Antagonistic Activity of Trichoderma viride and ...

J. Soil Biol. Ecol. 34 (1&2) : 60-69, 2014

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Antagonistic Activity of Trichoderma viride and Pseudomonas fluorescens Isolated From Bt and Non Bt Cotton Rhizosphere Against Rhizoctonia solani M. SIVAJI* and R. SRIDAR Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India Email: [email protected] Received: 27-01-2014; Accepted: 12-05-2014

Abstract Bt cotton which confers resistance to important insect pests of cotton, occupy around 86% of total cotton area in India. This study was conducted to check whether Bt protein released by Bt cotton affect the population of Trichoderma viride and Pseudomonas fluorescens and their antagonistic efficiency against Rhizoctonia solani. The isolates were collected at different intervals from the Bt and not Bt cotton rhizosphere i.e., 15, 30 and 45 DAS. The rhizosphere population of the tested microbes Trichoderma viride and Pseudomonas fluorescens did not significantly differ between Bt and non Bt cotton. The efficiency of these antagonistic strains was evaluated in vitro by dual plate method. The result showed that some variation was there in the pathogen inhibition percentage between isolates obtained from Non Bt and Bt cotton rhizosphere. Some of the isolates obtained from Bt rhizosphere showed better inhibition than its counterpart. However, the efficiency did not significantly vary between Bt and Non Bt isolates indicating, Bt protein does not have any impact on microbes of the cotton rhizosphere.

Introduction GM plants have the potential to significantly change the microbial dynamics and essential ecosystem functions such as nutrient mineralization, disease incidence, carbon turnover and plant growth through the products of introduced genes or modified rhizosphere chemistry or altered crop residue quality (Gupta et. al., 2000). A decrease in specific microbial populations would lead to a decrease in decomposition processes and have secondary effects on plant pathogen survival and build up, as well as soil organic matter level and composition (Termorshuizen and Lotz, 2002). However, little experimental data are available on the consequences of plant-microbe-soil interactions due to the sustained expression and/or presence of Bt toxin in the rhizosphere. Bt cotton, which confers resistance to important insect pests of

Antagonistic Activity of Trichoderma and Pseudomonas Against Rhizoctonia

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cotton was first adopted in India in 2002. From 2002 onwards Bt cotton area increased drastically and it reached 8.4 million hectares (occupied around 86% of total cotton grown area in India) in the year of 2009 (Choudhary and Gaur, 2010). The rhizosphere (the zone directly surrounding and influenced by plant roots) contains a large majority of the soils biota populations (>10-fold of that in the bulk soil) and the plant-microbe interaction in the rhizosphere is one of the major factors regulating the health and growth of plants. It is also widely acknowledged that root exudates govern the organisms in the rhizosphere (Lynch, 1994; Bardgett et. al., 1999). The exudates of Bt plants are found to be rich in endotoxin i.e., Cry protein. The well-justified caution that has been with regards to the introduction of Bt cotton and the development of insect resistance should also be applied to the preservation of soil sustainability to protect the soils biological function and diversity. Rhizoctonia solani is a plant pathogenic fungus with a wide host range and worldwide distribution. It is one cause of the condition known as damping off resulting in the death of seedlings. Biocontrol of soil-borne plant pathogens affecting agricultural plants can be controlled by the use of species of Trichoderma i.e., Trichoderma harzianum, Trichoderma viride and Trichoderma hamatum, and some antagonistic bacteria like Pseudomonas fluorescens, Bacillus subtilis, Enterobacter aerogenes, Streptomyces spp. (Chet, 1990). The use of these antagonists for suppression of R. solani has been demonstrated by several workers (Ali and Nadarajah, 2013; Rini and Sulochana, 2006 and Bautista et. al., 2007). A study was conducted to check the effect of Bt protein on Trichoderma viride and Pseudomonas fluorescens on the population and its antagonistic effect against R. solani in cotton.

Material and Methods Preparation of inoculum and seed treatment: The cultures of Trichoderma viride and Pseudomonas fluorescens (biocontrol agents) for cotton and R. solani (plant pathogenic fungus) were obtained from Plant Pathology department, TNAU for the present research. Trichoderma viride discs were transferred to Potato Dextrose Broth and kept in room temperature for 4 Days. Pseudomonas fluorescens was inoculated in KB broth, kept for incubation on incubator cum shaker for 72 hours at 32 0C. After three days the T. viride mat formed on the surface of the medium was homogenized and these microbial cultures were used to treat the RCH-2 (Non-Bt) and RCH-2 BG II (Bt cotton) seeds. Seeds were mixed with microbial culture containing 10

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CFU/ml culture. Bt and non Bt cotton seeds were obtained from Rasi Seeds Pvt. Ltd., Attur, Tamil Nadu. Cotton seeds were surface sterilized for 2 min with 70% ethanol followed by 2% sodium hypochlorite (10 min) and rinsed in sterile distilled water. A Quantity of 10 ml of culture

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M. SIVAJI and R. SRIDAR

was mixed with 0.2 g of CMC (2 %) to treat 5 gram each Bt and Non-Bt cotton seeds. Seeds were mixed inocula and kept for incubation for half an hour. After incubation treated seeds were shade dried for half an hour before sowing in pots. Biocontrol agents with Rhizoctonia solani: Completely Randomized Design (CRD) was adopted for pot culture study. A total of 6 treatments (Table 1) were chosen for the pot culture with four replications for each treatment. Bt and Non Bt cotton seeds treated with Trichoderma viride and Pseudomonas fluorescens was sown in the presence of plant pathogen Rhizoctonia solani (causal agent for root rot in cotton). The plastic pots (12 cm dia.) filled with sterilized clay loam soil were inoculated with one-week-old culture of R. solani, prepared on corn meal sand medium as described by Mathew and Gupta (1998) @ 2g/kg soil and allowed to stabilize for 5 days, then surface sterilized seeds were sown@ 2 seeds per pot. Trichoderma viride and P. fluorescens were isolated from Bt and Non Bt cotton rhizosphere at 15 Days interval i.e., 15, 30 and 45 DAS. This experiment was conducted in completely randomized design with four replications and the data were subjected to analysis of variance with 5 percent probability. Analysis of the antagonistic activity of Trichoderma viride and P. fluorescens: All the Trichoderma viride and P. fluorescens isolates obtained at 15 days intervals were evaluated for their antagonistic activity against the pathogen under in vitro conditions. The assay for antagonism was performed on Potato Dextrose Agar (PDA) adopting dual culture method (Dennis and Webster, 1971). A 5 mm diameter disc of Trichoderma viride was placed individually at one end of the Petri plate containing PDA and just the opposite end a 5 mm diameter disc of the pathogen (R. solani) was placed. In control, only pathogen was inoculated and incubated at 28± 2o C temperatures in BOD incubator. Each treatment was replicated thrice. Inhibition of R. solani growth was recorded after 72 h. A 5mm of mycelia agar disc from test fungal pathogen cultures was placed on the one side of a Petri plate containing PDA: KB (1:1, v/v) media. The plates were then incubated at 250C for 24 hrs. A loopful of test antagonistic bacterial culture was streaked on the opposite side of disc of pathogen on the same dish. PDA: KB (1:1, v/v) media plates inoculated only with pathogen were maintained as control. The zone of inhibition was recorded as the distance between the fungal pathogen and the area of antagonist growth after 72 h. The percentage inhibition of the growth of pathogen was calculated with the help of the formula given by Whipps (1997). This experiment was conducted in completely randomized design with three replications and the data were subjected to analysis of variance.


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CT PI =

__ x 100 C

where, PI = Percent inhibition of mycelial growth C = Radial growth of pathogen in control plates (mm) T = Radial growth of pathogen in dual culture (mm)

Results and Discussion The population of Trichoderma viride and P. fluorescens recovered from the soil in Bt and Non Bt cotton rhizosphere in the presence of R. solani at three different sampling times was estimated. The fungal and bacterial count in rhizosphere soil was determined after 15, 30 and 45 DAS (Table 2). The recovered populations of both biocontrol agents (Trichoderma viride and P. fluorescens) were high on 30 DAS in Bt and Non Bt cotton rhizosphere. Biocontrol agent P. fluorescens population was high in Bt cotton (8.34 Log 10 CFU. g-1) and Non Bt cotton rhizosphere (8.20 Log10 CFU. g-1) at 30 DAS rhizosphere soil samples. T. viride population was slightly higher i.e., 4.84 Log10 CFU. g-1 in non Bt cotton and 4.81 Log10 CFU. g-1 in Bt cotton of 30 DAS. Population variation was observed at all intervals in both Bt and non Bt cotton rhizosphere soil samples. However, statistical analysis showed that there was no significant difference in the beneficial microbial population in Bt and Non Bt cotton rhizosphere soil samples analyzed at different intervals. Inhibition percentage of T. viride and P. fluorescens isolated from different stages (15 DAS interval) of Non Bt and Bt cotton rhizosphere were tested for their efficacy against Rhizoctonia solani by dual culture method. The details of inhibition percentage are given in Table 3. T. viride isolated from Bt cotton rhizosphere at 15 DAS recorded the highest inhibition (62.8%) compared to T. viride obtained from Non Bt cotton (61.1%). P. fluorescens registered the highest inhibition of 51.6% and 50.4% from Non Bt and Bt cotton respectively at 45 DAS. Inhibition percentage was high in Bt cotton T. viride isolates at all sampling times than non Bt cotton isolates. In vitro condition inhibition pattern varied between isolates recovered from Bt and Non Bt cotton rhizosphere but the variation was not statistically significant.

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There has been strong debate on the safety of genetically modied plants ever since the introduction onto the market of plant products deriving from transgenic crops. This debate is still very much alive, and several issues have been raised, including the safety of transgenic food and the environmental impact of transgenic plants. The res of the debate are mainly fed by the insufficient knowledge available on biological systems, and by the potential danger that specic genetic manipulations could give unexpected effects (Saxena et. al., 2002). The rhizosphere environment hosts a complex microorganism network which interacts very closely with plant roots; the outcome of such interaction being seen in the strong inuence the plant type has on the microbial ecosystem of the soil. On considering the different exudates pattern of Bt cotton with respect to non transgenic cotton, we were prompted to evaluate whether this unexpected characteristic could affect the T. viride and P. fluorescens, in terms of abundance and functional activity. T. viride and P. fluorescens population variation between Bt and the Non Bt cotton rhizosphere was not significant in the present study. The results are in agreement with a previous study of Saxena and Stotzky (2001) who did not nd signicant differences in the colony-forming units of culturable bacteria, actinomycetes, fungi, protozoa and nematodes in the rhizosphere of transgenic Bt and non transgenic maize. Koskella and Stotzky (2002) conrmed that several Cry toxins from B. thuringiensis have no microbicidal or microbiostatic activity against selected bacteria. Brusetti et. al. (2005) detected no differences in the rhizosphere bacterial communities between transgenic Bt 176 maize and its non-transgenic counterpart. Petras and Casida (1985) observed slight increases in populations of bacteria, actinomycetes, fungi, and nematodes after the addition of B. thuringiensis subsp. kurstaki to the soil. A significant but transient increase in the populations of culturable bacteria and fungi was observed in soil with leaves of Bt cotton (Gossypium hirsutum L.) expressing the Cry1Ac protein (Donegan et. al. 1995). Rui et. al. (2005) found higher numbers of functional bacteria in the rhizosphere soil of Shiyuan321 (non-Bt cotton) than NuCOTN99B (Bt cotton); after adding pure Bt toxin to soil and also indicated that Bt toxin may not directly affect the numbers of functional bacteria in the rhizosphere. Inhibition of R. solani in this study ranged from 59.2 to 62.8 and 49.4 to 51.6 by T. viride and P. fluorescens respectively. Similar results were obtained for suppression of R. solani with the use T. viride and P. fluorescens by several research workers (Chaube and Sharma, 2002; Rini and Sulochana, 2007; Singh et. al., 2008; Dev and Dawande, 2010). However no significant difference was observed between Bt and non Bt cotton. In the present investigation there was no significant difference in the antagonistic activity of T.viride and P. fluorescens recovered from Bt and non Bt cotton rhizosphere at different intervals. This might be due to null impact of Bt protein on


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the population level of biocontrol agents. So the recovered population and inhibition per cent result showed that Bt protein did not have any adverse action on the biocontrol efficacy of these antagonists with in the period of 45 days.

Acknowledgement The authors are thankful to Department of Biotechnology, Government of India, New Delhi for the financial assistance to carry out this work.

References Ali, H.Z. and Nadarajah, K., 2013. Evaluating the efficacy of Trichoderma isolates and Bacillus subtilis as biological control agents against Rhizoctonia solani. Res. J. Appl. Sci., 8: 72-81 Bardgett, R.D., Dentin, C.S. and Cook, R., 1999. Below-ground herbivory promotes soil nutrient transfer and root growth in grassland. Ecol. Lett., 2: 357-360 Bautista, G., Mendoza, H. and Uribe, D., 2007. Biocontrol of Rhizoctonia solani in native potato (Solanum phureja) plants using native Pseudomonas fluorescens, Acta. Bioi. Colomb., 12: 19-32 Brusetti, L., Francia, P., Bertolini, C., Pagliuca, A., Borin, S., Sorlini, C., Abruzzese, A., Sacchi, G., Viti, C., Giovannetti, L., Giuntini, E., Bazzicalupo, M. and Daffonchio, D., 2005. Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart. Plant Soil, 266: 11-21 Chaube, H.S. and Sharma, J., 2002. Integration and interaction of solarization and fungal and bacterial bioagents on disease incidence and plant growth response of some horticultural crops. Pl. Dis. Res., 17: 201 Chet, I. 1990. Biological control of soil borne plant pathogens (Ed) Hornby, D., CAB International, Wallingford, UK, pp. 274-277 Choudhary, B. and Gaur, K., 2010. Bt Cotton in India: A Country Profile. ISAAA Series of Biotech Crop Profiles. ISAAA: Ithaca, NY. Dennis, C. and Webster, J., 1971. Antagonistic properties of species groups of Trichoderma III, hyphae interaction. Trans. Br. Mycol. Soc., 57: 363-369


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Dev, N. and Dawande, A.Y., 2010. Biocontrol of soil borne plant pathogen Rhizoctonia solani using Trichoderma spp. and Pseudomonas fluorescens. Asiatic J. Biotech. Res., 01: 39-44 Donegan, K.K., Palm, C.J., Fieland, V.J., Porteous, L.A., Ganio, L.M., Schaller, D.L, Bucao, L.Q. and Seidler, R.J., 1995. Changes in levels, species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstaki endotoxin. Appl. Soil Ecol., 2: 111124 Gupta, V.V.S.R., Roberts, G. and Putcha, S., 2000. Soil health: The role of microbes in crop productivity. Proceedings of the 10th Australian Cotton Conference held during August 2000 at Brisbane, Australia. pp. 639-643 Koskella, J. and Stotzky, G., 2002. Larvicidal toxins from Bacillus thuringiensis subsp. kurstaki, morrisoni (strain tenebrionis) and israelensis have no microbicidal or microbiostatic activity against selected bacteria, fungi, and algae in vitro. Can. J. Microbiol., 48: 262-267 Lynch, J., 1994. The rhizosphere - form and function. Appl. Soil Ecol., 1: 193-198 Mathew, K. A. and Gupta, S. K. 1998. Biological control of root rots of French bean caused by Rhizoctonia solani. J. Mycol. Pl. Pathol., 28: 202-205 Petras, S.F. and Casida, L.E., 1985. Survival of Bacillus thuringiensis spores in the soil. Appl. Environ. Microbiol., 50: 2835. Rini, C.R. and Sulochana, K.K., 2006. Management of seedling rot of chilli (Capsicum annuum L.) using Trichoderma spp. and fluorescent pseudomonads (Pseudomonas fluorescens). J. Trop. Agric., 44:7982 Rini, C.R. and Sulochana, K.K., 2007. Usefulness of Trichoderma and Pseudomonas against Rhizoctonia solani and Fusarium oxysporum infecting tomato. J. Trop. Agric., 45: 2128 Rui, Y.K., Yi, G.X., Zhao, J., Wang, B.M., Li, Z.H., Zhai, Z.X., He, Z.P. and Li, Q.X., 2005. Changes of Bt toxin in the rhizosphere of transgenic Bt cotton and its influence on soil Functional bacteria. World J. Microbiol. Biotechnol., 21: 1279-1284 Saxena, D. and Stotzky, G. 2001., Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem., 33: 1225-1230


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Saxena, D., Flores, S. and Stotzky, G., 2002. Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events. Soil Biol. Biochem., 34: 133137 Singh, S., Chand, H. and Varma, P.K., 2008. Screening of bioagents against root rot of mung bean caused by Rhizoctonia solani. Legume Res., 31:75 - 76 Termorshuizen, A.J. and Lotz, L.A.P., 2002. Does large-scale cropping of herbicide-resistant cultivars increase the incidence of polyphagous soil-borne plant pathogens? Outlook Agr., 31: 51-54 Whipps, J.M., 1997. Developments in the biological control of soil borne plant pathogens. Adv. Bot. Res., 26, 1-34

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Table 1 : Details of different treatments used for pot culture

Table 2 : Microbial population count from rhizosphere soil of Bt cotton and non Bt cotton at 15 days interval in presence of R. solani

Values are (averages of four replication) given in Log 10 CFU g-1dry soil D-days, O-microorganism, C- cotton variety


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Table 3 : In vitro evaluation of recovered biocontrol agents from Bt and non Bt rhizosphere soil against Rhizoctonia solani

D-days , O-microorganism, C- cotton variety