ANTAGONISTIC TRICHODERMA ISOLATES TO CONTROL BAKANAE PATHOGEN OF RICE

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1 Agric. Sci. Digest., 33 (2) : , 2013 AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com ANTAGONISTIC TRICHODERMA ISOLATES TO CONTROL BAKANAE PATHOGEN OF RICE S. Bhramaramba* and A. Nagamani Mycology and Plant Pathology Laboratory, Department of Botany, Post Graduate College of Science, Osmania University, Hyderabad ,India Received: Accepted: ABSTRACT Bakanae disease caused by Fusarium fujikuroi Nirenberg is one of the important diseases affecting the rice crop at different stages of the growth. Twenty eight Trichoderma isolates were collected from the major rice growing areas of Nalgonda, Krishna and Khammam districts of Andhra Pradesh belonged to seventeen species. The isolates found effective invitro against the Fusarium fujikuroi for their antagonistic property in dual plate culture were selected to study for their ability to reduce growth inhibitory volatile and non-volatile metabolites against the pathogen. Keywords: Antagonism Bakanae disease, Fusarium fujikuroi, Trichoderma. INTRODUCTION Rice is grown in many regions across India and Andhra Pradesh is one of the major rice producing state. The rice yield is affected by several biotic and abiotic factors. Among biotic factors, diseases caused by pathogens reduce the rice yield considerably (Janaiah et al., 2002). Foot rot and bakanae caused by Fusarium fujikuroi is one of the important disease of rice causing 20-50% yield losses ( Ou,1985). The crop losses caused by the disease may reach more than 20% in outbreak cases. The most conspicuous and common symptoms are the bakanae symptoms, i.e. the abnormal elongation of the plant. Infected seedlings in the seedbed are thin, yellowish green and up to several inches taller than normal plants. Severely diseased seedlings die before transplanting, and those which survive may die after transplanting. The bakanae disease is primarily seed and soil borne. Seed infection occurs via air borne ascospores and also from conidia that contaminate the seed during harvesting. The fungus infects plants through roots or crowns. The disease is managed by using various physical and chemical methods such as seed treatment with organo-mercury compounds, thiram, thiophanate-methyl, or benomyl. H owever, fungicides and fumigants commonly have drastic effects on the soil biota. * Corresponding author s bhramara8@gmail.com Further, these methods are time-consuming, uneconomical, and environmentally harmful. Hence, biological control is the best alternative control measure for the long-term sustainability and effective management of soil borne disease, which can minimize the disease (Mathre et al., 1999; Harman, 2000; Howell, 2003). Several Trichoderma species have been found effective in suppressing the soil borne diseases caused by Fusarium (Sivan and Chet, 1986), Pythium (Naseby et al., 2000) and Rhizoctonia (Lewis and Papavizas, 1987) species through competition, parasitism or antibiosis (Brotman et al., 2010). The characterization and application of effective Trichoderma isolates to control the soil borne diseases especially the bakanae disease is least studied to date. Hence, a study was conducted to isolate, identify the effective strains of Trichoderma from rice growing areas. The effective antagonistic isolates were evaluated for the production of volatile and non-volatile metabolites which known to play an important role in controlling the pathogen. MATERIALS AND METHODS Thirty-two soil samples were collected from the major rice growing areas of Andhra Pradesh using the method of Dhingra and Sinclair (1985). A total of fourteen soil samples were collected from

2 the Nalgonda district, ten and eight samples from Krishna and Khammam districts respectively. Soil ph was recorded by using ph meter (Elico LI 613). Trichoderma was isolated from soil samples by serial dilution plate method and soil plate techniques using Potato dextrose agar (PDA) and Trichoderma selective medium C (TSM) (Elad and Chet, 1983). Trichoderma colonies obtained on agar plates were purified, subcultured, preserved and identified (Bisset 1984, 1991 a, b, c, 1992; Samuels et al., 2006, Nagamani et al., 2006).The texture of the soil was analyzed by sedimentation method. Screening of Trichoderma isolates against Fusarium fujikuroi:twenty-eight isolates of Trichoderma obtained in this study were tested in vitro against Fusarium fujikuroi (MTCC No. 4649), which was obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India for their antagonistic property in dual plate technique (Aneja, 2003). The mycelial disc of 5 mm diameter of Trichoderma as well as the pathogen grown on PDA were cut with sterile cork borer from the periphery of an actively growing three day old culture and then placed on opposite side of Petri plate containing PDA at a distance of 7 cm. The inoculated Petri plates were incubated at 28 ± 2 C for three days. The Petri plate incubated with pathogen alone were served as control. Three replicates were maintained for all the treatments and average of the results is presented for analysis. Evaluation of volatile metabolites of Trichoderma species: This study was carried out following the procedure of Dennis and Webster (1971 c). The Petri-plates of PDA medium were inoculated with a 5 mm disc of Fusarium fujikuroi. After three days, when the mycelium had attained some growth, the bottom Petri plate (pathogen) was removed and placed on another PDA plate containing inoculated with 5 mm discs of Trichoderma spp. and taped together by adhesive tape. In the control, Fusarium fujikuroi plates were removed and placed over another PDA plate without Trichoderma spp. All of the plates were incubated at 25 ± 1ºC for 7 days and growth inhibition percentage of test pathogen was recorded at regular intervals by comparing growth of Fusarium fujikuroi in control with Vol. 33, No. 2, Trichoderma treated plates using the following equation (Vincent, 1947). C T Percentage inhibition = X 100 C Where C = Colony growth in control (Fusarium fujikuroi) plates T = Colony growth in Trichoderma treated plates. Evaluation of non - volatile metabolites of Trichoderma species: The effect of non-volatile metabolites from Trichoderma species against Fusarium fujikuroi was studied following Dennis and Webster (1971a). Initially, mycelial agar plugs (4 mm diameter) were removed with a cork borer from the edge of a young culture of Trichoderma species and transferred to the center of Petri dishes (90 mm diameter) containing PDA, which is overlaid with a sterilized cellophane disc (Courtauld Films: 0.5 mm thick). After three days of incubation, the cellophane along with the inoculated Trichoderma was removed. On the same agar plate, the pathogen was inoculated where the antagonist was grown earlier on the cellophane. The control treatments had Fusarium fujikuroi growing similarly on PDA medium where previously there was a cellophane disc without antagonist. The set up was incubated at C for 72 hours. The growth rate was recorded at regular intervals by measuring colony diameter of Fusarium fujikuroi. The inhibition percentage was calculated by using the formula cited above. The studies on evaluation of volatile and non-volatile metabolites were conducted in three replicates and whole experiment was repeated twice. The average of the replicates is presented in the data. RESULTS AND DISCUSSION The soil samples taken in present study were of clay soil with ph ranging from 7 to 8. The Trichoderma population varied from 2 to 8.5 x 104 cfu /g of dry soil.the percentage occurrence of the Trichoderma species ranged from 40-50% (Table 1). Highest percentage of occurrence was recorded in soils from the Khammam district (50%) followed by Nalgonda (42.8%) and Kri shna (40%) respectively. A total of twenty-eight Trichoderma isolates were obtained. The identification was confirmed on the basis of morphological characters. A total of eighteen

3 106 AGRICULTURAL SCIENCE DIGEST TABLE 1. Occurrence of Trichoderma species in rice cultivated soils from three districts of Andhra Pradesh Place of soil collection Nalgonda Krishna Khammam Total no. of soil samples % occurrence of Trichoderma species species were recorded in this study and identified as T. atroviride Karsten (F2), T. aureoviride Rifai (F8), T. austrokoningii Samuels & Druzhinina (F20), T. caribbaeum var. caribbaeum Samuels & Schroers (F4), T. eucorticioides Overton (F27), T. harzianum Rifai (F3,7,22,23,25), T. intricatum Samuels & Dodd (F6), T. koningii Oudem (F5), T. koningiopsis Samuels, C. Suarez & H.C. Evans( F12), T. konilangbra Samuels, O. Petrini & Kubicek (F14), T. longibrachiatum Rifai (F1), T. minutisporum Bisset (F17,19), T. neokoningii Samuels & Soberanis (F26,28), T. parceramosum Bisset (F11,13,15), T. piluliferum Webster & Rifai (F16,18,21), T. pseudokoningii Rifai (F9), T. strictipilis Bisset (F10) and Trichoderma sp (F 24). T. harzianum is found to be common and dominant in our study and is followed by T. piluliferum and T. parceramosum. The species namely T. intricatum, T. koningiopsis, T. minutisporum, T. austrokoningii, T. neokoningii, T. eucorticioides, T. caribbeaum var caribbeaum, T. parceramosum are new records to India. The data revealed that out of twenty eight isolates, twenty isolates were recorded above 50% of growth inhibition of F. fujikuroi. The isolates namely, T. atroviride (F2), T. strictipilis (F10) and T. neokoningii (F28) showed highest growth rate and significantly inhibited the growth of the pathogen (Fig. 1). T. pseudokoningii (F9), T. parceramosum (F13) recorded least inhibition of the growth of the pathogen. The percentage of inhibition is ranged between The Trichoderma isolates showed over growth on the Fusarium fujikuroi in dual plates indicating the variation of species in controlling the growth of the pathogen. The effective twenty Trichoderma isolates i.e, F2, F3, F5, F7, F8, F10, F11, F15, F16, F17, F18, F19, F20, F21, F22, F23, F24, F25, F27 and F28 which showed above 50% of inhibition of test pathogen in the dual plate method were selected for further investigation to identify the production and inhibitory effect of volatile and nonvolatile compounds against the pathogen (Fig. 2). Out of twenty isolates, T. atroviride (F2), T. harzianum (F3), T. koningii (F5), T. strictipilis (F10), T. parceramosum (F15), T. harzianum (F23), T. eucorticioides (F27) and T. neokoningii (F 28) showed the inhibitory effect of the pathogen by producing the volatile compounds. Similarly, the T. strictipilis (F10), T. parceramosum (F11), T. FIG.1. Screening of Trichoderma isolates against Fusarium fujikuroi in dual plate method

4 Vol. 33, No. 2, FIG.2. Effective Trichoderma isolates in dual plate method tested for the presence of volatile and non volatile compounds piluliferum (F21), T. harzianum (F22) showed the effect of non volatile compounds. The growth inhibition of the pathogen varied up to 30.2% with volatile and up to 25% with non-volatile metabolites respectively. Of all the isolates, the isolate T. strictipilis (F10) recorded the highest percentage of inhibition in all treatments. In the present study, the isolates which are able to produce volatile compounds were unable to produce non-volatile metabolites. Prakkola (1992) and Harman (2000) also observed an array of mechanisms by which Trichoderma Spp exerts biocontrol activity and thus they show strain or species variation in controlling the pathogen. The species or strain, which showed maximum antibiosis with non volatiles, may not produce volatiles and vice versa. The isolates T. atroviride (F2) and T. neokoningii (F28) showed complete over growth on the pathogen in the dual plate with 28 and 30.2% growth inhibition of the pathogen with volatile metabolites respectively, whereas with non-volatile metabolites only 3% of inhibition was recorded. In the present study intra species and inter species variation was clearly observed in controlling the growth of the test pathogen as well as in the formation of secondary metabolites. Ghisalberti et al., (1990) too stated that the production of secondary metabolites by Trichoderma spp is strain dependent and includes antifungal substances belonging to a variety of classes of chemical compounds. Deacon (2006) reported that the differences in antibiotic production between the Trichoderma strains led as commercial biological control agents against plant pathogenic fungi. Although higher rate of growth inhibition was found in many isolates namely F11, F16, F17, F19, F20, F24 in dual plate, these results were not correlated with volatile and non-volatile metabolite production. The isolate T. austrokoningii (F20) showed no effect of volatile and nonvolatile metabolites on the growth of the test pathogen but inhibited pathogen growth by more than 60% in dual plate. It clearly elucidated that the inhibition of the pathogen growth was controlled by different mechanisms in dual plate, which has to be investigated. CONCLUSION In this preliminary study, the effective antagonistic Trichoderma isolates selected in dual plate studies indicated that some isolates showed either the efficacy of volatile or non-volatile compounds in inhibiting the pathogen whereas in some isolates it was not recorded. The isolate (F20) is effective in controlling the growth of F. fujikuroi in dual plate is unable to produce either of the metabolites. It clearly indicates that some other mechanisms are operating in antagonizing the test pathogen. Our study demonstrated that there is a

5 108 AGRICULTURAL SCIENCE DIGEST variation in the effect of volatile and non volatile metabolites when compared with the results obtained on the growth of test pathogen in dual plate. It is essential to identify the involvement of other mechanisms in the biocontrol of the pathogen Fusarium fujikuroi with the effective Trichoderma isolates as an array of mechanisms are effecting the growth of the test pathogen. ACKNOWLEDGEMENT The authors are thankful to the UGC, New Delhi for providing financial assistance through Minor research project. REFERENCES Aneja K R. (2003). Experiments in Microbilogy plant pathology and Biotechnology,New Age International Pvt.Ltd, India, 607 pp. Bisset J. (1991b). A revision of the genus Trichoderma III.Sect. Pachybasium, Can. J. Bot, 69: Bisset J. (1991a). A revision of the genus Trichoderma II. Infrageneric classification, Can. J. Bot, 69: Bisset J. (1991c). A revision of genus Trichoderma IV. Additional notes on section Longibrachiatum, Can. J. Bot, 69: Bisset J. (1992). Trichoderma atroviride, Can. J. Bot, 70: Bisset J. (1984). A revision of the genus Trichoderma I. Sect. Longibrachiatum sect. nov, Can. J. Bot, 62: Brotman Y, Kapuganti J G and Viterbo A. (2010). Trichoderma. Curr Biol, 20(9) R Deacon J. (2006). Fungal interactions; Mechanisms and Practical exploitation,in Fungal biology. 4th Edition, Blackwell publishing ltd, 240 pp. Dennis C and Webster J. (1971). Antagonistic properties of species group of Trichoderma I. Production of non-volatile antiobitics. Trans Bri. Mycol Soc, 57: Dennis C and Webster J. (1971c). Antagonistic properties of species group of Trichoderma II. Production of volatile antibiotics. Trans. Bri. Mycol. Soc, 57: Dhingra O D and Sinclair J B. (1985). Basic Plant Pathology Methods. CRC Press, Boca Raton, Florida. 859 pp. Elad Y and Chet I. (1983). Improved media for isolation of Trichoderma SPP. OR Fusarium SPP. Phytoparasitica, 11 (1) : Ghisalberti E L, Narbey M J, Dewan M M and Sivasithamparam K. (1990). Variability among strains of Trichoderma harzianum in their ability to reduce take -all and to produce pyrones, Pl Soil, 121: Harman G E. (2000). Myths and dogmas of biocontrol changes in perceptions derived from research on Trichoderma harzianum T 22. Plant Dis, 84: Howell C R. (2003). Mechanisms employed by Trichoderma spp. in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis, 87: Janaiah A, Singh M and Hassain M. (2002). Constraints to sustaining rice yield in irrigated system \ an assessment of farm level experiences in Andhra Pradesh, India.in constraints to rice production in Asia: insights from farmer s perception, IRRI Philippines EDS Hossain M and others. Lewis J A and Papavizas G C. (1987). Application of Trichoderma and Gliocladium in alginate pellets for control of Rhizoctonia damping off. Pl Pathol, 36: Mathre D E, Cook R J and Challan N W. (1999). From discovery to use; Traversing the world of commercializing bio control agents for plants disease control, Plant Dis 83: Nagamani A, Kunwar I K and Manoharachary C. (2006). Handbook of Soil Fungi. I.K. International Pvt. Ltd, New Delhi. 477 pp. Naseby D C, Pascual J A and Lynch J M. (2000). Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum populations, soil microbial communities and soil enzyme activities. J Appl. Micro, 88: Ou S H. (1985). Rice Diseases. 2 nd ed. Commonwealth Mycological Institute, Kew, Surrey, England. 380 pp. Prokkala S. (1992). Antagonistic properties of Trichoderma species against Mycocentrospora acerina, Bull OlLB Srop, 15: Samuels G J, Dodd S, Lu B S, Petrini O, Schroers H J, Druzhinia IS. (2006). The Trichoderma koningii aggregate Species. Studies in Mycology, 56 : Sivan A and Chet I. (1986). Biological control of Fusarium spp. in cotton, wheat and muskmelon by Trichoderma harzianum. J Phytopathol, 116: Vincent J H. (1947). Distortion of fungal hyphae in the presence of certain inhibitors. Nature, 15: 850.