The present study investigated the effects of various pH levels on the growth, sporulation, and antagonistic effectiveness of T. viride isolates, a fungus known for its ability to combat plant pathogens. The experiments were conducted at the Biocontrol Laboratory in the Department of Plant Pathology at C.S. Azad University of Agriculture and Technology, Kanpur, Uttar Pradesh, India.

Collection of soil samples and isolation and purification of T. viride
Fifty soil samples were collected from the rhizosphere of chickpea fields across various districts of Uttar Pradesh, each taken at a depth of approximately 5 to 6 cm. The samples were carefully placed in polythene bags, with each bag clearly labeled to indicate the location and date of collection. The samples were then transported to the laboratory for further analysis. Trichoderma species were isolated using the Trichoderma selective medium (TSM), which consisted of the following components: MgSO₄.7H₂O (0.2 g), K₂HPO₄ (0.9 g), KCl (0.15 g), NH₄NO₃ (1.0 g), glucose (3.0 g), chloramphenicol (0.20 g), Apron 35SD (0.01 g), Captan (0.2 g), Rose Bengal (0.15 g), agaragar (20 g), and distilled water (1 liter).⁷ The soil samples were processed using the serial dilution technique,⁸ followed by incubation of the plates at 28°C ± 1°C for 5 days. The appearance of colonies was noted and recorded between the third and fifth days. Individual colonies were selected and maintained in pure culture for further examination. T. viride isolates were purified using the single spore isolation method, with subculturing performed from the growing tip of each new colony. A small quantity of spores was aseptically taken and streaked onto potato dextrose agar (PDA) plates using a sterilized inoculating needle.

Morphological characterization of Trichoderma sp
The Trichoderma genus can be identified when grown on TSM by observing specific colony characteristics, such as growth pattern, growth rate, odor, and color.⁹

Microscopic features
Identifying Trichoderma species typically involves examining these characteristics under a light microscope, as described.^10-12 Key morphological features used for identification include the branching pattern of conidiophores, the elongation and shape of conidiophore tips (whether coiled, straight, or undulating), the structure, size, and shape of phialides, and the morphology of conidia.

Coding of T. viride isolates
Chickpea rhizospheric soil samples were collected from various districts of Uttar Pradesh, including Etawah, Kaushambi, Faizabad, Sultanpur, Mirzapur, and Bhadohi. These samples were coded as 02CP, 10CP, 13CP, 21CP, 25CP, 45CP, 49CP, 64CP, 70CP, and 117CP, respectively.

Reconfirmation of cultures by ITCC, New Delhi
The Trichoderma pure culture was obtained using the single spore technique. Microscopic observation of mounted slides of the cultures was performed, and the cultures were sent to the ITCC in New Delhi, India, for accurate identification and to obtain accession numbers for the pure culture.

Collection of disease samples, isolation, and purification of pathogen
PDA medium was prepared for isolating the pathogen, following the method outlined.¹³ The medium consisted of 200 g of peeled potato, 20 g of dextrose, 20 g of agar, and 1000 mL of distilled water. Pathogen identification involved comparing the cultural and morphological characteristics of the fungus with those described¹⁴ for Fusarium oxysporum f. sp. ciceri. The color nomenclature was based on the standards provided.¹⁵ The growth habit, cultural traits, and morphological features of the fungus were examined on the PDA medium.

Effect of various pH levels on growth and sporulation of T. viride isolates
The potato dextrose broth (PDB) medium was prepared with different pH levels (4.5, 5.5, 6.5, and 7.5) by adding the appropriate amount of citrate phosphate buffer and adjusting the pH accordingly. Each 250 mL of conical flask was filled with 100 mL of PDB. The flasks were tightly sealed with nonabsorbent cotton plugs and covered with aluminum foil secured by rubber bands. The medium was sterilized by autoclaving at 15 psi (121.6°C) for 20 minutes. Under aseptic conditions in a laminar airflow, the PDB medium in the flasks was inoculated with 5 mm discs from 7-day-old cultures of T. viride isolates, with three replicates for each treatment. The inoculated flasks were then incubated in a biological oxygen demand (BOD) chamber at 28°C ± 2°C for 7 days. After incubation, the intensity of growth and sporulation of the different isolates of T. viride was assessed. The mycelial mats were harvested by filtering the culture through sterilized Whatman Filter Paper No. 4 and then dried in a hot air oven at 35°C for 48 h to obtain their dry weight. The biomass was calculated using the methods described.^16,17

Antagonistic efficacy of T. viride isolates against soil borne pathogens by dual culture assay
The effectiveness of T. viride isolates was evaluated against Fusarium oxysporum f. sp. ciceri, Sclerotium rolfsii, Sclerotinia sclerotiorum, Rhizoctonia bataticola, and Pythium sp. using the dual culture technique. Both the target pathogens and the fungal antagonist (T. viride) were cultured on the PDA medium. Five-millimeter-diameter bits were cut from the edges of five-day-old cultures of both the pathogens and the antagonist, and these were placed diametrically opposite each other on the Petri dish, 5 mm from the edge. A control Petri dish was inoculated with each target pathogen alone without an antagonist. Each treatment was replicated three times and incubated at 28°C ± 2°C. After 7 days, the presence of inhibition zones between T. viride and the pathogens was observed. The radial growth of the pathogens on both the dual culture and control plates was measured, and the inhibition percentage of the pathogens was calculated using the methodology outlined.¹⁸

Percent inhibition (I) = C-T/C x 100

Where,
C represent the mycelial growth of pathogens in control.
T is the mycelial growth of pathogens in the dual culture plate.