Int. J. LifeSc. Bt & Pharm. Res. 2013 K C Deepthi, 2013 ISSN 2250-3137 www.ijlbpr.com Vol. 2, No. 2, April 2013 © 2013 IJLBPR. All Rights Reserved Research Paper EFFECT OF POTENTIAL BIOCONTROL AGENTS AGAINST SCLEROTIUM ROLFSII CAUSING STEM ROT OF GROUNDNUT K C Deepthi1* *Corresponding Author: K C Deepthi, [email protected] Total of 40 antagonists were isolated from groundnut rhizosphere and root endophytes of Regional agricultural research station of tirupati region. When tested in vitro, the potential biocontrol agents GRE-9 (100%) and GRB-16 (85%) were predominantly aggressive in inhibiting the mycelial growth of the pathogen in dual culture against Sclerotium rolfsii. Among the fungicides tested, Mancozeb was most compatible with all antagonists. Keywords: Groundnut, Stem rot, Sclerotium rolfsii, Biocontrol agents INTRODUCTION sclerotia which overwinter in soil and on plant Groundnut (Arachis hypogaea L.) is a major debris and can survive in a long period causing legume and oil seed crop in India, covering nearly disease in the following season (Punja, 1985). half of the area under oilseeds. It is grown in over Thus, the control of the disease is very difficult. 100 countries with a total estimated area of 21.8 Different control methods have been investigated m. ha and with production of 28.5 MT. In India it is before including cultural practices (Gurkin and grown over an area of 4 lakhs/hectare, with an Jenkins, 1985; Punja et al., 1986), chemical annual production of 5.5 million tonnes and yield control (Bowen et al., 1992; Minton et al., 1993; was 1007 kg/hectare in the year 2009-10 Culbreath et al., 1992), biological control (Elad (Economic survey 2010-11). Sclerotium rolfsii et al., 1984; Papavizas and Lewis, 1989; Latunde- Sacc. is an important soil-borne pathogen and Dada, 1993; and integrated control (Bicici et al., causes disease in numerous crops including 1994; Cilliers et al., 2003). It is widely recognised peanut (Punja, 1988; Krupa and Dummergues, that biological control of plant pathogens is the 1979). The loss of yield caused by pathogen most important alternative for the future and can infection generally is 25%, but sometimes it be exploited within the frame work of integrated reaches 80-90% in some cases (Grichar and disease management. The present study is Bosweel, 1987). The disease causes damage mainly focussed to study and to isolate and identify on root and stem of plant. The pathogen produces potential antagonists of S.rolfsii and their 1 Department of plant pathology, S.V. Agricultural College, Tirupati-517502. This article can be downloaded from http://www.ijlbpr.com/currentissue.php 58 Int. J. LifeSc. Bt & Pharm. Res. 2013 K C Deepthi, 2013 compatibility with the commonly used fungicides. Suitable controls were kept without antagonist. Growth of antagonists, pathogen and zone of MATERIALS AND METHODS inhibition were measured after recording full Isolation and Identification of Pathogen growth in control plate. Percent inhibition of The pathogen was isolated from stem rot infected mycelial growth of test pathogen was calculated groundnut plants showing typical symptoms of Efficacy and Compatibility of Native stem rot disease by tissue segment method Potential Antagonists with Different (Rangaswami and Mahadevan, 1999). Fungicides Under In Vitro The pathogen was identified based on mycelial The selected native potential biocontrol agents and sclerotial characters based on standard were tested for their compatibility with the mycological keys (Barnett and Hunter, 1972) and fungicides generally recommended for soil maintained on Potato dextrose agar (Dhingra and drenching viz., copper oxychloride (0.1%), Sinclair, 1995) for further studies. mancozeb (0.2%), carbendazim (0.2%), thiophanate methyl (0.1%), Hexaconazole (0.1%) Isolation of Root and Endophytes and Propiaconazloe (0.1%). Fungal isolates were For isolation of root endophytes and rhizosphere tested for their compatibility by poisoned food bacteria, 5 g of root were surface sterilized for 5 technique (Nene and Thapliyal, 1993) and min. with 70.0 per cent ethanol and homogenized spectrophotometric method (Kishore et al., 2005) in 20 ml of sterilized phosphate buffer (20mM for bacterial isolates under in vitro. Na2HPO4+20mM NaH2PO4) using mortar and Statistical Analysis pestle. Appropriate dilutions (10-4 for fungi and 10-6 for bacteria) of these suspensions were Completely Randomized Design (CRD) was used plated on PDA and NA for the isolation of fungi for radial growth percent disease incidence, and bacteria respectively. The plates were poisoned food technique and dual cultural incubated for 72 hr at 28±2°C (Kishore et al., technique (Gomez and Gomez,1984) 2005). RESULTS AND DISCUSSION Screening of Native Potential Biocontrol A total of 40 antagonists were obtained from Agents by Dual Culture Technique groundnut rhizosphere bacteria and root The antagonistic activity of microflora isolates endophytes. 5 fungi and 40 bacterial isolates were against S.rolfsii was determined by dual culture obtained. The bacteria was designated as GRE technique (Johnson et al., 1959). Mycelial discs 1 to GRE 20 (from Groundnut Root endophytes) measuring 6 mm diameter from four day old and GRB 1 to GRB 20 (from Rhizosphere soil) cultures of both fungal antagonist and the test similarly the fungi were named as GREF and pathogen were placed at equidistant on sterile GRBF and these were identified as Trichoderma Petri plate containing PDA medium. One day old spp. in which GRHF 75% from rhizosphere was cultures of bacteria were streaked on opposite side of the pathogen on PDA medium. The Petri found to be superior when compared to other plates were then incubated at 28 ± 2°C. Three isolates in inhibiting the growth of S.rolfsii in dual replications were maintained in each treatment. culture (Table 1 and Figure 1). Fungi in the genus This article can be downloaded from http://www.ijlbpr.com/currentissue.php 59 Int. J. LifeSc. Bt & Pharm. Res. 2013 K C Deepthi, 2013 Table 1: In Vitro Efficacy of Rhizosphere and Root Antagonistic by Trichoderma Isolates Against S. rolfsii in Dual Culture Technique Isolate *Linear Growth of S. rolfsii (cm) Percent of Inhibition of Mycelial Growth of S. rolfsii GREF-1 4 55.55 (48.18) GREF-2 4.5 50 (45.00) GREF-3 5 44.44 (41.80) GRHF-4 2.2 75.55 (60.36) GRHF-5 4.8 46.66 (43.08) Control 9 0 CD (0.05) - 7.99 S.Em± - 2.59 Note: * Mean of three replications. Figure 1: In Vitro Efficacy of Rhizosphere and Root Antagonistic Trichoderma Isolates Against S. Rolfsii in Dual Culture Trichoderma have been known since 1920s for isolates GRE 9 Completely inhibited (100%) the ability to act as biocontrol agents against plant followed by GRB 16 (85%) the growth of pathogen pathogens (Harman, 2006). In case of 40 bacterial (Table 2). This article can be downloaded from http://www.ijlbpr.com/currentissue.php 60 Int. J. LifeSc. Bt & Pharm. Res. 2013 K C Deepthi, 2013 Table 2: In Vitro Evaluation of the Antagonistic Activity of Bacterial Root Endophytes and Rhizosphere Bacteria by S. Rolfsii in Dual Culture Technique Isolate *Linear Growth Percent Inhibition of Isolate *Linear Growth Percent Inhibition of of S. rolfsii (cm) Mycelial Growth of S. rolfsii of S. rolfsii (cm) Mycelial Growth of S. rolfsii GRE-1 2.1 77.77 (61.86) GRB-1 2.1 77.77 (61.86) GRE-2 2.2 75.55(60.36) GRB-2 4.3 52.22 (46.27) GRE-3 2.0 76.66 (61.11) GRB-3 3.8 57.77 (46.89) GRE-4 3.7 58.88 (46.53) GRB-4 3.0 65.55 (54.05) GRE-5 3.1 66.66(54.73) GRB-5 3.1 66.66 (54.73) GRE-6 2.8 68.89 (56.09) GRB-6 2.6 71.11(57.48) GRE-7 2.43 72.96 (58.83) GRB-7 3.5 61.11 (51.26) GRE-8 2 77.78 (61.88) GRB-8 2 77.78 (61.88) GRE-9 0.0 100 (90.00) GRB-9 3.2 64.44 (53.39) GRE-10 2.83 68.52 (55.87) GRB-10 2.83 68.52 (55.87) GRE-11 3.96 55.93 (48.41) GRB-11 3.9 55.93 (48.41) GRE-12 3.13 65.19 (53.84) GRB-12 3.1 65.19 (53.84) GRE-13 2.5 72.22 (58.19) GRB-13 2.5 72.22 (58.19) GRE-14 3.4 62.00 (51.94) GRB-14 3.4 62.00 (51.94) GRE-15 3.6 60 (50.76) GRB-15 3.6 60 (50.76) GRE-16 2.2 75.55 (60.36) GRB-16 1.3 85.55 (67.65) GRE-17 3.0 66.66 (54.73) GRB-17 3.0 66.66 (54.73) GRE-18 4.0 57.23 (49.34) GRB-18 2.03 77.41 (61.66) GRE-19 2.43 72.96 (58.83) GRB-19 3.3 63.33 (52.73) GRE-20 1.4 84.44 (66.76) GRB-20 1.8 80.00 (63.43) Control 9 0 Control 9 0 CD(0.05) - 10.21 CD(0.05) - 7.97 S.Em± - 3.46 S.Em± - 2.70 Note: * Mean of three replications. The use of biological control methods to being used in variety of crops. By including reduce disease incidence caused by plant biocontrol agents for controlling S.rolfsii as a part pathogens is continually being developed and is of integrated disease management will help in This article can be downloaded from http://www.ijlbpr.com/currentissue.php 61 Int. J. LifeSc. Bt & Pharm. Res. 2013 K C Deepthi, 2013 managing disease as well as delaying the compatible with Mancozeb followed by copper oxy development of tolerance/resistance to chloride.