- more than 70 biochemical constituents including feedent, anti-filarial, anti-stress, anti-tuberculosis triterpenoid saponins (Jiang et al., 2005; Kuroda et al., (Chakraborty et al., 1996; Srivastava et al., 1997) and anti- 2001), polyacetylenes (Schulte et al., 1973), flavones viral activities (Yoosook et al., 2000). The drugs from this (Prum et al., 1983), and sterols (Kapoor et al., 2003). plant possess anti-epileptic (Moharana and Moharana, Asiaticoside, a trisaccharide triterpene in this plant has 1994), anti-tumor (Babu et al., 1995) and anti-hepatoma been identified as the most active compound associated activities (Lin et al., 2002). with the healing of wounds and duodenal ulcers, whilst the A survey of literature indicated that C. asiatica is triterpene saponins are reported to possess Immune infected by fungal pathogens like Pseudocercospora centelli and Cercospora centellae that cause considerable Corresponding Author damage to plants in cultivated land (Dubey and Pandey, 2008; Manoharachary et al., 2003). The plant species is Shivanna Manchanahally Byrappa affected by C. centellae during different seasons of the Email: [email protected] year in Bhadra Wildlife sanctuary (Parashurama et al., 65 Parashurama TR et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 64-71. 2007). These studies are limited to the symptomatological leaf-bioassay technique (Shivanna and description of C. centellae disease in C. asiatica in certain Mallikarjunaswamy, 2009). The apparently healthy leaves seasons, however a detailed study of foliar diseases in were surface disinfected and prick-inoculated with sterile varying severity in this plant althrough the year and disease water or spore suspension (4x106 or 6x106 spores ml-1) of distribution pattern in this area is lacking. Hence, an the fungal colony culture on PDA and incubated as attempt was made to determine Cercospora leaf spot described above. disease incidence and severity in the entire sanctuary through different growing stages and seasons and the Seasonal occurrence and distribution of foliar disease in spatial distribution of foliar disease. The effect of foliar the sanctuary disease on the content of certain pharmaceutical important The data of disease incidence (DI %) and severity secondary metabolites was also determined. (DS %) were recorded in an interval of 30 days for a period of 36 months during 2006-09. The disease incidence and MATERIALS AND METHODS severity was determined as described previously (Shivanna Selection of study area and Mallikarjunaswamy, 2009). Bhadra Wildlife sanctuary (130 341 to 130 461 N The spatial distribution of Cercospora leaf spot lat. and 750 291 to 750 451 E long.) is located in the disease in the study area during 2006-09 was determined southern central part of the Western Ghats region of by Taylor’s power law (Taylor, 1984) using the disease Karnataka over an area of 492.46 sq km. Eight forest incidence data. Taylor’s power law relates the observed regions- Hebbegiri, Gangegiri, Madla, Lakkavalli, variance (Vobs) to the population mean (m) for count data b Madhuguni, Muthodi, Kemmannugundi and Kagemanegiri with no upper limit by Vobs=Am (Equation 1), where A that run across Bhadravathi and Tarikere taluks of Shimoga and b are parameters, binomial distribution [log (Vr)] and Chikmagalur districts, respectively, were selected as equals the mean and estimated by regression after study regions. The study area consisted of dry- and moist- logarithmic transformation of Equation 1 to log (Vobs) = deciduous and semi evergreen forests and receives an log (A) + b log (Vr) (Equation 2) annual rainfall of 1600-2000 mm. In each study region, The parameter b is considered an index of three study sites were identified randomly; each of the aggregation, providing a measure of the degree of ‘disease study site in turn consisted of three quadrates (10x10 sq m) incidence dependence of aggregation’ (Taylor & Taylor, representing three replicates. Plant taxa of the species were 1977). For count data, vobs = m (i.e. a = b = 1) indicates a collected from the study area and identified based on their random pattern of disease incidence as represented by a morphology (Yoganarasimha et al., 1982; Gamble, 1995) binomial distribution. Parameter estimates of a and b > 1 and in comparison with herbarial specimens in the indicate heterogeneity as an aggregation at the scale of the department. The species identity was kindly confirmed by sampling unit is dependent on disease incidence. Least Prof. emeritus Dr. G. R. Shivamurthy, University of square regression was used to estimate the intercept and Mysore, Mysore. slope parameters. Significance in relationship between log (vobs) and log (Vr) was determined with F test. The co- Isolation and characterization of causal organism and its efficient of determination (R2) and the mean square error pathogenicity were used to determine the goodness of fit of the model. The symptoms of disease in infected C. asiatica Differences between the observed and predicted variance plants in different growing stages and seasons were were plotted against predicted values to evaluate the recorded. The foliages of C. asiatica with disease symptom appropriateness of the model. For comparative purpose and were collected from different area in sterilized because of the importance of obtaining accurate estimate of polypropylene bags, surface disinfected (0.2%, NaOCl, 2 A and b, two other approaches were evaluated for min), segmented and placed on moistened blotter discs estimating parameters, namely geometric mean and contained in Petri dishes and incubated under light- resistant line regression. For geometric mean regression, darkness cycle of 12/12 hr at 23±2°C for 5-7 days. The the slope is always greater than the least squares slope associated fungal species on incubated diseased plant (Madden et al., 1995; Gent et al., 2008). Least squares materials were characterized based on the morphological regression was used to estimate the intercept and slope characteristics of colony and spores (Barnett et al., 1998; parameters of Taylor’s power law using MINITAB version Sivanesan, 1983 and Subramaniyan, 1983; Booth, 1977) 15. and by visiting index Fungorum (www.indexfungorum.org). The fungal species that Assay of phytochemical compounds in infected foliages expressed up on incubation were cultured on PDA and The leaves of C. asiatica were grouped into three their identity was confirmed by comparing with the fungal infection categories - i) apparently healthy leaf sample (no colony characteristics on incubated diseased parts. visible symptom when collected or zero fungal incidence), The fungal species associated with disease ii). partially infected leaves (≤50% DS), and iii) totally symptoms were tested for their pathogenicity by detached infected leaves (100% DS). The DS was determined as 66 Parashurama TR et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 64-71. described previously. The above samples were dried and Hillis, 1959), sterols (Sanchez et al., 1972) and alkaloids powdered (0.5-mm particles) and determinedquantitatively (Ikan, 1969).