Eco-friendly management of wilt caused by Fusarium oxysporum f.sp. Ciceri in chickpea

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1 Legume Research, 39 (1) 2016: Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Eco-friendly management of wilt caused by Fusarium oxysporum f.sp. Ciceri in chickpea Chandar Kala*, S. Gangopadhyay, and S.L. Godara Department of Plant Pathology, College of Agriculture, Swami Keswanand Rajasthan Agricultural University, Bikaner , India. Received: Accepted: DOI: /lr.v0iOF.6789 ABSTRACT Antagonistic potentiality of Trichoderma viride, T. harzianum and Pseudomonas fluorescens were evaluated against Fusarium oxysporum f. sp. ciceri under in vivo conditions. The effect of organic amendments viz; farm yard manure, vermicompost and mustard cake on disease control potentiality of test antagonists against chickpea wilt and on population dynamics of the antagonists and pathogen in soil was also studied. Maximum inhibition of mycelial growth of F. o. f. sp. ciceri was recorded in presence of P. fluorescens (%) followed by T. harzianum (%) and T. viride (%). Seed treatment with P. fluorescens was more effective in suppressing the disease incidence as compared to T. harzianum and T. viride. The disease control efficacy and population dynamics of all the three test antagonists was enhanced in response to application of organic amendments. Among the three organic amendments tested, mustard cake was most effective in enhancing the disease control potentiality of these antagonists. Key words: Biocontrol agents, Cicer arietinum, Fusarium oxysporum f. sp. ciceri, Organic amendments. INTRODUCTION Chickpea or gram (Cicer arietinum L.) is traditionally grown in different parts of the world covering Asia, Africa, Europe and North and South America. India is the largest producer of chickpea in the world, sharing 65 and 70 per cent of the total global area and production, respectively (Paroda and Chadha, 1996). In India, the low productivity of chickpea is due to several biotic and abiotic factors. Fusarium wilt is a serious problem in most of the chickpea growing areas in India and world. This disease causes considerable yield losses which may reach up to 100 per cent under conditions favourable for high disease severity (Navas-Cortes et al., 2000). Management of Fusarium wilt of chickpea is difficult as the pathogen is soil-borne and may attack at various stages of crop growth. No single control measure is fully effective against the disease. However, the desired level of disease control may not be achieved under certain circumstances since the fungicide remains effective for a limited period. While the disease may appear at later stages of crop growth. Management of wilt, root rot diseases. of various field crops including chickpea using microbial antagonists such as Trichoderma spp., Pseudomonas fluorescens, etc. as seed treatment and soil application have drawn the attention of scientists throughout the world (Mukhopadhyay et al., 1992; Hervas et al., 1998, Laskin and Fravel, 1998; Gholve and Kurund Ker, 2002). Management of soil borne pathogens with fungicides has been attempted for long time. However, it is difficult to manage these diseases economically with fungicides alone because of their soil borne nature and wide host range. Biological control of these pathogens therefore, is an alternative possibility. Mass multiplication, efficacy of the bioagents and delivery system of bioagents have been considered to be the major constraints in the success of the bioagents. The present investigations deals with antagonistic potentiality and population dynamics of the antagonists and test pathogen under in vitro conditions. MATERIALS AND METHODS A virulent isolate of Fusarium oxysporum f. sp. ciceri (Padwick) Synd. & Hans. isolate from wilt infested chickpea plants was used in the present studies. Chickpea variety RSG-44 was used as a host crop. Three antagonists viz., Trichoderma viride, Trichoderma harzianum and Pseudomonas fluorescens obtained from culture collection of the Department of Plant Pathology. The experiment was conducted in vitro and green house conditions during rabi 2006 in the Department of Plant Pathology, College of Agriculture, S.K.R.A.U., Bikaner, Rajasthan. Evaluation of antagonistic potentiality of bioagent in vitro; Antagonistic potentiality of Trichoderma viride, T. harzianum *Corresponding author s chandarkalagodara@gmail.com.

2 130 LEGUME RESEARCH - An International Journal and Pseudomonas fluorescens was tested against Fusarium oxysporum f. sp. ciceri under laboratory conditions. For two Trichoderma isolates dual culture technique (Skidmore and Dickinson, 1976) was used. Mycelial discs of 5 mm diameter taken from actively growing cultures of the test pathogen and respective antagonists were placed on opposite points at 4 cm apart in petridishes containing PDA medium. In case of control, petridishes containing PDA medium were inoculated only with mycelial disc of Fusarium oxysporum f. sp. ciceri. The inoculated petridishes were incubated at 25 0 C. Observations were recorded after seven days of incubation. For each treatment five replications were kept. The bacterial antagonist P. fluorescens was grown on Pseudomonas agar fluorescens slants for 24 hours at 27 0 C. Ten milliliter sterilized distilled water was added to each slant and bacterial suspension was prepared by gently scrapping the bacterial growth with the help of sterilized inoculating needle. The bacterial suspension so prepared was transferred to sterile petridishes. In order to determine the antagonistic potentially of P. fluorescens against Fusarium oxysporum f. sp. ciceri, two different inoculation methods were followed which are paper disc inoculation method and streaking method. Radial growth of Fusarium oxysporum f. sp. ciceri was recorded and inhibition percentage was calculated by using the following formula: C T C C= Radial growth of Fusarium oxysporum f. sp. ciceri in control (mm) T = Radial growth of Fusarium oxysporum f. sp. ciceri in presence of antagonist (mm). Pot experiments: The inoculum of Fusarium oxysporum f. sp. ciceri was prepared on sand maize meal media (1 part partially broken maize grain + 3 part sand + distilled water to moisten the media). The flasks containing the sterilized media were inoculated with mycelial disc of Fusarium oxysporum f. sp. ciceri (5 mm diameter) and incubated at 25 0 C for 10 days. This inoculum was used for soil inoculation at 40 g kg -1 soil in all the pot experiments. Preparation of talc based formulation of bioagents: In this case Trichoderma viride and T. harzianum were grown on potato dextrose broth media. Pseudomonas fluorescens was grown on Kings B broth medium. The flasks containing sterile potato dextrose broth media were inoculated with fresh culture of Trichoderma viride and T. harzianum separately and incubated at 25 0 C for 10 days. The flasks containing the sterile Kings B broth media was inoculated with fresh culture of Pseudomonas fluorescens and incubated at 27 0 C for 72 hours. Potato dextrose broth containing the fungal biomass and Kings B broth containing the bacterial growth were homogenized and mixed with talc powder in 1:2 ratio and dried in shade. The mixture was sieved to obtain a fine powder. Carboxy methyl cellulose was added at 6 g kg -1 talc preparation as sticker and mixed thoroughly. Efficacy of antagonists used as seed treatment against chickpea wilt: Two different levels of talc based formulations of three test bioagents i.e. 4 and 6 g kg -1 seed were used as seed treatment before sowing. Ten seeds were sown in each pot keeping three replication untreated seeds served as control. The experiment was conducted in soils containing Fusarium oxysporum f. sp. ciceri inoculum at 40 g kg -1 soil. Observations on disease incidence were recorded at 15 days interval up to 60 days of sowing. Effect of organic amendments on efficacy of antagonists against chickpea wilt: Soil was first mixed thoroughly with the three organic amendments viz., farm yard manure (FYM) at 10 and 20 g kg -1 soil, vermicompost 10 and 20 g kg -1 soil and mustard cake 1 and 2 g kg -1 soil. After 24 hours, sand maize meal inocula of Fusarium oxysporum f. sp. ciceri was added in soil and allowed to stabilize for 48 hours. Seeds treated with talc based formulations of bioagents were sown in amended soils. In case of control, untreated seeds were sown in soil without organic amendments. For each treatment, three replications were kept. An observation on wilt incidence was recorded up to 60 days after sowing. Population of Fusarium oxysporum f. sp. ciceri and respective antagonists were determined just before sowing and at 30 and 60 days after sowing. Enumeration of population of Fusarium oxysporum f. sp. ciceri and bioagents in soil: Population of Fusarium oxysporum f. sp. ciceri and bioagents enumerated immediate before sowing and at 30 and 60 days after sowing of chickpea seeds. Soil samples were collected using cork borer from each pot, mixed thoroughly and air dried in shade for 4-6 hours. Ten gram soil was added in 90 ml sterilized distilled water in Erlenmeyer flask and stirred thoroughly. Serial dilutions were made from stock soil suspension upto For pathogen a 0.2 ml soil suspension having 10 4 dilution was added on surface of Fusarium selective medium (Papavizas, 1967) in petridishes uniformly spread with the help of glass spreader. In case of Trichoderma spp. a 0.2 ml soil suspension having 10 5 dilutions was transferred to the surface of Trichoderma selective medium (Elad and Chet, 1983) and spread uniformly. These inoculated petridishes were incubated at 25 0 C for 5-6 days. The colonies of Fusarium oxysporum f. sp. ciceri and Trichoderma spp. developed on petridishes were counted. In case of P. fluorescens a 0.2 ml

3 suspension of suitable dilution (10 8, 10 9 and ) was added on surface of Pseudomonas agar fluorescens (PAF) medium in petridishes and spread uniformly with the help of glass spreader. The inoculated petridishes were incubated at 27 0 C for 24 hours and colonies appeared were counted. RESULTS AND DISCUSSION In vitro testing of antagonists: Mycelial growth of Fusarium oxysporum f. sp. ciceri was significantly inhibited by the three test antagonists (Table 1). Maximum inhibition was recorded in presence of Pseudomonas fluorescens followed by T. harzianum and T. viride. The colony diameter of the test pathogen was statistically at par in presence of T. viride and T. harzianum. Whereas, the colony diameter of the pathogen was significantly less in presence of Pseudomonas fluorescens as compared to T. viride and T. harzianum. Singh et al (1997) tested antagonistic potentiality of two isolates of Trichoderma and one isolate of Epicoccum purpurascens against Fusarium oxysporum f. sp. ciceri causal agent of chickpea wilt. The mode of actions of these bioagents was investigated. Trichoderma harzianum inhibited the growth of Fusarium oxysporum f. sp. ciceri through mycoparasitism, while E. purpurascens exhibited antibiosis against the test pathogen. Inam et al (2001) evaluated different strains of P. fluorescens against chickpea wilt pathogen alone and in combinations showed that all the isolates were capable of suppressing Fusarium oxysporum f. sp. ciceri when used individually or in combination in vitro. Godwin-Egein and Arinzae (2001) revealed the mode of antagonisms of T. harzianum against Volume 39 Issue 1 (2016) 131 TABLE 1: Effect of antagonists on the growth of Fusarium oxysporum f. sp. ciceri on potato dextrose agar medium Antagonists Inoculation method Colony diameter of Fusarium Inhibition of growth (%) oxysporum f. sp. ciceri (mm) Trichoderma viride Dual inoculation Trichoderma harzianum Dual inoculation Pseudomonas fluorescens Paper disc Streaking Control (without antagonist) CD (P=0.05) 1.28 TABLE 2: Efficacy of antagonists used as seed treatment against chickpea wilt under pot culture conditions Antagonists Dose(g kg -1 seed) incidence (%) control (%) Trichoderma viride (53.27)* (49.41) Trichoderma harzianum (51.48) (47.44) Pseudomonas fluorescens (49.99) (46.05) Control (without antagonist) (74.62) - CD (P=0.05) Antagonist 2.56 Dose 1.62 Antagonist x Dose NS *Figures in parenthesis are angular transformed values F. oxysporum were competition, lysis and hyperparasitism. Haq et al. (2001) demonstrated that certain isolates of P. fluorescens were capable of suppressing Fusarium oxysporum f. sp. ciceri under laboratory condition. Efficacy of bioagents used as seed treatment against chickpea wilt; The wilt incidence was significantly reduced in response to seed treatment with bioagents as compared to control (Table 2). The study also showed that P. fluorescens was more effective in suppressing the wilt incidence as compared to Trichoderma spp. used in the present test. Again, performance of T. harzianum in controlling the disease was slightly better than T. viride. With increase in dose of the respective bioagents the disease incidence was reduced in all the three bioagent treatments. Deshmukh and Raut (1992) observed that T. harzianum and T. viride were effective against Fusarium oxysporum when present in 1:2 mixtures in the soil under pot culture conditions. Raghuchander et al. (1997) reported that dipping of banana suckers in the suspension of Pseudomonas fluorescens or T. viride effectively checked the incidence of banana wilt (Fusarium oxysporum f. sp. cubense) and simultaneously enhanced the yield. Singh et al (1997) reported the Trichoderma harzianum gave maximum control of chickpea wilt in soil infested with Fusarium oxysporum f. sp. ciceri. Effect of organic amendments on disease control efficacy and population dynamics of antagonists: The results given in Table 3 showed that wilt incidence was significantly reduced when three bioagent treated seeds were sown in soils

4 132 LEGUME RESEARCH - An International Journal TABLE 3: Effect of organic amendments on disease control efficacy of biogents used as seed treatment (6 g kg -1 seed) against chickpea wilt Treatment Farm yard manure Vermicompost Mustard cake Control(Without organic amendments and biogents) Dose (g/kg soil) Trichoderma viride Trichoderma harzianum Pseudomonas fluorescens Incidence (%) (42.01)* (39.79) (36.73) (35.25) (36.33) (32.66) (74.62) Control (%) Incidence (%) (40.86)* (38.74) (36.08) (34.23) (32.59) (31.44) (74.62) Control (%) Incidence (%) (40.09) * (38.13) (35.13) (33.36) (31.49) (28.86) (74.62) Control (%) CD (P= 0.05) Treatment Dose - NS - NS Treatment Dose - NS - NS - NS - *Figures in parenthesis are angular transformed values amended with organic amendments i.e. FYM, vermicompost and mustard cake at two different levels tested. The study showed that the mustard cake was most effective in enhancing the disease control potentiality of all three tested bioagent. The other two organic amendments also exhibited positive influence towards disease control efficacy of bioagents. The disease incidence recorded in mustard cake treatment was significantly less as compared to vermicompost and farm yard manure. The three amendments positively influenced the population of these antagonists (Table 4). Population of T. harzianum, T. viride and P. fluorescens was enhanced in amended soils. Mustard cake was most effective in increasing the population of three antagonists in soil followed by vermicompost and farm yard manure. Bora et al (2000) reported that application of Pseudomonas fluorescens in combination with soil amendment of Sesbania provided most effective protection against tomato wilt caused by Fusarium oxysporum f. sp. lycopersici under field conditions. Ram Gopal (2002) also observed the disease control efficacy of Trichoderma viride and T. harzianum was significantly enhanced in response to FYM under pot culture conditions used as seed treatment. Effect of organic amendments and bioagents seed treatment on population of F.oxysporum f. sp. ciceri in soil: The population of F. oxysporum f. sp. ciceri in inoculated TABLE 4: Effect of organic amendments on population of bioagents in soil Organic Dose g kg -1 Trichoderma viride Trichoderma harzianum P. fluorescens amendment soil CFU ( x 10 5 g -1 soil) CFU ( x 10 5 g -1 soil) CFU ( x g -1 soil) 30 DAS 60DAS 30 DAS 60DAS 30 DAS 60DAS FYM Vermicompost Mustard cake Control CD (P=0.05) Organic amendment Dose NS organic amendment x dose NS NS NS NS NS NS Population of T. viride, T. harizanum and P. fluorescens in soil just before sowing of treated seeds were 1 x 10 3 g -1 soil, 0.66 x 10 3 g -1 soil and 8.33 x 10 5 g -1 soil respectively. -

5 Volume 39 Issue 1 (2016) 133 TABLE 5: Effect of organic amendments and bioagents seed treatment on population of F. oxysporum f. sp. ciceri in soil Organic Dose g kg -1 Trichoderma viride Trichoderma harzianum P. fluorescens amendment soil CFU ( x 10 4 g -1 soil) CFU ( x 10 4 g -1 soil) CFU ( x 10 4 g -1 soil) 30 DAS 60DAS 30 DAS 60DAS 30 DAS 60DAS FYM Vermicompost Mustard cake Control CD (P=0.05) Organic amendment Dose NS organic amendment x dose NS NS NS NS NS NS Population of F. oxysporum f. sp. ciceri just before sowing of chickpea seeds was x 10 5 g -1 soil. soil was x 10 5 g -1 soil just before sowing of the crop. Population of F. oxysporum f. sp. ciceri was significantly reduced when bioagents treated seeds were sown in organic amended soil (Table 5). The population of F. oxysporum f. sp. ciceri in soil was least when Trichoderma viride treated seeds were sown in soils amended with vermicompost. In case of T. harzianum and P. fluorescens treatments, the population of the pathogen was least in soils amended with mustard cake. Srivastava and Singh (1991) observed that neem and mustard cake had reduced the population of Fusarium solani under pot culture conditions. Diyora and Khandar (1995) observed that Neem cake was more effective than mustard cake in checking the Fusarium population in soil. In case of cumin wilt (Fusarium oxysporum f. sp. cumini) application of mustard cake proved to be most effective under pot culture conditions. Under field condition, Patel and Anahosur (1998) observed that farm yard manure significantly reduced the population of Fusarium oxysporum f. sp. ciceri and Macrophomina phaseolina in chickpea field. It may be concluded that maximum inhibition of mycelial growth of Fusarium oxysporum f. sp. ciceri was recorded in presence of Pseudomonas fluorescens followed by Trichoderma harzianum and T. viride. Seed treatment with Pseudomonas fluorescens was more effective in suppressing the disease incidence as compared to Trichoderma harzianum and T. viride. Among three organic amendments tested, mustard cake was most effective in enhancing the disease control potentiality of all the three antagonists. REFERENCES Aghnoom, R.; Falahati, Rastegar, M. and Jafarpour, B. (1999). Comparison of chemical and biological control of cumin wilt (Fusarium oxysporum f. sp. cumini) in laboratory and green house conditions. Indian J. Agril. Sci. 30: Bora, L.C.; Das, B.C. and Das, M. (2000). Influence of microbial antagonists and soil amendments on bacterial wilt severity and yield of tomato (Lycopersicon esculentum). Indian J. Agril. Sci. 70: Deshmukh, P.P. and Raut, J.G. (1992). Antagonism by Trichoderma spp. on five plant pathogenic fungi. New Agriculturist 3: Diyora, P.K. and Khandar, R.R. (1995). Management of wilt of cumin (Cuminum cyminum L.) by organic amendments. J. Spices and Aromatic Crops. 4: Gholve, V.M. and Kurundkar, B.P. (2002). Efficacy of Pseudomonas fluorescens isolates against wilt of pigeonpea. J. Maharashtra Agril. Univ. 27: Godwin-Egein, M.I and Arinzae, A.E. (2001). Antagonism between Trichoderma harzianum Rifai and Fusarium oxysporum Schlecht emend Sny. & Hans. J. Mycol. Pl. Pathol. 31: Haq, M.I.; Ahmed, R., Khan, S.M. and Javed, N. (2001). Use of different strains of Pseudomonas fluorescens alone and in combination to suppress chickpea wilt. Pakistan J. Phytopath. 13: Inam, Ul.; Haq. M.I., Riaz, A., Khan, S.M. and Javed, N. (2001). Use of different strains of Pseudomonas fluorescens alone and in combination to suppress chickpea wilt. Pakistan J. Phytopath. 13:

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