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Biochemical Characterization of Defense Response of Vigna Aconitifolia Against the Fungal Pathogen Macrophomina Phaseolina

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Biochemical Characterization of Defense Response of Vigna Aconitifolia Against the Fungal Pathogen Macrophomina Phaseolina International Journal of Advanced Scientific Research and Management, Special Issue I, Jan 2018. www.ijasrm.com ISSN 2455-6378 Biochemical Characterization of Defense Response of Vigna aconitifolia Against the Fungal Pathogen Macrophomina phaseolina 1 2 1 Shruti shree Pareek , Indu Ravi and Vinay Sharma 1Department of Bioscience and Biotechnology, Banasthali University, Banasthali, Rajasthan 304022, India; Regional Centre 2Indira Gandhi National Open University, Jaipur, Rajasthan 302020, India Abstract 1. Introduction Charcol root rot disease of Vigna aconitifolia Plant pathogens are a real threat to worldwide (mothbean) caused by pathogen Macrophomina agriculture. Significant yield losses phaseolina is the most destructive disease. The due to fungal attacks occur in most of the agricultural investigation was carried out to know the and horticultural species. More than 70% of all major biochemical basis of defense mechanism of three crop diseases are caused by fungi (Agrios, 2005). (two susceptible RMO-40, CZM-3 and one resistant Plants provide long-lasting systemic immunity to FMM-96 varieties) of Vigna aconitifolia inoculated secondary infection by a range of biotrophic and with Macrophomina phaseolina. The activity of necrotrophic pathogens (Grant and Lamb, 2006). The Polyphenol, Flavonoid, PAL, Total protein content, interaction between plants and pathogens induces a Proline content and oxidative enzymes involved in variety of defense mechanisms which includes cell the removal of reactive oxygen species (ROS) such wall strengthening (Bradley et al., 1992), de novo as, peroxidase (POD), catalase (CAT) and superoxide production of antimicrobial compounds such as dismutase (SOD) and histochemical detection of pathogenesis response proteins and secondary hydrogen peroxide (H2O2) using DAB were recorded metabolites (Gupta et al., 2010a) and rapid localized at different time intervals after pathogen inoculation. cell death etc (Alvarez, 2000). Two age groups of plants that are 15 days and one Plant defense responses have been broadly grouped month were taken for experiments. The increase in as localized acquired response (LAR) and systemic production of these biochemical parameters was acquired response (SAR). The LAR is characterized higher in the inoculated plants compared to control by localized cell death and necrosis at the site of plants and their response in the resistant cultivar was infection. In addition to cell death, LAR is faster and higher than in the susceptible cultivars. characterized by an increase in the production of cell Resistant variety FMM-96 exhibited maximum wall phenolics, release of active oxygen species - increase in these parameters. The finding revealed (AOS) such as superoxide anion radical (O2 ), that FMM-96 is comparatively more resistance hydroxyl radical (OH-) and hydrogen peroxide against infection of Macrophomina phaseolina then (H2O2), production of phytoalexins and synthesis of other observed cultivar. salicylic acid (Hahlbrock and Scheel, 1987). In Keywords: Vigna aconitifolia, 3,3’-Diamino addition to causing accumulation of antimicrobial benzidine (DAB), phenylalanine ammonia lyase compounds, such as phenolic compounds and (PAL), polyphenol, flavnoid, Total protein content, phytoalexins (Ortega et al., 2005), the LAR also Proline content, Reactive Oxygen Species (ROS), leads to an increase in the activity of antioxidant peroxidase (POD), catalase (CAT) and superoxide enzymes such as peroxidases (Kortekamp and dismutase (SOD), Macrophomina phaseolina. Zyprian, 2003) and polyphenol oxidases enzymes (Agrios, 1997) involved in defense responses (Thipyapong et al., 2004). LAR is also known as 158 International Journal of Advanced Scientific Research and Management, Special Issue I, Jan 2018. www.ijasrm.com ISSN 2455-6378 hypersensitive response (HR). HR leads to SAR plants with the pathogen in 15 days and one month against a wide range of pathogens (Metraux, 2001). old plants. SAR is a mechanism of induced resistance in plants that provides them with extended temporal and Preparation of spore suspension and mode of spatial protection against a wide range of microbes. inoculation Moth bean (Vigna aconitifolia) is an important kharif The fungal strain of Macrophomina phaseolina pulse as well as a fodder crop in arid and semiarid (MTCC NO. 2165) was procured from IMTECH, regions, particularly in north western states. It Chandigarh. The lyophilized fungal strain was belongs to the family leguminaceae and subfamily activated on potato dextrose broth (PDB) under papillionaceae. Moth bean is drought resistant, hard proper aseptic conditions in the laminar flow. The and is able to survive under dry conditions. The plant flasks were incubated in incubator shaker (28±2˚C) conserves moisture, protects soil erosion and fixes for 120 h at 120 rpm. Activated fungal strain was atmospheric nitrogen through symbiosis with nodule then streaked on potato dextrose agar (PDA) slants. bacteria. The main contribution of moth bean as a Fungal spore suspension was prepared in sterilized food is based on its protein content, which ranges water at a concentration of105 spores ml-1 under from 20 to 30%. This plant is infected by fungi like aseptic conditions and kept in the incubator shaker Fusarium sp., Macrophomina phaseolina, Phythium (28±2˚C) at 120 rpm for 1 h to obtain a uniformly sp. etc. Macrophomina phaseolina is a common root distributed spore suspension. For the plant infection parasite found in warmer lands. It is omnivorous and the leaf and stem surfaces of the plants were injured is a widely infecting pathogen. Mycelium spreads mildly with an abrasive to facilitate entry of spores through soil causing seedling blight, stem and root rot on spraying with fungal spores using a sprayer. The of herbaceous plant and also decay of woody plant. plants sprayed with autoclaved distilled water without Macrophomina phaseolina is responsible for causing fungal spores were served as control. charcoal root rot disease in mothbean cultivars. The aim of this study is to evaluate the defense Extraction and Assay of Polyphenols mechanisms of Vigna aconitifolia in response to plant The determination of polyphenols was done using the fungal pathogens, by analyzing the occurrence of the method of (Mc Donald et al., 2001). One g tissue i.e. hypersensitive response, changes in the levels of total leaf and stem portions of the plants were phenolic compounds and the activity of the homogenized in 10 ml of 50% methanol. The Phenylalanine ammonia-lyase (PAL) enzyme. supernatant was filtered and centrifuged at 5000 rpm Susceptible and resistant mothbean cultivars in a cooling centrifuge (Remi cooling compufuge, inoculated with Macrophomina phaseolina, were CPR 24) for 25 min at 4˚C. The pellet was discarded used for understanding biochemical mechanism of and the supernatant was used for further assay. 0.5 ml disease resistance. of the plant extract was mixed with 5 ml of Folin’s reagent and 4 ml sodium carbonate. The mixtures 2. Material and Methods were allowed to stand for 15 min and the total phenols were determined by colorimetry at 765 nm. Plant material and growth conditions (ECIL, UV-VIS spectrophotometer). The standard Three different cultivars of Mothbean viz., var. curve was prepared using gallic acid in methanol: FMM-96 (resistant) and RMO-40, CZM-3 water (50:50, v/v). (susceptible) were procured from SKN College of Agriculture, Jobner (Rajasthan, India) and from Extraction and Assay of Flavonoid Central Zone Research Institute (CAZRI), Jodhpur The determination of flavonoids was done using the (Rajasthan, India). Seeds were surface sterilized in method of (Chang et al., 2002). The flavonoid 0.1% HgCl2 and grown in pots containing steam- content was determined by aluminum chloride sterilized garden soil in plant growth chamber with a colorimetric method. One g tissue i.e. leaf and stem photoperiod of 14 hr light and 10 hr dark with portions of the plants were homogenized in the conc. (28±2˚C) temperature and 60% relative humidity of (1:25 v/v) with 95% ethanol. The supernatant was under control conditions. Two age groups of plants filtered and centrifuged at 5000 rpm in a cooling that are 15 days and one month were taken for centrifuge (Remi cooling compufuge, CPR 24) for 25 experiments. The Polyphenol , Flavnoid, PAL, Total min at 4˚C. The pellet was discarded and the protein content, Proline content and oxidative supernatant was used for further assay. 0.5 ml of enzymes activity was determined at 0, 2, 4, 24, 48, extract was mixed with 1.5 ml of 95% ethanol, 0.1 ml 72, 96, 120, 144 and 168 h after inoculation of moth of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. After incubation 159 International Journal of Advanced Scientific Research and Management, Special Issue I, Jan 2018. www.ijasrm.com ISSN 2455-6378 at room temperature for 30 min, the absorbance of the reaction mixture was read at 415 nm with a Shimadzu Enzyme activity was expressed in terms of rate of UV-2450 spectrophotometer (Japan). The amount of increased absorbance per unit time per mg protein. 10% aluminum chloride was substituted by the same amount of distilled water in blank. Quercetin was Histochemical detection of hydrogen peroxide used to make the calibration curve. using 3, 3’-Diamino benzidine (DAB) The presence of hydrogen peroxide (H2O2) was done Extraction and Assay of Phenylalanine ammonia histochemically by the method of Christensen et al. lyase (PAL) (1997). Leaves of 15 days and one month old control Phenylalanine ammonia lyase (PAL) activity was and inoculated plants of the var. viz., FMM-96, determined using the method of (Camm and Towers, RMO-40 and CZM-3 were placed in 20 ml solution 1973). 1 gm plant tissue was homogenized in 15 ml of DAB of 1 mg/ml concentration in petri-plates for 8 0.05 M borate buffer, pH 8.8. The homogenate was h. These were then subsequently shifted in boiling filtered and centrifuged at 10,000 rpm (Remi cooling 96% ethanol for 10 min. Then samples were compufuge, CPR 24) for 15 min at 4˚C.
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