Biocontrol potential of Trichoderma Sp. against plant pathogens

Forty two strains of Trichoderma sp. were isolated from cultivated lands around Bangalore and analyzed for their antagonistic potential against Sclerotium rolfsii and Fusarium ciceri. The potential of biocontrol agents ultimately lies in their capacity to control pathogens in vivo. Bioefficacy studies were hence conducted using chickpea (Cicer argentums c.v. Annigeri) as an experimental plant by the roll paper towel method. Overall the isolates T40, T35, T30 and T25 showed better antagonistic potential in addition to enhancing plant growth. The production of chitinases to break down the mycelial cell walls of fungal plant pathogens has been implicated as a major cause of biocontrol activity (Inbar and Chet, 1995). In order to study the mechanism of biocontrol, ten better performing strains were plated on media, amended with colloidal chitin and Sclerotium rolfsii cell wall extract. All the isolates showed chitinolytic activity on day three as well as day five. Production of endochitinase and exochitinase were assayed in liquid media using colloidal chitin amended broth. Strains T35 and T6 displayed maximum endochitinase and exochitinase activity. Although all strains exhibited cellulase activity, the quantum of enzyme produced was higher in T35 and T6. The results also indicate a positive correlation between enzyme production and bioefficacy. Keywordsbiocontrol, bioefficacy, cellulase, chitinase Introduction The fungal pathogens play a major role in the development of diseases on many important field and horticulture crops; resulting in severe plant yield losses. Intensified use of fungicides has resulted in accumulation of toxic compounds potentially hazardous to humans and environment and also in the buildup of resistance of the pathogens. In order to tackle these national and global problems, effective alternatives to chemical control are being employed [9]. Biological control is a nature friendly approach that uses specific microorganisms, which interfere with plant pathogens and pests to overcome the problems caused by chemical methods of plant protection. Commercial preparations of plant disease biocontrol agents are based on the practical application of rhizosphere competent species of bacteria or fungi. Although biological control occurs naturally, and is the principal reason diseases are not usually catastrophic, sufficient knowledge in many cases is not available to explain how biological control operates or how abiotic and biotic factors can be manipulated to effect the economic control of a pathogen [17]. Fungi in the genus Trichoderma are among the most promising biocontrol agents against plant pathogenic fungi. Specific strains have the ability to control a range of pathogens under a variety of environmental conditions. Moreover, they may be rhizosphere competent which allows them to colonize and protect plant roots. Among the action mechanisms proposed is mycoparasitism, with concomitant production of enzymes that degrade cell walls. Chitinolytic enzymes, together with ß-glucanase or cellulases are the enzymes most frequently considered critical in biocontrol. In addition, chitinolytic enzymes may be important industrially for decomposing chitinous wastes from Shellfish [11]. In spite of enormous scientific research on biological control of plant pathogens with Trichoderma sp., the most effective species against a wide range of pathogens is yet to be identified. With this in view, the present investigation was undertaken to examine the efficacy of selected Trichoderma isolates against common soil borne fungal pathogens of chickpea. Methodology Dual Plating Forty two isolates of Trichoderma (T1 to T42) from rhizosphere soil collected around Bangalore were plated in replicates against the pathogens Fusarium ciceri and Sclerotium rolfsii to test their antagonistic potential on Potato Dextrose Agar (PDA). The colony diameters of the pathogens and the antagonists, pigmentation and overgrowth of either organism if any, were recorded periodically. Bioefficacy (Roll Paper Towel method) Seedling vigor testing, by the Roll Paper Towel Method (ISTA, 1976) was used for testing bioefficacy of all Trichoderma isolates. Pathogens Fusarium ciceri and Sclerotium rofsii, known to cause wilt, stem rot, respectively in Chickpea (Cicer arietinum cv. Annigeri) and other commercially important crops were grown and maintained on PDA plates. Surface disinfected seeds were first inoculated with mycelial suspension of pathogens followed by various talc preparations of Trichoderma isolates separately. In one treatment, fungicide (Captan at 2.5g/kg seeds) was used to treat seeds without any of the bioagents. Seeds treated with pathogen mycelium alone served as a check. For each treatment two replicates were maintained. Observations on seed germination Biocontrol potential of Trichoderma Sp. against plant pathogens International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 31 and disease incidence were recorded after 10 days of incubation by following the method of Abdul-Baki and Anderson (1973) [1]. A disease grading key as described by Srivastava et al., 2002 [20] was followed to rate the efficacy of bicontrol agents, based on seed rotting and infection occurring on roots and shoots. Screening of Trichoderma isolates for chitin utilization Ten strains were selected based on performance in dual Plating and in vivo bioefficacy. They were screened for their capacity to grow on colloidal chitin amended solid medium (CCAM) and S. rolfsii cell wall amended solid medium (SrCWAM). The amended media contained 0.2% colloidal chitin and Sclerotium rolfsii cell wall chitin respectively. The cultures were gown on Potato Dextrose Agar also for comparison. The colony growth was measured as colony diameter on the 3 rd and 5 th day after inoculation. For inoculation of isolates, mycelial discs of 48 hr. old cultures were used. Assay of chitinases in liquid cultures The crude culture filtrates of the selected ten Trichoderma isolates grown on colloidal chitin amended broth for seven days were used for the assay of endochitinase and exochitinase activity. The assay mixture contained 0.5ml of 0.2% colloidal chitin, 0.5ml of enzyme solution (crude culture filtrate) and 0.5ml of 0.1M citrate buffer (pH 5.1). The reaction mixture was incubated for 4-6hrs at 37°C in a water bath. The reaction was stopped by centrifugation (5000 rpm for 10 mins). 0.5ml of the reaction mixture was taken, to which 0.1ml of 0.2M Potassium tetra borate buffer (pH 9.2) was added, followed by boiling in a water bath for 3 mins. The reaction mixture was then cooled and 5ml of p-dimethylaminebenzaldehyde (DMAB) solution was added to it. This was incubated in a waterbath at 37°C, for 20mins and cooled prior to recording absorbance. Similarly, blanks were prepared for each sample in which the samples were boiled to inactivate the enzyme activity and rest of the treatments was performed as previous. The absorbance was recorded at 585 nm. Assay of Exochitinase The assay mixture contained 0.5ml of 0.2M Sodium acetate buffer (pH 4.7), 0.3ml of enzyme solution (crude culture filtrate) and 0.2ml of 0.5mM p-Nitrophenyl nacetyl β-D-glucosaminide (pNP). Blanks were prepared for each sample in which the samples were boiled to inactivate the enzyme activity and rest of the treatments was performed as previous. The reaction mixture was incubated for 4-6hrs at 37°C, in a water bath and the absorbance was read at 400nm. Assay of Cellulases The crude culture filtrates of the ten Trichoderma isolates grown on Carboxy Methyl Cellulose Amended Broth were used for the assay of Cellulase activity. Substrate 1% carboxy methylcellulose was prepared in 0.1M Sodium Citrate buffer. To 0.45 ml of this substrate, 0.05 ml of enzyme extract (culture filtrate) was added and incubated at 55 0 C for 15 minutes. To this 0.5 ml of DNS reagent was added, mixed well and boiled on a water bath (100 0 C) for exactly 5 minutes. After incubation 1 ml of 40% Rochelle’s salt solution was added. This was cooled to room temperature & made upto 5 ml with distilled water. Controls were made by following the same procedure as described above, except that, the tubes were boiled after adding enzyme extract to inactivate the enzyme. The intensity of yellow colour was measured at 540ηm against blank. The results were expressed as μM ml -1 of culture filtrate. Discussion In recent years, research on biological control has gained momentum for controlling serious soil born plant pathogens like Fusarium, Rizoctonia, Macrophomina, Sclerotium, Pythium and Phytophthora spp, employing Trichoderma and Gliocladium species and varied success has been achieved.[7,19,18,14]. In the current study investigations were carried out on the forty two Trichoderma strains, isolated from rhizosphere soil samples from cultivated lands around Bangalore, for their biocontrol potential against the pathogens Sclerotium rolfsii and Fusarium ciceri. All strains were subjected to dual plating on Potato Dextrose Agar, against two pathogens viz, F. ciceri and S. rolfsii. Observing the zone of inhibition at the point of contact of pathogen and the antagonist and measuring their colony diameter on the third and fifth day after inoculation, served as an indicator of their in vitro biocontrol activity. The percentage reduction of growth over control for the pathogen F. ciceri, was calculated and the isolate T40 (153.892%) was found to be the most effective on the third day after inoculation. On the fifth day of inoculation, T40 (186.173%) followed by T27 (152.099%) were the best performers. The lowest percentage reduction of F. ciceri over control was recorded by T38 on the third day (9.98%) and on the fifth day (12.346) (Table 1). Against S. rolfsii, T30 (66.05%) and T6 (63.19%), showed maximum antagonistic activity on the third day of incubation. On day five, maximum percentage reduction of the pathogen growth over control indicated T30 (43.22%) and T6 (40.0%) to be the best performers while T13 (-41.22%) recorded the least (Table 2). Similar antagonistic ability of Trichoderma isolates against the plant pathogen S. rolfsii, indicated by formation of lytic zones along the point of contact is corrobated by Haran Anand S and Jayarama Reddy Copyright © 2009, Bioinfo Publications, International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 32 et al, 1993 [10]. The potential of biocontrol agents ultimately lies in their ability to control plant pathogens in vivo. Therefore, the isolates were tested for their bioefficacy in vivo, using RollPaper Towel method, with Chickpea (Cicer arietinum cv. Annigeri), as the experimental plant. High percentage of germination was observed in the isolates T40, T35, T30 and T25, and the disease incidence was found to be nil, compared to the pathogen check. A disease grading key, was used for rating efficacy of the bioagents used, based on the seed rotting, infection occurring on roots and shoots. All bioagent treatments and fungicide treatments recorded significantly less disease incidence, compared to the pathogen check (Tables 3 & 4). These results of improved plant growth are well supported by earlier works of Abdul-Baki and Anderson, 1973; and Srivastava et al, 2002[ 1, 20]. The mechanism of biocontrol activity is a subject of great curiosity. The production of chitinases, to breakdown the mycelial cell wall of the fungal plant pathogens have been implicated as a major cause of biocontrol activity. Both endochitinases and exochitinases have been considered to be responsible for the biocontrol activities exhibited. Inbar and Chet, 1995 indicated that the production of these enzymes by species of Trichoderma might be associated with their antagonistic behaviour against other soil fungi. Studies conducted by de la Cruz et al. (1993)[6]; and Lorito et al. (1996)[16] showed that the endochitinases were among the most effective for both antifungal and lytic activities when compared with other chitinolytic enzymes. On the solid media, amended with colloidal chitin and S. rolfsii cell wall extract, all of the strains exhibited strong chitinolytic activity, as determined by the formation of clearing zones on the third as well as the fifth day (Table 5). The results obtained indicate a positive correlation between chitinolytic activity and bioefficacy. These findings however, differ from the results obtained by Cotes et al., (1994) [5], which showed that the protective ability of Trichoderma isolates was unrelated to the in vitro enzyme activities in culture filtrates, regardless of the carbon source used. Further, endochitinase produced by isolates T35, T6 and T7 and exochtinase produced by isolates, T35, T6 and T30, also associate well with their bioefficacy [Figs. (1) & (2)]. Production of exochitinase by efficient Trichoderma isolates, for biocontrol of Rhizoctonia solani, which causes root rot of Soya bean, has been observed and documented by Bertagnolli, Dal Soglio and Sinclair (1995) [2]. The above observations indicate a strong correlation between bioefficacy and chitinase production, which is consistent with the findings of Elad et al. (1982)[8], Cook and Baker (1983) [4], Chet, I. (1987, 1990) [3], Inbar and Chet (1995) [12], Haran et al., (1996) [10], Krishnamurthy et al., (1999) [15], and StephenJebakumar et al., (2000) [13]. All strains exhibited cellulolytic activity. The activity was seen to be high in T35 followed by T6. T27 being a moderate performer in dual plating and bioefficacy studies, recorded high cellulolytic activity [Fig. (3)]. However, a positive correlation was seen between the cellulolytic activity and in vivo bioefficacy for the isolate T35. In the present study the Trichoderma isolates, T35 and T6, stand out with respect to their biocontrol potential followed by T25 and T30. However, these isolates must be evaluated under the full range of conditions that are experienced by the farmers. Field trials need to be conducted across different agro-ecological zones to utilize the potential exhibited by them. Bibliography [1] Abdul-Baki A. A. and Anderson J. D. (1973) Crop Sci, 13: 63-633. [2] Bertagnolli B. L., et al., (1995) Physiological and Molecular Plant Pathology, 48: 145160. [3] Chet I. (1990) In: Biological control of soilborne plant pathogens, D. Hornby(Ed.); Wallingford : CAB International. [4] Cook R.J. and Baker K.F. (1983) Amer. Phytopathol. Soc., Minnesota, 539. [5] Cotes A., Thonart P. and Lepoivre P. (1994) Med. Fac. Landbouww. Univ. Gent. 59 : 931 – 941. [6] De La Cruz. J., Rey M., Lora J.M., HidalgoGallego A., Domingrrez F. and PintorToro J.A. (1993) Arch. Microbiol. 159: 316 – 322. [7] Dwivedi, R., (1984) Indian Journal of Agricultural Sciences. 54: 513-514. [8] Elad Y., Chet I. and Henis Y. (1982) Can. J. Microbiol. 28: 719 – 725. [9] El-Katatny M. H., Somitsch W., Robra K. H., El-Katatny M. S. and Gubitz G. M. (2000) Food Technol. Biotechnol. 38(3): 173-180. [10] Haran S., Schickler H., Oppeheim, A.B. and Chet I., (1995) Mycol. Res. 99: 441 – 446. [11] Harman G.E., Hayes C.K., Lorito M., Broadway R.M., Di Pietro A., Peterbauer C. and Tronsmo A., (1993) Phytopathology 83: 313-318. [12] Inbar J. and Chet I. (1995) Microbiology 141: 2823 – 2829. [13] Jebakumar R.S., George G. L., Babu S., Raghuchander T., Gopalaswamy G. Vidhyasekharan P., Samiyappan R. and Raja J. A. J. (2000) Indian Phytopathology 53: 2, 157 – 161. [14] Jeyarajan, R., Ramakrishanan, G., Dinakaran, D. and Sridar, R. (1994) In: Dwivedi, R (Ed.) Biotechnology in India. Bioved research Society, Allahabad, 2536. Biocontrol potential of Trichoderma Sp. against plant pathogens International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 33 [15] Krishnamurthy J., Vidhyasekharan, P., Samiyappan, R., Nakkeeran, S., Rajeswari, E. and Raja, J. A. J., (1999) Journal of Biosciences, 24(2): 207 – 213. [16] Lorito M., Peterbauer, C., Sposato, P., Mach, R. L., Strauss, J., and Kubicek, C. P., (1996) Proc. Nat. Acad. Sci(USA) 93: 14868 – 14872. [17] Milton N. Schroth and Joseph G. Hanock. (1981) Annual Review of Microbiology, vol 35: 453-476. [18] Raghuchander T., Samiyappan R. and Arjunan G. (1993) Indian Phytopathology, 46: 379-382. [19] Singh D. (1991) Tropical Pest Management, 37: 374-378. [20] Srivastava R. K., Prasad R. D., Rangeshwaran R., Wasnikar A. R., Singh S. P. and Rao N. S. (2002) Journal of Biological Control, 16(2): 173176. Anand S and Jayarama Reddy Copyright © 2009, Bioinfo Publications, International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 34 Table 1: Dual plating of Trichoderma against F. ciceri Culture 3rd day 5th day T Fc Fc (% reduction over control T Fc Fc (% reduction over control T1 10.58 8.54 40.719 10.6 9.12 36.543 T2 10.57 5.09 109.381 10.64 5.2 134.321 T3 5.9 3.53 47.305 6.2 4.2 49.383 T4 6.45 4.56 37.725 6.7 4.79 47.16 T5 5.67 3.94 34.531 6.12 4.2 47.407 T6 10.18 3.95 124.351 7.76 4.1 90.37 T7 7.69 5.36 46.507 7.9 5.9 49.383 T8 8.09 6.57 30.339 8.2 6.7 37.037 T9 7.7 4.56 62.675 7.775 4.6 78.395 T10 6.8 5.4 27.944 7 5.8 29.63 T11 7.6 5.31 45.709 7.8 5.9 46.914 T12 10.56 8.6 39.122 10.6 9 39.506 T13 9.56 7.34 44.311 9.6 7.59 49.63 T14 10.73 5.81 98.204 10.8 6.38 109.12 T15 9.09 6.59 49.9 9.1 7.14 48.395 T16 7.78 6.36 28.343 7.8 6.4 34.568 T17 7.73 5.98 34.93 7.8 6.2 39.506 T18 7.23 4.84 47.705 7.74 5.65 51.605 T19 7.25 5.38 37.325 7.69 5.69 49.383 T20 7.14 3.01 82.435 7.775 3.1 115.432 T21 6.23 5.4 16.567 6.3 5.64 16.296 T22 5.48 3.21 45.309 5.56 3.64 47.407 T23 3.68 2.2 29.541 3.7 2.48 30.123 T24 4.9 3.78 22.355 5.01 3.82 29.383 T25 7.78 3.03 94.81 7.825 3.14 115.679 T26 7.89 6.72 23.35 7.92 6.9 25.185 T27 11.48 4.63 136.73 11.59 5.43 152.099 T28 9.58 8.21 27.345 9.61 8.5 27.407 T29 9.26 7.98 25.549 9.35 8.12 30.37 T30 10.95 5.14 115.968 11.02 5.26 142.222 T31 10.92 9 38.323 10.97 9.7 31.358 T32 11.32 9.56 35.13 11.45 9.87 39.012 T33 9.58 8.24 26.747 9.75 8.56 29.383 T34 8.76 7.21 30.938 8.89 7.64 30.864 T35 10.5 5.85 92.814 10.62 5.94 115.556 T36 10.23 7.79 48.703 10.35 8.42 47.654 T37 9.08 8.57 10.18 9.28 8.75 13.086 T38 9.12 8.62 9.98 9.2 8.7 12.346 T39 4.6 3.3 25.95 5 3.89 27.407 T40 12.62 4.91 153.892 12.75 5.21 186.173 T41 5.89 4.52 27.345 5.92 4.69 30.37 T42 6.56 5.32 24.751 6.68 5.8 21.728 F. ciceri-control 5.01 4.05 SD 2.1239 1.9126 2.0627 1.9403 SEM 1.5018 1.3524 1.4586 1.372 Biocontrol potential of Trichoderma Sp. against plant pathogens International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 35 Table 2: Dual plating of Trichoderma against S. rolfsii Culture 3rd day 5th day T Sr Sr (% reduction over control) T Sr Sr (% reduction over control) T1 6.44 4.65 36.61 7.75 5.74 22.33 T2 7.95 5.59 48.26 7.96 5.81 23.89 T3 7.81 6.14 34.15 7.93 6.31 18 T4 8 5.58 49.49 7.65 5.2 27.22 T5 7.28 6.28 20.45 7.96 7.04 10.22 T6 7.98 4.89 63.19 8.01 4.41 40 T7 7.91 5.68 45.6 7.93 5.69 24.89 T8 8.14 5.81 47.65 8 5.48 28 T9 7.95 5.71 45.81 7.64 8.26 -6.89 T10 8.14 4.91 66.05 8.19 4.3 43.22 T11 7.18 6.64 11.04 7.2 7.6 -4.44 T12 7.88 6.73 23.52 7.94 6.53 15.67 T13 4.65 7 -48.06 4.68 8.39 -41.22 T14 7.31 5.95 27.81 7.79 6.59 13.33 T15 5.59 6.73 -23.31 5.25 8.04 -31 T16 7.36 5.94 29.04 7.23 6.41 9.11 T17 7.29 6.1 24.34 8.36 7.22 12.67 T18 7.29 7.06 4.7 6.29 7.8 -16.78 T19 7.41 6.29 22.9 7.71 6.48 13.67 T20 6.8 6.9 -2.04 9 9 0 T21 7.39 6.01 28.22086 7.42 6.2 13.55556 T22 6.5 6.43 1.431493 6.78 6.65 1.444444 T23 6.66 6.79 -2.65849 6.75 6.85 -1.11111 T24 7.56 6.65 18.60941 7.68 6.78 10 T25 7.88 6.73 23.52 7.94 6.53 15.67 T26 6.89 5.9 20.2454 6.92 6.01 10.11111 T27 7.91 5.68 45.6 7.93 5.69 24.89 T28 6.56 7.01 -9.20245 6.7 7.1 -4.44444 T29 6.77 6.8 -0.6135 6.8 6.82 -0.22222 T30 8.14 4.91 66.05 8.19 4.3 43.22 T31 7.2 6.9 6.134969 7.89 6.99 10 T32 7.6 7.3 6.134969 7.88 7.56 3.555556 T33 6.9 5.67 25.15337 7.3 6.07 13.66667 T34 7.4 7.04 7.361963 7.48 7.13 3.888889 T35 8.14 5.81 47.65 8 5.48 28 T36 8.01 7.88 2.658487 8.2 7.95 2.777778 T37 7.77 6.89 17.99591 7.99 7.12 9.666667 T38 8 7.79 4.294479 8.01 7.83 2 T39 6.92 7.2 -5.72597 6.95 7.6 -7.22222 T40 8 5.58 49.49 7.65 5.2 27.22 T41 7.07 6.92 3.067485 7.12 6.97 1.666667 T42 7.17 7.02 3.067485 7.22 7.08 1.555556 S. rolfsii control 4.89 9 SD 0.7235 0.7741 0.7871 1.0962 SEM 0.1116 0.1731 0.176 0.2451 Anand S and Jayarama Reddy Copyright © 2009, Bioinfo Publications, International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 36 Table 3: Bioefficacy of Trichoderma isolates Vs F.ciceri disease incidence Treatment % Germination Disease Incidence % Rating Mean Study % T1 Vs F.c 84 4 efficient T2 Vs F.c 95 Nil highly efficient T3 Vs F.c 86 8 efficient T4 Vs F.c 78 22 moderately efficient T5 Vs F.c 90 8 efficient T6 Vs F.c 96 Nil highly efficient T7 Vs F.c 92 6 efficient T8 Vs F.c 90 7 efficient T9 Vs F.c 96 Nil highly efficient T10 Vs F.c 77 21 moderately efficient T11 Vs F.c 79 24 moderately efficient T12 Vs F.c 81 12 efficient T13 Vs F.c 80 24 moderately efficient T14 Vs F.c 96 Nil highly efficient T15 Vs F.c 80 20 moderately efficient T16 Vs F.c 88 4 efficient T17 Vs F.c 90 4 efficient T18 Vs F.c 79 16 moderately efficient T19 Vs F.c 83 4 efficient T20 Vs F.c 88 4 efficient T21 Vs F.c 87 9 efficient T22 Vs F.c 89 8 efficient T23 Vs F.c 82 16 moderately efficient T24 Vs F.c 92 8 efficient T25 Vs F.c 97 Nil highly efficient T26 Vs F.c 72 25 moderately efficient T27 Vs F.c 94 Nil highly efficient T28 Vs F.c 81 19 moderately efficient T29 Vs F.c 72 26 moderately efficient T30 Vs F.c 98 Nil highly efficient T31 Vs F.c 71 27 moderately efficient T32 Vs F.c 66 31 moderately inefficient T33 Vs F.c 32 68 highly inefficient T34 Vs F.c 79 18 moderately efficient T35 Vs F.c 96 Nil highly efficient T36 Vs F.c 54 39 moderately inefficient T37 Vs F.c 69 30 moderately efficient T38 Vs F.c 77 9 efficient T39 Vs F.c 80 16 moderately efficient T40 Vs F.c 97 Nil highly efficient T41 Vs F.c 87 10 efficient T42 Vs F.c 74 23 moderately efficient F. ciceri-control 54 Biocontrol potential of Trichoderma Sp. against plant pathogens International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 37 Table 4: Bioefficacy of Trichoderma isolates Vs S. rolfsii disease incidence Treatment Disease Incidence % Mean Study % T1 Vs S.r 88 4 efficient T2 Vs S.r 98 Nil highly efficient T3 Vs S.r 86 8 efficient T4 Vs S.r 85 7 Efficient T5 Vs S.r 89 8 Efficient T6 Vs S.r 92 Nil highly efficient T7 Vs S.r 90 9 Efficient T8 Vs S.r 85 13 Efficient T9 Vs S.r 89 4 Efficient T10 Vs S.r 82 15 Efficient T11 Vs S.r 78 20 moderately efficient T12 Vs S.r 79 18 moderately efficient T13 Vs S.r 81 8 moderately efficient T14 Vs S.r 96 Nil highly efficient T15 Vs S.r 80 20 moderately efficient T16 Vs S.r 88 4 Efficient T17 Vs S.r 89 4 Efficient T18 Vs S.r 92 8 moderately efficient T19 Vs S.r 79 20 moderately efficient T20 Vs S.r 96 Nil highly efficient T21 Vs S.r 85 13 Efficient T22 Vs S.r 85 9 Efficient T23 Vs S.r 80 14 Efficient T24 Vs S.r 90 10 Efficient T25 Vs S.r 92 Nil highly efficient T26 Vs S.r 75 21 moderately efficient T27 Vs S.r 92 Nil highly efficient T28 Vs S.r 79 20 moderately efficient T29 Vs S.r 70 24 moderately efficient T30 Vs S.r 96 Nil highly efficient T31 Vs S.r 69 31 moderately inefficient T32 Vs S.r 68 30 moderately inefficient T33 Vs S.r 30 70 highly inefficient T34 Vs S.r 82 9 Efficient T35 Vs S.r 96 Nil highly efficient T36 Vs S.r 49 48 Inefficient T37 Vs S.r 70 22 moderately efficient T38 Vs S.r 75 10 Efficient T39 Vs S.r 84 10 Efficient T40 Vs S.r 92 Nil highly efficient T41 Vs S.r 84 16 moderately efficient T42 Vs S.r 72 25 moderately efficient S. rolfsii-control 56 Anand S and Jayarama Reddy Copyright © 2009, Bioinfo Publications, International Journal of Agriculture Sciences, ISSN: 0975-3710, Volume 1, Issue 2, 2009 38 Table 5: Growth of Trichoderma isolates on PDA, SrCWAM, CCAM study on solid media Table 5: Growth of Trichoderma isolates on PDA, SrCWAM, CCAM study on solid media 3rd day observation 5th day observation PDA SrCWAM CCAM PDA SrCWAM CCAM Treatments MEAN MEAN MEAN MEAN MEAN MEAN T2 8.275 5.4875 5.65 9 8.475 8.4625 T40 8.3 5.7125 4.6875 9 8.5875 8.275 T6 6.65 5.35 5.0875 9 8.225 7.4125 T27 5.4 4.75 5.125 9 8.4625 8.4625 T35 8.275 5.475 6.4125 9 8.4875 8.4625 T9 8.3875 4.575 4.0875 9 8.55 7.4 T30 5.1375 5.45 4.525 9 8.7125 8.4375 T20 7.9625 5.4 4.2125 9 8.6625 8.1125 T25 6.275 4.625 4.2 9 8.35 7.425 T14 8.6375 4.2 5.475 9 8.375 8.3875 SD 1.3359 0.5137 0.7494 0 0.1477 0.4758 SEM 0.2987 0.1149 0.1676 0 0.033 0.1064 Endochtinase Activity 9.867 8.8 14.8914.5615.2 13.6 12.267 10.4 9.067 8.267 0 2 4 6 8 10 12 14 16 T2 T4 T6 T7 T35 T9 T30 T20 T25 T14 Trichoderma isolates (μ g /m l) Endochtinase Activity(μg/ml)