INTRODUCTION Food and nutritional security of the growing population amid raised concerns for a safe environment and injudicious use of chemical pesticides is a great challenge for plant protection scientists. The issue has resulted in the need to develop and validate alternative disease management strategies for reducing crop losses due to biotic stresses. Biological control of plant pathogens is one such strategy and involves the use of effective biocontrol agents like Trichoderma spp., Pseudomonas fluorescens, etc. The genus Trichoderma is a fungal biocontrol agent, with potential for management of soil-borne pathogens like Rhizoctonia solani, Fusarium spp., Aspergillus niger etc. The fungus is cosmopolitan in soils and on decaying wood and organic matter. Species of Trichoderma are frequently dominant components of the soil microflora in widely varying habitats. This may be attributable to the diverse metabolic capability of Trichoderma species and their aggressive competitive nature. For achieving effective biocontrol activity isolation and characterization of Trichoderma spp. from an agro-ecology is required. Cultural characters can be distinctive and may be characteristic of a species; these characters may be used for differentiating between isolates obtained from same agro-ecology. Similarly, in vitro growth rates in culture can be useful in distinguishing isolates with higher competitive ability due to vigorous growth. Thus, these descriptive characteristics may be instrumental in identification of the isolates with higher biocontrol and plant growth promotion activity. Furthermore, the native/ local isolate of the Trichoderma spp. may have higher ecological adaptability and exhibit higher biocontrol potential. Therefore, the present study was conducted to isolate and characterize native Trichoderma spp. from Chhattisgarh, India from the rhizospheres of diverse crops and their cultural characteristics were enumerated. MATERIAL AND METHODS Collection of soil samples and isolation of Trichoderma spp. Soil samples were collected from the rhizospheres of various crops (Table 1) from different locations in Chhattisgarh state of India for isolation of the Trichoderma spp. The soil samples were brought to the State Biocontrol Laboratory, Indira Gandhi Krishi Vishwavidyalaya, Barrister Thakur Chhedilal College of Agriculture and Research Station, Chorbhatti, Bilaspur, Chhattisgarh and stored at 4°C till further processing. Trichoderma spp. was isolated using soil dilution technique (Ben-David et al., 2014) using serial dilution of the soil sample. 100 µl of fivefold dilution was poured aseptically on the Trichoderma Selective Medium (TSM), Himedia, India in three replications and incubated at 27±2°C for 72 hours in BOD incubator, Sanco, India. The colonies were observed after 72 hours and morphologically distinct colonies were purified on Potato Dextrose Agar (PDA), SRL, India and incubated at 27±2°C. The purified colonies were transferred aseptically to PDA slants and preserved at 4°C for further studies. Further studies were conducted at Microbial Biocontrol Laboratory, ICAR-National Research Centre for Integrated Pest Management, New Delhi. Phenotypic Characterization. The isolates (Table 1) were revived from the slants by transferring aseptically to PDA in Petri plates followed by incubation at 27±2°C. The cultural characteristics of all the isolates under investigation were observed on 20 ml of sterilized Potato Dextrose Agar (PDA) poured aseptically in sterilized 90 mm glass Petri plates. A mycelial disc of approximately 4 mm diameter was transferred aseptically at the centre of each Petri plate in three replicates and incubated at 27±2°C for 5 days in a BOD incubator (SANCO, India). The colony characteristics observed were: - a) Colour of the fungus b) Growth of the fungus; Growth patterns; Appearance: - Ringed/sectoral/uniform/smooth/rough. The cultural characteristics were photographed. Diffusible pigments and characteristic odour were also observed and recorded (Gams and Bissett 1998). Effect of growth media on Trichoderma isolates. The following media were evaluated for maximum growth of Trichoderma spp. under in vitro conditions: Potato Dextrose Agar (PDA), Malt Extract Agar Base (MEAB), and Oatmeal Agar (OMA). Radial growth in ‘mm’ & growth rate (mm/day) was measured after 72 hrs. followed by 120 hrs. of inoculation (Hoyos-Carvajal and Bissett 2011). Determination of optimum temperature for in vitro growth and sporulation. Isolates were inoculated in three replicates on PDA medium and incubated at 15°C, 25°C, and 37°C (Leo et al., 2011). Radial growth, growth rate (mm/day), initiation of sporulation, and sporulation rate after 5 days of inoculation were observed for cultures incubated at 25°C and 37°C, and after 7 days for cultures incubated at 15°C. The sporulation rate was calculated using the Neubauer counting chamber of the Haemocytometer (ROHEM, India). After five days in the case of 25°C and 37°C, and after 7 days in the case of 15°C, the spores were harvested from the plates and diluted following the serial dilution technique. The 50 µl spore suspension was transferred between the cover slip and chamber, and the number of spores in the central square was counted using a compound light microscope (Nikon 80i) at 40X. The number of spores was calculated using the formula (Nirmalkar et al., 2020): (Area of each square = 1mm, Depth = 0.1mm, Volume = 0.1mm3 = 0.1µl occupied by each of the squares. So, 1ml (1000 µl) will have: Determination of optimum pH for in vitro growth and sporulation. Isolates were assessed for growth on PDA media in three replicates at pH levels 5, 7 & 9. The lower pH was adjusted using 10N HCl acid while the higher pH was maintained by 1N NaOH. Radial growth in ‘mm’, growth rate (mm/day), initiation of sporulation, and sporulation rate (no. of spore/ml) was measured after 72 hours of inoculation (Omar et al., 2012). The number of spores/ml was calculated using the Neubauer counting chamber of the Haemocytometer (ROHEM, India) as explained earlier after five days of inoculation. RESULT AND DISCUSSION The cultural characteristics of all the isolates under study were examined on PDA and are presented in Table 1 & Fig. 1. Trichoderma isolates’ colony colour ranged from white, yellowish green to green, dark green upon sporulation. 1-2 concentric rings were observed in many isolates. A coconut-like odour was detected in isolates IGKV (T2, T20, T24, T28). A white colour colony and yellow pigmentation were seen in isolate IGKV T18 in the PDA medium. A dark green sporulation centre was observed in several isolates IGKV (T5, T11, T13, T19, T17, T21, T24, T27, T28, T29, T30). Effect of growth media on Trichoderma isolates. Growth and sporulation of Trichoderma spp. were studied on three synthetic media PDA, MEAB and OMA after 72 hrs. of inoculation at 27±2° in BOD incubator (Sanco, India) (Table 2, Fig. 2). On MEAB, the maximum radial growth observed was 90 mm in the four isolates IGKV -T4, T5, T6 & T29 with a growth rate of 30.00 mm/day followed by 87.5 mm in IGKV -T2, T11-24, & T31 with a growth rate of 29.17 mm/day. The least radial growth (80 mm) and growth rate (26.67 mm/day) were observed were the isolates IGKV T7 and IGKV T8. On OMA, the maximum radial growth observed was 80 mm in the isolates: IGKV -T11-32; with a growth rate of 26.67 mm/day followed by 75 mm by IGKV -T2 & T3 (growth rate 25.00 of mm/day). The least radial growth observed on OMA was by the isolate IGKV T6 (65mm, growth rate 21.67 mm/day) preceded by IGKV T1 (67.5, growth rate 22.50 mm/day). On PDA, the maximum radial growth observed was 90 mm by all isolates, except IGKV T3, T4, T5, T6, T7, T8, T9 & T10. Least radial growth observed was 67.5 mm, growth rate of 22.50 mm/day in isolate IGKV T4, preceded by IGKV T3 (radial growth 70 mm, growth rate 23.33 mm/day), IGKV T5, T8, T9, T10 (radial growth 80 mm, growth rate 26.67 mm/day) and IGKV T6, T7 (radial growth 85 mm, growth rate 28.83 mm/day). Amongst the different media under study, PDA supported maximum radial growth with a mean radial growth of 87.10 mm and growth rate of 29.03 mm/day followed by MEAB (86.25 mm, 28.74 mm/day) & OMA (76.73 mm, 25.57 mm/day) (Table 5). The isolates of Trichoderma were slow growing on OMA whereas, on MEAB, the sporulation was very fast as compared to OMA and PDA. Previously, various authors have studied the cultural and morphological characteristics of Trichoderma spp. on different media. Malt extract agar is reported to be appropriate for conidia production and the observation of conidiophore branching, or potato dextrose agar, which is reported useful for observing pigment production (Hoyos-Carvayal and Bissett 2011). T. harzianum formed 1-2 concentric rings with green conidial production on PDA while white cottony mycelium with green conidiation was produced on Malt Extract Agar (MEA) (Shah et al., 2012). The authors also reported that density of conidia production is higher at the centre compared to the margins of the colony. On PDA T. viride produced granular colonies with green conidia distributed throughout. An irregular yellow zone without conidia was present around the inoculum in T. harzianum, some white pustules were also found growing on the green mat of conidia. T. pseudokoningii formed white mycelia with no conidial formation on PDA as well as MEA. The growth of T. viride was faster on MEA than the other two species, showing complete growth on 3rd day. Shah et al. (2012), uniformly flat and velvety colonies, with aerial mycelium consisting of short hyphae in one lawn over the colony, were observed on the MEAB medium (Carvalho et al., 2018). Colony showed dull green tufts or pustules, colony reverse was discoloured was identified as Trichoderma aureovirid; colony showed dark green producing tufts or pustules fringed by sterile white mycelium, colony reverse showed dull yellowish was identified as Trichoderma harzianum; colony showed scattered in minute tufts, pale yellowish-green in colour, colony reverse was pale yellowish was identified as Trichoderma reeseii; colony showed dull green to bluish green sporulation, colony reverses colourless to pale yellow were identified as Trichoderma koningii; colony showed dark green to dark bluish green sporulation, colony reverse was amber or uncoloured were identified as Trichoderma viride (Shekhar et al., 2017). Effect of temperature on radial growth and growth rate. Different isolates were inoculated on PDA media and incubated at 15±2°C, 25±2°C, and 37±2°C, observations on RG, GR, initiation of sporulation, sporulation rate were recorded (Table 3, Fig. 3). At 25°C, isolate IGKV T22 exhibited a maximum RG of 90 mm with a GR of 30.00 mm/day followed by IGKV T3 (RG 88 mm, GR 29.33 mm/day) and IGKV-T1, T4, T6, T11, T18, T27 & T31 (RG85 mm, GR 28.33 mm/day). The least growth was observed in the isolates IGKV-T5, T12, T13, T14, T15, T16, T17, T19, T20, T21, T23, T24, T25, T26, T28, T29, T30 & T32 as 80 mm with a GR of 26.67 mm/day. At 37°C, the maximum RG of 80 mm was shown by the isolates IGKV -T24, T27, and T29 with a GR of 26.67 mm/day; while, least RG was recorded as 20 mm in the isolates IGKV -T3, T4, T7, T18, T23 with a GR of 6.67 mm/day. At 15°C, IGKV- T2, T5 & T8 showed a maximum RG of 50 mm with a GR of 16.67 mm/day. The least RG was observed in IGKV -T19, T21, T23, T24, T29 & T30 as 20 mm with a GR of 6.67 mm/day. Interestingly the least RG at 25°C was the same as the maximum RG at 37°C. Amongst the different temperatures, 25±2°C was found to be optimum for in vitro growth of the fungus with a mean value of 82.03 mm followed by 37±2°C (44.68 mm) & 15±2°C (36.75 mm) (Table 5). IGKV T24 & T29 showed similar growth at 25±2°C and 37±2°C (RG 80 mm and GR 26.67 mm/day). No isolates exhibited similar growth rates across the temperature suggesting variation in biocontrol potential of the BCA when exposed to different environmental conditions. At 25±2°C and 37±2°C sporulation started after 72 hours (03 days) of incubation, however, at 15±2°C sporulation was delayed and was observed after 168 hours (Fig. 3). Mishra (2016) reported Trichoderma strain number T1, T4, T7, and T8 was not affected at different temperature i.e., 20°C, 25°C, 30°C, and 35°C whereas in Trichoderma strain number T2, T5, T18, T28 less growth was recorded at 20°C and 35°C, suggesting variation in the temperature optima among strains of Trichoderma isolated from same agroecology. Tolerance to range of temperature is reported in T. viride and T. harzianum (20°C to 40°C) & T. asperellum and T. hamatum (25 °C -35°C) is reported (Ali et al., 2015). However, the author reported slower growth at 15°C. Temperature significantly affected the radial growth and sporulation of Trichoderma spp. T. viride and T. harzianum showed a high range of temperature tolerance. It grew and sporulated well between temperatures 20 to 40°C. T. asperellum and T. hamatum grew and sporulated well between temperatures (25-35°C). But at low temperature (15°C), the growth was slow and all the Trichoderma spp. failed to sporulate even after 7 days of incubation. A similar trend was also observed at high temperatures for all four spp. of Trichoderma (Zehra et al., 2017). Three morphologically similar species, T. harzianum, T. aggressivum, and T. atroviride, can be distinguished by growing them at 35°C. After 96 h, T. harzianum grows well and sporulates at 35°C, but the remaining two, T. aggressivum and T. atroviride, can have a colony radius of more than 5 mm (Gorai et al., 2020). Bastos (2001) studied the effect of temperature (10, 15, 20, 25, 30 & 35 °C), pH (3.5-9.5), and liquid medium composition on the growth and sporulation of T. stromatium and observed that a temperature range of 20 and 30°C was optimum for growth and sporulation. No growth occurred at 10 and 35°C. Effect of pH. Isolates were assessed for growth on amended PDA media in three replicates at pH 5, 7 & 9. RG in ‘mm’, GR (mm/day), initiation of sporulation, and sporulation rate (no. of spore/ml) were measured after 72 hours of inoculation at 27±2° in BOD incubator, (Table 4, Figure 4). At pH 5, IGKV T3 recorded the max RG of 90.00 mm with a GR of 30.00 mm/day followed by IGKV T6 (RG 86.00 mm, GR 28.67 mm/day). The least RG was noted in isolate IGKV 26 as 50.00 mm with a GR of 16.67 mm/day. The isolates produced greenish cottony colony at pH 5. At pH 9, a maximum RG of 90.00 mm was observed in isolates IGKV -T6, T8, T12, T14, T16, and T18 with a GR of 30.00±00 mm/day whereas the least RG of 60.00±00 mm was recorded in isolate IGKV T7. At pH 7, a maximum RG of 90.00 mm with a GR of 30.00 mm/day was shown by isolate- IGKV T22 followed by IGKV T3 (RG 88.00 mm GR 29.33). The minimum RG of 80.00 mm with a GR of 26.67 mm/day was observed in 18 isolates IGKV-T5, T12, T13, T14, T15, T16, T17, T19, T20, T21, T23, T24, T25, T26, T28, T29, T30 & T32. Amongst the different pH levels, pH 7 was found optimum with a mean value of 82.03 mm followed by pH 9 (80.31 mm) & pH 5 (70.93 mm) (Table 5). Higher growth of IGKV T3 and IGKV T6 at different pH levels indicates that these isolates may be useful in highly acidic or alkaline crop fields.Optimum growth and sporulation of four species of Trichoderma were recorded between pH 4.1 to 8.6. Growth and sporulation of all the isolates decreased significantly with either decrease or increase in the pH below 4.6 or above 7.6. At pH 4.6, the growth was still good but sporulation was poor. At pH 8.6, the growth completely ceased (Zehra et al., 2017). Lejeune et al. (1995) using glucose-limited continuous culture showed that between pH 4 and 5 the specific growth rate, the maximum tip extension rate, and the branching frequency were highest, whereas the average hyphal diameter was smallest (2.1 µm vs. 2.5–2.7). Their data are in contrast to similar studies by Brown and Halsted (1988), who reported that the maximal specific growth rate of T. reesei under glucose limitation increased with increasing H+ concentration, whereas the biomass yield Y remained constant at 0.4.