Assessment of the morphological, molecular and environmental adaptability of the native Trichoderma isolates for biocontrol applications

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This study aimed to evaluate the morphological, cultural, molecular, and antagonistic variability of native Trichoderma isolates under laboratory conditions. Six isolates were collected from different crops and agro ecological zones, and their growth characteristics were assessed using Trichoderma Selective Medium (TSM). Morphological characterization was conducted based on colony colour, morphology, pigmentation, concentric ring formation, and spore size, while molecular characterization using ITS marker analysis confirmed the genetic identity of the isolates. The results revealed considerable morphological and cultural variability among the isolates. Colony colours ranged from dark green to light green, with most showing rough, spongy, and raised morphology; two isolates (Tr 1 and Tr 2) exhibited prominent concentric ring formation, indicating higher sporulation. Reverse side pigmentation varied from dark brown to whitish creamy and pinkish, reflecting metabolic diversity. Spore sizes ranged from 3.06 × 2.92 µm (Tr 6) to 3.74 × 3.46 µm (Tr 3), showing moderate intraspecific variation. Despite these differences, PCR amplification of the ITS region consistently yielded a ~ 600 bp product across all isolates, confirming genetic uniformity within the Trichoderma genus. Environmental adaptability studies indicated that Trichoderma isolates thrived best at 30°C and in a pH range of 4.5–6.5, with significantly reduced growth under extreme conditions. In vitro antagonistic assays using the dual culture technique demonstrated that all isolates significantly inhibited the growth of major soil-borne pathogens including Sclerotium rolfsii , Macrophomina phaseolina , Alternaria alternata , Fusarium oxysporum , and Colletotrichum spp. The highest inhibition percentages were recorded with isolates Tr 1, Tr 2, and Tr 3, indicating their strong biocontrol efficacy. These findings underscore the potential of morphologically and genetically diverse native Trichoderma isolates as promising biocontrol agents under variable agro-climatic conditions, and support their integration into sustainable plant disease management strategies. Biological sciences/Biotechnology Biological sciences/Microbiology Biological sciences/Plant sciences Colony colour Morphology Pigmentation Concentric ring formation spore size Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Rapid growth of the global population is expected to reach 9.10 billion by 2050, necessitating 70% increase in food production to ensure food security 1 . However, plant diseases remain a significant challenge to agricultural productivity, leading to an estimated 16% reduction in the global crop yields 2 . Among these threats, soil-borne plant pathogens have emerged as a major concern, affecting the crops across tropical, sub-tropical, and temperate regions 3 . To combat plant diseases and enhance agricultural output, synthetic agro-chemical pesticides have been extensively used world-wide. Currently, ~ 2.0 million tons of pesticides applied annually, with herbicides comprising 47.5%, insecticides 29.5%, fungicides 17.5%, and other pesticides accounting for 5.5% 4 . Despite their effectiveness, extensive use of chemical pesticides poses serious environmental and health risks, including soil degradation, water contamination, biodiversity loss, and food safety concerns 5 . As a result, there is a pressing need to explore the sustainable alternatives for plant disease management. Biological control i.e. biocontrol has emerged as an eco-friendly and effective strategies to mitigate plant diseases while reducing reliance on chemical pesticides. Among various bio-control agents, Trichoderma sps. have gained prominence due to their remarkable ability to suppress soil-borne pathogens, enhance the plant growth, and improve soil fertility 6 . Trichoderma -based bio-fungicides constitutes over 60% of all the commercially available bio-fungicides worldwide, underscoring their significance in modern sustainable agriculture 7 . These fungi predominantly inhabit rhizosphere of plants, where they colonize plant roots, leaves, grain, acting as biocontrol agents and plant growth promoters 8 . Effectiveness of Trichoderma in plant disease management stems from its multifaceted mechanisms of action, including mycoparasitism, competition for nutrients, and production of antifungal metabolites 9, 10 . Through mycoparasitism, Trichoderma sps. invade and parasitize plant pathogens, effectively reducing their spread. Additionally, their competitive ability enables them to outcompete harmful fungi for essential resources such as nutrients and space 11 . Moreover, Trichoderma sps. produce variety of secondary metabolites, including pep-taibols, gliotoxin, and trichodermin, which exhibit strong antifungal properties 12 . Beyond pathogen suppression, Trichoderma enhances plant growth by promoting root development, improving nutrient uptake, inducing systemic resistance against various plant stresses 13 . Adaptability of Trichoderma sps. to diverse environmental conditions further enhances their potential as biocontrol agent. Unlike synthetic fungicides, which have detrimental ecological effects, Trichoderma -based solutions align with sustainable farming practices by minimizing chemical inputs while maintaining high crop yield 14 . In addition, their ability to solubilize nutrients like phosphorus and produce plant hormones such as auxins, gibberellins, cytokinin’s contributes to improved plant health and productivity. Trichoderma sps. exhibit the significant morphological, cultural, molecular variability, influencing their biocontrol efficacy. Morphologically, species differentiation is based on conidial structure, size and pigmentation. Culturally, different Trichoderma strains exhibit the distinct growth patterns on various media, reflecting their adaptability to the environmental conditions. Molecular techniques, particularly analysis of the Internal Transcribed Spacer (ITS) region of ribosomal RNA genes, have proven essential in accurately identifying Trichoderma isolates and selecting the most effective strains for biocontrol applications 15 . Environmental factors such as temperature and pH significantly impact Trichoderma growth, sporulation, and biocontrol efficiency. Studies indicate that temperature variations influence mycelial growth and antifungal metabolite production, while pH levels affect colonization and pathogen suppression 16, 17 . Understanding these environmental parameters is a crucial for optimizing Trichoderma applications in sustainable modern crop production. Given its exceptional biocontrol capabilities and plant growth-promoting properties, Trichoderma represents a promising alternative to chemical pesticides. Its integration into agricultural management practices can significantly contributes to the sustainable food production, ensuring crop protection while safeguarding environmental and human health. Thus, present study focuses on Trichoderma isolates collected from crop field in Kishanganj (26.0976° N, 87.9420° E) and Purnea districts (25.7771° N, 87.4753° E) of Bihar, India. Kishanganj and Purnea districts are situated in the northeastern part of Bihar, India where agriculture is the primary occupation. Region experiences diverse climatic conditions, with varying temperatures and humidity levels throughout year. These environmental variations provide an ideal setting for isolation of Trichoderma sps. that are adapted to different ecological niches. By investigating morphological and molecular diversity of Trichoderma isolates from these districts, the study aims to identify the strains that are particularly effective against the locally prevalent plant pathogens, such as Fusarium, Rhizoctonia , and Pythium , which cause root rot and complex wilt diseases in major field crops. 2. Materials and Methods 2.1 Collection and Isolation of Trichoderma Isolates Rhizospheric soil samples were collected from maize, sugarcane, jute, potato, and wheat fields across different location in Kishanganj & Purnea districts of Bihar, India. Trichoderma spp. was isolated using Trichoderma Selective Medium (TSM), and their growth patterns were recorded properly. Antagonistic activity was tested on Potato Dextrose Agar (PDA), while mycelial mats were grown in Potato Dextrose Broth (PDB) for DNA extraction. Media were prepared by dissolving components in distilled water and autoclaving at 121°C for 20 min. Soil suspensions were prepared using serial dilution method, plated on TSM and PDA, and incubated at 28 ± 1°C. Sub-culturing was performed on PDA, and isolates were identified based on conidiophore branching, phialide formation, and spore characteristics. 2.2 Morphological Characterization Morphological identification was based on cultural characterization (colony appearance and growth rate) and microscopic examination. Trichoderma isolates were transferred from slants to PDA plates and incubated at 28°C for 24–48 hours. A 5-mm mycelial disc from an actively growing colony (before conidial production) was inoculated at centre of a fresh PDA plate, which was then incubated at 30°C with intermittent light. Each isolate was tested in triplicate, and all procedures were conducted under the aseptic conditions. Colony traits, growth rate, & sporulation patterns were recorded followings 18 . Colony diameter was measured from third to seventh day to assess growth rate. 2.3 Spore Size Measurement Spore size was measured using a cilika digital microscope. Spores were first focused at 10X, observed at 40X, and measured at 100X using a scale. Diameter of five spores per isolate was recorded and the average was calculated. 2.4 Fungal Spore Count Fungal spore count was determined using a haemocytometer. Drop of conidial suspension was placed on engraved grid, allowed to settle for 1–2 minutes, covered with a cover slip to prevent air bubbles. Spores were counted in central square (E), consisting of 25 groups of 16 tiny squares (0.2 mm each). Spore concentration (spores/ml) was calculated using formula: Spores/ml = N×104, Where N is the number of spores counted in the middle square. 2.5 Morphological Characterization The colony colour, morphology, ring formation, pigmentation, and spore size were assessed. The observations were made to categorize the isolates based on distinct morphological traits. 2.6 Fungal Culture and Mycelial Preparation Trichoderma isolates cultured in Potato Dextrose Broth (PDB) within 250 mL Erlenmeyer flasks containing 100 mL of medium. A 5 mm fungal disc from actively growing cultures was inoculated into each flask. Cultures were incubated at 25 ± 1°C with continuous shaking at 120 rpm for 3–5 day. Mycelial mats harvested using Whatman filter paper, washed three times with sterile distilled water, ground in liquid nitrogen for further processing. 2.7 DNA Extraction and Purification Genomic DNA was extracted using a modified CTAB method 19 . The ground mycelial tissue was mixed with DNA extraction buffer consisting of 100 mMTris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 1.4 M NaCl, and 2% CTAB, followed by incubation at 65°C for 60 min. After extraction with chloroform: isoamyl alcohol (24:1), supernatant was precipitated with isopropanol and centrifuged at 12,000 rpm for 10 min. Resulting DNA pellet was washed twice with 70% ethanol, air-dried, resuspended in 80 µL Tris-EDTA buffer, stored at-20°C. RNase treatment performed at 37°C to remove RNA contamination. 2.8 DNA Quantification Extracted DNA quantified using a Nano-Drop Spectrophotometer by measuring absorbance at 260 nm (OD260). Purity was assessed by calculating the OD260/OD280 ratio, and DNA integrity verified through 0.8% agarose gel electrophoresis stained with ethidium bromide, using a 100 bp DNA ladder as a molecular marker. 2.9 PCR Amplification of ITS Region ITS1-ITS4 region, including the 5.8S rDNA gene, was amplified using primer pair ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) 20 . PCR reaction mixture consisted of 4 µL 10X reaction buffer, 0.5 µL 2 mM dNTP mix, 0.25 µL 1 U Taq DNA polymerase, 2 µL of each primer (5 µM), 2 µL of 25 ng template DNA, and 32 µL of deionized water, making a total volume of 38 µL. PCR was performed with an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds, and extension at 70°C for 2 minutes, with a final extension at 72°C for 7 min. Amplified PCR products analysed using 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV trans illumination. The DNA was extracted from six Trichoderma isolates, and ITS-PCR analysis was conducted using ITS1 and ITS4 primers. The PCR amplification was performed to confirm the genetic variability. 2.10 Effect of pH Levels and Temperature Effect of various pH levels and temperatures on growth of Trichoderma isolates was studied using Completely Randomized Design (CRD) & replicated thrice. For pH evaluation, Potato Dextrose Broth (PDB) was prepared, and its pH was adjusted between 4.0 & 8.0 using 1 N HCl or 1 N NaOH. Medium was then dispensed into sterile conical flasks, autoclaved, and inoculated with a 5 mm-diameter mycelial disc from 5-day old culture. Flasks were incubated at 28 ± 1°C for seven days, after which cultures were filtered using Whatman filter paper no. 42, dry mycelial weight was measured. For temperature evaluation, six Trichoderma isolates were inoculated into the Potato Dextrose Agar (PDA) plates and incubated at temperatures ranging from 15°C to 40°C. Growth was assessed by recording radial growth measurements after 48 hours and subsequently at 24-hour intervals for 7-days. Cumulative colony diameter was computed to analyse impact of pH and temperature on Trichoderma growth. 2.11 In Vitro Evaluation of Antagonistic Potential of Trichoderma spp Six isolates of Trichoderma spp. were evaluated for their antagonistic activity against five phyto pathogenic fungi: S. rolfsii , M. phaseolina , A. alternata , F. oxysporum , and Colletotrichum spp. The dual culture technique was employed, where a 5 mm disc of the test pathogen and the respective Trichoderma isolate were inoculated on opposite sides of a PDA (Potato Dextrose Agar) plate. The plates were incubated at 25 ± 1°C, and radial growth inhibition of the pathogens was recorded after seven days. The percentage inhibition of mycelial growth was calculated using the formula: Percent Inhibition (I%) = [(C - T)/C] × 100 where, C = radial growth of pathogen in control plate T = radial growth of pathogen in dual culture plate 3. Results 3.1 Morphological and Cultural Variability Morphological and cultural characteristics of six Trichoderma isolates were studied using monoconidial cultures grown on Trichoderma Selective Medium (TSM). Observations were recorded on colony colour, morphology, concentric ring formation, pigmentation on lower side of culture plates, and spore size (Table 1 & Fig. 1). Results indicated noticeable variation among isolates, highlighting intraspecific diversity within the genus. Detailed analysis of morphological traits revealed distinct characteristics among the isolates: Colony colour: The isolates exhibited the various shades of green, a characteristic traits of Trichoderma sps. Trichoderma 1 & 3 showed dark green colonies, while Trichoderma 2, 4, and 6 displayed a transition from light to dark green. Trichoderma 5 was characterized by a uniform light green coloration. Colony morphology: Most isolates, including Trichoderma 1, 2, 4, Tr 5, showed a rough, spongy, and raised morphology, suggesting active mycelial growth and aeration. In contrast, Trichoderma 3 and 6 presented smooth, flat colonies with dense sporulation localized at the border, indicating potential differences in sporulation patterns and radial growth behaviour. Concentric ring formation: Concentric rings, indicative of the distinct sporulation phases or mycelial growth cycles observed in Trichoderma 1 & 2. Their presence may be associated with periodic nutrient availability or intrinsic sporulation rhythms. The remaining isolates ( Trichoderma 3, 4, 5 and 6) did not exhibit ring formation. Pigmentation (lower side of plate): Significant differences in pigmentation were observed on reverse side of culture plates. Trichoderma 1, 2, and 4 exhibited dark brown pigmentation, potentially indicating robust secondary metabolite production. Trichoderma 3 and 5 showed whitish-creamy pigmentation, whereas Trichoderma 6 demonstrated a distinctive pinkish hue, suggesting variability in metabolic activity among isolates. Spore size: Spore size analysis revealed minor yet distinct differences across isolates. The largest spores were recorded in Trichoderma 3 (3.74×3.46 µm), while smallest were observed in Trichoderma 6 (3.06 × 2.92 µm). Such variations could affect spore germination, dispersal efficiency, and potential biocontrol performance. 3.2 Molecular Identification The PCR amplification of ITS region yielded a 600 bp fragment in all six isolates, indicating genetic similarity within Trichoderma genus. No inter-or intra-species ITS length diversity was detected, suggesting conservation of ITS region among isolates (Fig. 2). This result implies a high degree of genetic homogeneity among isolates, reinforcing their classification within Trichoderma g enus. Use of ITS-PCR as a molecular identification tool confirmed that these isolates belong to same genetic cluster, highlighting reliability of molecular methods in fungal taxonomy and genetic differentiation. 3.3 Evaluation of Trichoderma isolates for pH and temperature tolerance The growth and development of Trichoderma isolates are significantly influenced by pH and temperature, as these factors regulate metabolic and enzymatic activities essential for biomass accumulation and sporulation. Effects of pH and temperature on mycelial growth The mycelial growth was assessed across six Trichoderma isolates at pH levels ranging from 4.0 to 8.0 (in 0.5-unit increments) and at six temperature levels (15°C, 20°C, 25°C, 30°C, 35°C and 40°C). Results revealed that the significant variability among isolates, emphasizing influence of these factors on fungal physiology (Tables 2 and 3; Figure 3 and 4). 3.4 Optimal growth conditions Highest biomass production was recorded at pH levels between 4.5 & 6.5, with Trichoderma 2 achieving highest mycelial weight (1.09 g) at pH 6.5, followed by Trichoderma 6 (1.08 g). A progressive increase in biomass was observed from pH 4.0 to 6.5, but growth declined significantly at extreme pH levels (4.0 & 8.0). While mycelial growth remained satisfactory at these extremes, sporulation notably poor, indicating that suboptimal pH conditions might inhibit the reproductive processes in Trichoderma isolates (Fig. 5). Temperature played a crucial role in colony expansion, with 30°C identified as optimal temperature. Trichoderma 3 exhibited largest colony diameter (88.00 mm), followed by Tr2 (87.00 mm) and Trichoderma 6 (86.33 mm). Growth declined at temperatures below 30°C, with Trichoderma 3 maintaining superior growth at 25°C (84.67 mm). At 20°C and 15°C, colony diameters were significantly reduced, with Trichoderma 4 consistently showing lowest growth across all temperatures. At elevated temperatures (35°C and 40°C), growth progressively declined, with severe inhibition at 40°C, where Trichoderma 3 was recorded the highest colony diameter (21.67 mm). These results indicate that Trichoderma isolates thrive best within moderate temperature conditions and that extreme temperatures adversely affect fungal proliferation and survival. 3.5 Effects of Antagonistic Potential of Trichoderma spp. Against Major Plant Pathogenic Fungi All six isolates of Trichoderma spp. showed significant inhibitory effects against the tested pathogens. However, the degree of antagonism varied among isolates and pathogens(Table 4 and Figure 6). Antagonism Against Alternaria alternata Alternaria alternata is a foliar pathogen responsible for causing leaf spots and blights in various crops. The inhibition percentage ranged between 17.37% and 60.86% . Among the isolates, Tr 2 demonstrated the highest inhibition ( 60.86% ), indicating its strong antagonistic potential, followed by Tr 1 (58.69%) , Tr 3 (56.51%) , Tr 6 (52.16%) , and Tr 5 (47.81%) . Tr 4 recorded the lowest inhibition ( 17.37% ), suggesting its limited efficacy against A. alternata . Antagonism Against Colletotrichum spp. Colletotrichum spp. cause anthracnose disease, which affects fruits, flowers, and stems. All six Trichoderma isolates effectively inhibited Colletotrichum spp. growth. The maximum inhibition was observed with Tr 1 (79.39%) , followed by Tr 6 (76.35%) , Tr 2 (71.62%) , Tr 5 (64.19%) , and Tr 3 (61.48%) . Tr 4 again showed the least inhibition ( 47.97% ). The strong performance of Tr 1 and Tr 6 suggests their potential utility in managing anthracnose in crops. Antagonism Against Fusarium oxysporum F. oxysporum is a notorious soil-borne pathogen responsible for vascular wilt in numerous crops. All Trichoderma isolates significantly suppressed its growth, with inhibition ranging from 28.70% to 65.74% . The highest inhibition was recorded with Tr 1 (65.74%) , followed by Tr 3 (60.19%) , Tr 5 (49.07%) , Tr 2 (44.44%) , and Tr 6 (38.89%) . The lowest inhibition was again observed with Tr 4 (28.70%) , suggesting that isolates Tr 1 and Tr 3 hold promise as biocontrol agents against Fusarium wilt. Antagonism Against Macrophomina phaseolina M. phaseolina , the causal organism of charcoal rot, was effectively suppressed by all isolates of Trichoderma . The percent inhibition ranged from 47.10% to 73.55% , with Tr 5 demonstrating the highest inhibition ( 73.55% ), followed by Tr 3 (71.61%) , Tr 1 (64.52%) , Tr 4 (60.65%) , and Tr 2 (56.78%) . The minimum inhibition was observed with Tr 6 (47.10%) . This suggests that Tr 5 and Tr 3 may be potent candidates for managing M. phaseolina in crops prone to dry root rot and stem rot. Antagonism Against Sclerotium rolfsii Sclerotium rolfsii causes collar rot, particularly in legume crops and vegetables. All Trichoderma isolates significantly reduced its growth. The maximum inhibition was shown by Tr 3 (69.33%) , followed by Tr 6 (64.00%) , Tr 2 (62.67%) , Tr 5 (60.00%) , and Tr 1 (57.33%) . Tr 4 recorded the minimum inhibition ( 36.00% ), suggesting its comparatively lower antagonistic potential against S. rolfsii . 4. Discussion Results align with previous studies by 16 , who observed similar colony growth rates and colour variations under different temperature conditions. Singh et al. (2014) also reported that Trichoderma sps. exhibit the optimal growth between 25°C and 30°C, which was consistent with the present observations. 21 confirmed the importance of molecular tools in species identification, supporting our findings that ITS-PCR is an effective method for genetic differentiation. Similar distinctions among Trichoderma species based on cultural and morphological characteristics were also observed by 22 , who identified difference between T. asperellum , T. harzianum, T. reesei, T. hamatum based on the radial growth, colony morphology, pigmentation and conidial shape. These morphological differences indicate that native Trichoderma isolates exhibit species-specific traits, which could influence their biocontrol potential. Presence of concentric rings in some isolates suggests that sporulation efficiency may vary based on environmental adaptation, as also observed by research findings of 23 in their genotypic and phenotypic classification of Trichoderma sps. The observed colony colour variations could be attributed to metabolic adaptations to the different soil conditions, as previously discussed by 24 , who reported pH & temperature significantly influence colony pigmentation and growth. The PCR amplification of ITS region yielded a 600 bp fragment in all six isolates, indicating genetic similarity within Trichoderma genus. These findings are in agreement with 25 , who reported 98.6–100% DNA similarity among Trichoderma isolates based on ITS barcode analysis. 26 also noted the high similarity among Trichoderma species using ITS sequencing, reinforcing genetic homogeneity observed in the present investigations. Molecular techniques had become reliable & highly suitable tools for identifying microbial sps & for assessing genetic variation within collections & population 27 . 28 emphasized impact of environmental factors on Trichoderma growth, which aligns with present observations that some isolates exhibited the morphological changes depending on culture conditions Despite variation in the colony colour and sporulation, ITS-PCR results indicated that genetic uniformity. This suggests that while phenotypic plasticity exists among isolates, their genetic identity remains conserved, which aligns with findings by 29 , who demonstrated that environmental conditions influence morphological traits while genetic markers remain stable. 30 analysed the genetic relatedness of these isolates using RAPD (Random Amplified Polymorphic DNA) profiling with six-random primers. RAPD profiles revealed genetic diversity among the isolates, forming two distinct clusters corresponding to Trichoderma viride & Trichoderma harzianum , each further divided into five subgroups. 15 identified Trichoderma strain morphologically, microscopically, and biochemically, after which it was further analysed and confirmed using ITS sequencing molecular technique. The study confirmed that growth was entirely inhibited at 10°C and 35°C, regardless of pH 29 . Similar findings were reported in previous studies by 31 , 32 where the optimal temperature range for Trichoderma growth was identified as 25°C to 30°C. Notably, a temperature of 25°C and a pH of 5.5 were not limiting factors for R. solani growth. According to 33 these conditions actually favoured its mycelial growth & survival, highlighting the potential competition between Trichoderma and R. solani under such conditions. These results reinforce the necessity of maintaining moderate temperature and pH conditions for the optimal fungal performance. According to our results, Trichoderma isolates demonstrated optimal growth at 30°C and a pH range of 4.5 to 6.5. Growth progressively declined beyond these condition, underscoring sensitivity of Trichoderma isolates to extreme environmental variations. These findings emphasize importance of maintaining optimal temperature and pH conditions to enhance the biocontrol potential of Trichoderma species in agricultural applications. The efficacy of Trichoderma spp. as biocontrol agents has been well-documented across various crops and pathogens. In the present study, native Trichoderma isolates exhibited significant in vitro antagonism against Sclerotium rolfsii , Macrophomina phaseolina , Fusarium oxysporum , Alternaria alternata , and Colletotrichum spp., with isolates Tr 1, Tr 2, and Tr 3 showing superior inhibition rates. These findings are consistent with those of 34 , who tested sixteen Trichoderma strains against Rhizoctonia solani , Sclerotinia sclerotiorum , and S. rolfsii , observing average inhibition rates of 60% for R. solani and S. sclerotiorum , and up to 70% for S. rolfsii . The study also highlighted considerable variation among strains, with certain isolates demonstrating more aggressive antagonism. Similarly, 35 reported that T. asperellum restricted the growth of multiple fungal phytopathogens by 65–74% and inhibited spore germination by 30–75%. Further supporting these outcomes, 36 demonstrated the efficacy of T. asperellum against a spectrum of phytopathogens in dual culture assays, with the highest inhibition observed against F. oxysporum (53.24%). Collectively, these studies reinforce the relevance of native Trichoderma isolates as promising agents for biological disease management. The observed variability in antagonistic potential underscores the need for precise selection and characterization of strains tailored to specific pathogens and agro ecological condition. 5. Conclusions This present study highlights the diverse morphological & molecular characteristics of native Trichoderma isolates, reinforcing their potential as an effective biocontrol agent. Variations in colony traits, pigmentation, and sporulation suggest adaptations to different environments, while ITS-PCR analysis confirmed that the genetic stability within genus. Despite their morphological differences, isolates share a conserved genetic identity, making them reliable candidates for agricultural applications. Given that Trichoderma ability to suppress the plant pathogens and promote plant growth, its role in sustainable farming production systems is undeniable. Future research should focus on large-scale field trials, exploring its interactions with other beneficial microbes, and enhancing its biocontrol potential through genetic and biochemical studies. Additionally, developing the stable bio-formulations and stress-tolerant strains will be key to maximizing its practical applications. Trichoderma thrives the best at a temperature of 30°C and within a pH range of 4.5 to 6.5. Growth was significantly reduced at extreme conditions, highlighting importance of maintaining optimal environmental factors for its application in modern crop production. All six Trichoderma isolates possessed significant antagonistic activity against the five tested plant pathogenic fungi under in vitro conditions. Among them, Tr 1, Tr 2, Tr 3, and Tr 5 consistently showed higher efficacy across most pathogens, indicating their potential for further evaluation under greenhouse and field conditions. In contrast, Tr 4 exhibited the least inhibition in all pathogen interactions and may not be ideal for biocontrol purposes. Ultimately, Trichoderma offers a promising, eco-friendly alternative to agro-chemical pesticides, contributing to the resilient and sustainable agricultural production systems. By further refining its use through integrated research, it can play a crucial role in improving crop health and soil fertility while minimizing environmental impacts. 6. Future scope of study Future of Trichoderma research lies in advancing its applications for biocontrol, plant growth promotion, and sustainable agricultural production systems in broad blew of climate change scenarios. Large-scale field trials will validate its efficacy across diverse environments, while molecular studies will elucidate its antifungal mechanisms and role in systemic resistance. Genetic improvements through selective breeding and CRISPR could enhance its adaptability and efficiency. Investigating its interactions within soil microbiomes will further its role in nutrient cycling and ecosystem balance. Additionally, developing stable bio-formulations and innovative application methods will improve its practicality for farmers. With the growing emphasis on climate resilience, Trichoderma also holds promise in mitigating abiotic stresses, ultimately supporting environmentally friendly and sustainable crop production. Declarations Author Contributions Sanjeev Kumar, Sardar Sunil Singh and Chandra Shekhar Azad conceptualized the study, guided the research framework, and supervised the manuscript preparation. Devendra Mandal and Mohammad Shamim were involved in data collection, field experimentation, and initial analysis. Rakesh Kumar, Pravin Kumar Upadhyay, Niru Kumari, Mahesh Kumar, Malkhan Singh Gurjar, Erayya, Satendra Singh and Subrat Keshori Behera contributed to data curation, statistical analysis, and interpretation of results. Achin Kumar, Rajeev Padbhushan, and Yalamareddy Kiranmai supported literature review, drafting of methodology, and technical validation. Brajendra Parmar contributed to reviewing and editing. Funding This research received no external funding. Acknowledgments The authors gratefully acknowledge Bihar Agricultural University, Sabour, for providing comprehensive institutional support and research assistance for the successful conduct of this study. Conflicts of Interest Authors declare no conflicts of interest. Permission for Soil Samples Collection We hereby confirm that permission has been obtained from the respective farmers for collecting soil samples from their fields for the isolation of Trichoderma spp. Data Availability Statement All data generated or analysed during this study are included in this article. References Singh, A. et al. Review on plant-Trichoderma-pathogen interaction. Int. J. Curr. Microbiol. Appl. 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Morphological and molecular characterization of Trichoderma isolates of North Bengal. J. Mycol. Plant Pathol., 41, 207–214 (2011). Pérez, A. A. et al. Selección de aislamientos de Trichoderma spp. in vitro como potenciales biofungicidas para el control de Rhizoctonia solani Kühn en papa. Agri. Scientia , 37, 21–33 (2020). Zin, N. A. et al. Biological functions of Trichoderma spp. for agriculture applications. Ann. Agric. Sci. , 65, 168–178 (2020). Raza, W. et al. Volatile and non-volatile antifungal compounds produced by Trichoderma harzianum SQR-T037 suppressed the growth of Fusarium oxysporum f. sp. niveum. Sci. Lett. , 1, 21–24 (2013). Manganiello G., Nicastro N., Caputo M., Zaccardelli M., Cardi T., and Pane C. Functional hyperspectral imaging by high-related vegetation indices to track the wide-spectrum Trichoderma biocontrol activity against soil-borne diseases of baby-leaf vegetables. Front. in Plant Sci., 12, 630059. https://doi.org/10.3389/fpls.2021.630059 (2021). Win, T.T. et al. Newly isolated strain of Trichoderma asperellum from disease suppressive soil is a potential bio-control agent to suppress Fusarium soil borne fungal phytopathogens. J. of Plant Path ., 103, 549-561. http://dx.doi.org/10.1007/s42161-021-00780-x (2021). Sehim, A.E., Hewedy, O.A., Altammar, K.A., Alhumaidi, M.S., Abd Elghaffar RY. Trichoderma asperellum empowers tomato plants and suppresses Fusarium oxysporum through priming responses. Front. in Microbio., 14, 1140378. https://doi.org/10.3389/fmicb.2023.1140378 (2023). Tables Table 1. Morphological characterization of Trichoderma isolates S.N. Isolate Colony Colour Concentric Rings Morphology Pigmentation (Lower Side) 1 Tr 1 Dark green Present Rough, spongy and raised Dark brown 2 Tr 2 Light to dark green Present Rough, spongy and raised Dark brown 3 Tr 3 Dark green Absent Smooth, flat and dense at border Whitish creamy 4 Tr 4 Light to dark green Absent Rough, spongy and raised Dark brown 5 Tr 5 Light green Absent Rough, spongy and raised Whitish creamy 6 Tr 6 Light to dark green Absent Smooth, flat and dense at border Pinkish Table 2. Mycelial weight (g) of Trichoderma isolates at different pH levels Trichoderma isolate pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 6.5 pH 7.5 pH 8.0 Tr 1 0.32 0.50 0.57 0.62 0.66 0.72 0.30 0.20 Tr 2 0.36 0.54 0.59 0.64 0.81 1.09 0.36 0.22 Tr 3 0.38 0.59 0.63 0.66 0.70 0.74 0.39 0.19 Tr 4 0.21 0.24 0.29 0.36 0.54 0.90 0.26 0.15 Tr 5 0.34 0.37 0.42 0.44 0.52 0.59 0.41 0.32 Tr 6 0.38 0.53 0.56 0.59 0.64 1.08 0.43 0.30 C.V. 1.42 1.78 3.21 4.12 2.53 1.93 3.08 4.58 Table 3. Colony diameter (in mm) of Trichoderma isolates at different temperature range Trichoderma isolates 15°C 20°C 25°C 30°C 35°C 40°C Tr 1 45.00 59.33 82.00 85.67 83.33 18.00 Tr 2 48.33 61.67 82.33 87.00 85.00 17.67 Tr 3 40.33 63.00 84.67 88.00 85.00 21.67 Tr 4 12.00 29.67 35.33 54.00 47.33 17.67 Tr 5 44.67 60.00 83.00 85.33 84.67 18.00 Tr 6 47.33 58.00 80.67 86.33 83.00 18.33 C.V. 1.05 2.585 1.954 3.033 4.772 3.517 Table 4. In vitro evaluation of antagonism of Trichoderma isolates against major plant pathogenic fungi. Trichoderma isolates A. alternata Colletotrichum spp. F. oxysporium M. phaseolina S. rolfsi Radial growth (mm) Per cent inhibition Radial growth (mm) Per cent inhibition Radial growth (mm) Per cent inhibition Radial growth (mm) Per cent inhibition Radial growth (mm) Per cent inhibition Tr 1 6.33 58.69 10.17 79.39 11.00 38.89 18.33 64.52 10.67 57.33 Tr2 6.00 60.86 14.00 71.62 10.00 44.44 22.33 56.78 9.33 62.67 Tr3 6.67 56.51 19.00 61.48 7.17 60.19 14.67 71.61 7.67 69.33 Tr 4 12.67 17.37 25.67 47.97 12.83 28.70 20.33 60.65 16.00 36.00 Tr 5 8.00 47.81 17.67 64.19 9.17 49.07 13.67 73.55 10.00 60.00 Tr 6 7.33 52.16 11.67 76.35 6.17 65.74 27.33 47.10 9.00 64.00 Control 15.33 --- 49.33 --- 18.00 --- 51.67 --- 25.00 --- C V 1.04 1.575 2.059 2.56 3.10 Additional Declarations No competing interests reported. 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Introduction","content":"\u003cp\u003eRapid growth of the global population is expected to reach 9.10\u0026nbsp;billion by 2050, necessitating 70% increase in food production to ensure food security\u003csup\u003e1\u003c/sup\u003e. However, plant diseases remain a significant challenge to agricultural productivity, leading to an estimated 16% reduction in the global crop yields\u003csup\u003e2\u003c/sup\u003e. Among these threats, soil-borne plant pathogens have emerged as a major concern, affecting the crops across tropical, sub-tropical, and temperate regions\u003csup\u003e3\u003c/sup\u003e. To combat plant diseases and enhance agricultural output, synthetic agro-chemical pesticides have been extensively used world-wide. Currently, ~\u0026thinsp;2.0\u0026nbsp;million tons of pesticides applied annually, with herbicides comprising 47.5%, insecticides 29.5%, fungicides 17.5%, and other pesticides accounting for 5.5% \u003csup\u003e4\u003c/sup\u003e. Despite their effectiveness, extensive use of chemical pesticides poses serious environmental and health risks, including soil degradation, water contamination, biodiversity loss, and food safety concerns\u003csup\u003e\u003cb\u003e5\u003c/b\u003e\u003c/sup\u003e. As a result, there is a pressing need to explore the sustainable alternatives for plant disease management.\u003c/p\u003e\u003cp\u003eBiological control i.e. biocontrol has emerged as an eco-friendly and effective strategies to mitigate plant diseases while reducing reliance on chemical pesticides. Among various bio-control agents, \u003cem\u003eTrichoderma\u003c/em\u003e sps. have gained prominence due to their remarkable ability to suppress soil-borne pathogens, enhance the plant growth, and improve soil fertility\u003csup\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sup\u003e. \u003cem\u003eTrichoderma\u003c/em\u003e-based bio-fungicides constitutes over 60% of all the commercially available bio-fungicides worldwide, underscoring their significance in modern sustainable agriculture\u003csup\u003e\u003cb\u003e7\u003c/b\u003e\u003c/sup\u003e. These fungi predominantly inhabit rhizosphere of plants, where they colonize plant roots, leaves, grain, acting as biocontrol agents and plant growth promoters\u003csup\u003e\u003cb\u003e8\u003c/b\u003e\u003c/sup\u003e. Effectiveness of \u003cem\u003eTrichoderma\u003c/em\u003e in plant disease management stems from its multifaceted mechanisms of action, including mycoparasitism, competition for nutrients, and production of antifungal metabolites \u003csup\u003e\u003cb\u003e9, 10\u003c/b\u003e\u003c/sup\u003e. Through mycoparasitism, \u003cem\u003eTrichoderma\u003c/em\u003e sps. invade and parasitize plant pathogens, effectively reducing their spread. Additionally, their competitive ability enables them to outcompete harmful fungi for essential resources such as nutrients and space\u003csup\u003e\u003cb\u003e11\u003c/b\u003e\u003c/sup\u003e. Moreover, \u003cem\u003eTrichoderma\u003c/em\u003e sps. produce variety of secondary metabolites, including pep-taibols, gliotoxin, and trichodermin, which exhibit strong antifungal properties\u003csup\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sup\u003e. Beyond pathogen suppression, \u003cem\u003eTrichoderma\u003c/em\u003e enhances plant growth by promoting root development, improving nutrient uptake, inducing systemic resistance against various plant stresses \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdaptability of \u003cem\u003eTrichoderma\u003c/em\u003e sps. to diverse environmental conditions further enhances their potential as biocontrol agent. Unlike synthetic fungicides, which have detrimental ecological effects, \u003cem\u003eTrichoderma\u003c/em\u003e-based solutions align with sustainable farming practices by minimizing chemical inputs while maintaining high crop yield \u003csup\u003e\u003cb\u003e14\u003c/b\u003e\u003c/sup\u003e. In addition, their ability to solubilize nutrients like phosphorus and produce plant hormones such as auxins, gibberellins, cytokinin\u0026rsquo;s contributes to improved plant health and productivity. \u003cem\u003eTrichoderma\u003c/em\u003e sps. exhibit the significant morphological, cultural, molecular variability, influencing their biocontrol efficacy. Morphologically, species differentiation is based on conidial structure, size and pigmentation. Culturally, different \u003cem\u003eTrichoderma\u003c/em\u003e strains exhibit the distinct growth patterns on various media, reflecting their adaptability to the environmental conditions. Molecular techniques, particularly analysis of the Internal Transcribed Spacer (ITS) region of ribosomal RNA genes, have proven essential in accurately identifying \u003cem\u003eTrichoderma\u003c/em\u003e isolates and selecting the most effective strains for biocontrol applications \u003csup\u003e\u003cb\u003e15\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEnvironmental factors such as temperature and pH significantly impact \u003cem\u003eTrichoderma\u003c/em\u003e growth, sporulation, and biocontrol efficiency. Studies indicate that temperature variations influence mycelial growth and antifungal metabolite production, while pH levels affect colonization and pathogen suppression \u003csup\u003e\u003cb\u003e16, 17\u003c/b\u003e\u003c/sup\u003e. Understanding these environmental parameters is a crucial for optimizing \u003cem\u003eTrichoderma\u003c/em\u003e applications in sustainable modern crop production. Given its exceptional biocontrol capabilities and plant growth-promoting properties, \u003cem\u003eTrichoderma\u003c/em\u003e represents a promising alternative to chemical pesticides. Its integration into agricultural management practices can significantly contributes to the sustainable food production, ensuring crop protection while safeguarding environmental and human health.\u003c/p\u003e\u003cp\u003eThus, present study focuses on \u003cem\u003eTrichoderma\u003c/em\u003e isolates collected from crop field in Kishanganj (26.0976\u0026deg; N, 87.9420\u0026deg; E) and Purnea districts (25.7771\u0026deg; N, 87.4753\u0026deg; E) of Bihar, India. Kishanganj and Purnea districts are situated in the northeastern part of Bihar, India where agriculture is the primary occupation. Region experiences diverse climatic conditions, with varying temperatures and humidity levels throughout year. These environmental variations provide an ideal setting for isolation of \u003cem\u003eTrichoderma\u003c/em\u003e sps. that are adapted to different ecological niches. By investigating morphological and molecular diversity of \u003cem\u003eTrichoderma\u003c/em\u003e isolates from these districts, the study aims to identify the strains that are particularly effective against the locally prevalent plant pathogens, such as \u003cem\u003eFusarium, Rhizoctonia\u003c/em\u003e, and \u003cem\u003ePythium\u003c/em\u003e, which cause root rot and complex wilt diseases in major field crops.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Collection and Isolation of Trichoderma Isolates\u003c/h2\u003e\u003cp\u003eRhizospheric soil samples were collected from maize, sugarcane, jute, potato, and wheat fields across different location in Kishanganj \u0026amp; Purnea districts of Bihar, India. \u003cem\u003eTrichoderma\u003c/em\u003e spp. was isolated using \u003cem\u003eTrichoderma Selective Medium\u003c/em\u003e (TSM), and their growth patterns were recorded properly. Antagonistic activity was tested on Potato Dextrose Agar (PDA), while mycelial mats were grown in \u003cem\u003ePotato Dextrose Broth\u003c/em\u003e (PDB) for DNA extraction. Media were prepared by dissolving components in distilled water and autoclaving at 121\u0026deg;C for 20 min. Soil suspensions were prepared using serial dilution method, plated on TSM and PDA, and incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Sub-culturing was performed on PDA, and isolates were identified based on conidiophore branching, phialide formation, and spore characteristics.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Morphological Characterization\u003c/h2\u003e\u003cp\u003eMorphological identification was based on cultural characterization (colony appearance and growth rate) and microscopic examination. \u003cem\u003eTrichoderma\u003c/em\u003e isolates were transferred from slants to PDA plates and incubated at 28\u0026deg;C for 24\u0026ndash;48 hours. A 5-mm mycelial disc from an actively growing colony (before conidial production) was inoculated at centre of a fresh PDA plate, which was then incubated at 30\u0026deg;C with intermittent light. Each isolate was tested in triplicate, and all procedures were conducted under the aseptic conditions. Colony traits, growth rate, \u0026amp; sporulation patterns were recorded followings\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e. Colony diameter was measured from third to seventh day to assess growth rate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Spore Size Measurement\u003c/h2\u003e\u003cp\u003eSpore size was measured using a cilika digital microscope. Spores were first focused at 10X, observed at 40X, and measured at 100X using a scale. Diameter of five spores per isolate was recorded and the average was calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Fungal Spore Count\u003c/h2\u003e\u003cp\u003eFungal spore count was determined using a haemocytometer. Drop of conidial suspension was placed on engraved grid, allowed to settle for 1\u0026ndash;2 minutes, covered with a cover slip to prevent air bubbles. Spores were counted in central square (E), consisting of 25 groups of 16 tiny squares (0.2 mm each). Spore concentration (spores/ml) was calculated using formula:\u003c/p\u003e\u003cp\u003eSpores/ml\u0026thinsp;=\u0026thinsp;N\u0026times;104, Where N is the number of spores counted in the middle square.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Morphological Characterization\u003c/h2\u003e\u003cp\u003eThe colony colour, morphology, ring formation, pigmentation, and spore size were assessed. The observations were made to categorize the isolates based on distinct morphological traits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Fungal Culture and Mycelial Preparation\u003c/h2\u003e\u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e isolates cultured in Potato Dextrose Broth (PDB) within 250 mL Erlenmeyer flasks containing 100 mL of medium. A 5 mm fungal disc from actively growing cultures was inoculated into each flask. Cultures were incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with continuous shaking at 120 rpm for 3\u0026ndash;5 day. Mycelial mats harvested using Whatman filter paper, washed three times with sterile distilled water, ground in liquid nitrogen for further processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 DNA Extraction and Purification\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted using a modified CTAB method \u003csup\u003e\u003cb\u003e19\u003c/b\u003e\u003c/sup\u003e. The ground mycelial tissue was mixed with DNA extraction buffer consisting of 100 mMTris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 1.4 M NaCl, and 2% CTAB, followed by incubation at 65\u0026deg;C for 60 min. After extraction with chloroform: isoamyl alcohol (24:1), supernatant was precipitated with isopropanol and centrifuged at 12,000 rpm for 10 min. Resulting DNA pellet was washed twice with 70% ethanol, air-dried, resuspended in 80 \u0026micro;L Tris-EDTA buffer, stored at-20\u0026deg;C. RNase treatment performed at 37\u0026deg;C to remove RNA contamination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 DNA Quantification\u003c/h2\u003e\u003cp\u003eExtracted DNA quantified using a Nano-Drop Spectrophotometer by measuring absorbance at 260 nm (OD260). Purity was assessed by calculating the OD260/OD280 ratio, and DNA integrity verified through 0.8% agarose gel electrophoresis stained with ethidium bromide, using a 100 bp DNA ladder as a molecular marker.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 PCR Amplification of ITS Region\u003c/h2\u003e\u003cp\u003eITS1-ITS4 region, including the 5.8S rDNA gene, was amplified using primer pair ITS1 (5\u0026rsquo;-TCC GTA GGT GAA CCT GCG G-3\u0026rsquo;) and ITS4 (5\u0026rsquo;-TCC TCC GCT TAT TGA TAT GC-3\u0026rsquo;) \u003csup\u003e\u003cb\u003e20\u003c/b\u003e\u003c/sup\u003e. PCR reaction mixture consisted of 4 \u0026micro;L 10X reaction buffer, 0.5 \u0026micro;L 2 mM dNTP mix, 0.25 \u0026micro;L 1 U Taq DNA polymerase, 2 \u0026micro;L of each primer (5 \u0026micro;M), 2 \u0026micro;L of 25 ng template DNA, and 32 \u0026micro;L of deionized water, making a total volume of 38 \u0026micro;L. PCR was performed with an initial denaturation at 94\u0026deg;C for 5 minutes, followed by 35 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 59\u0026deg;C for 30 seconds, and extension at 70\u0026deg;C for 2 minutes, with a final extension at 72\u0026deg;C for 7 min. Amplified PCR products analysed using 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV trans illumination. The DNA was extracted from six \u003cem\u003eTrichoderma\u003c/em\u003e isolates, and ITS-PCR analysis was conducted using ITS1 and ITS4 primers. The PCR amplification was performed to confirm the genetic variability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Effect of pH Levels and Temperature\u003c/h2\u003e\u003cp\u003eEffect of various pH levels and temperatures on growth of \u003cem\u003eTrichoderma\u003c/em\u003e isolates was studied using Completely Randomized Design (CRD) \u0026amp; replicated thrice. For pH evaluation, Potato Dextrose Broth (PDB) was prepared, and its pH was adjusted between 4.0 \u0026amp; 8.0 using 1 N HCl or 1 N NaOH. Medium was then dispensed into sterile conical flasks, autoclaved, and inoculated with a 5 mm-diameter mycelial disc from 5-day old culture. Flasks were incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for seven days, after which cultures were filtered using Whatman filter paper no. 42, dry mycelial weight was measured. For temperature evaluation, six \u003cem\u003eTrichoderma\u003c/em\u003e isolates were inoculated into the Potato Dextrose Agar (PDA) plates and incubated at temperatures ranging from 15\u0026deg;C to 40\u0026deg;C. Growth was assessed by recording radial growth measurements after 48 hours and subsequently at 24-hour intervals for 7-days. Cumulative colony diameter was computed to analyse impact of pH and temperature on \u003cem\u003eTrichoderma\u003c/em\u003e growth.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 \u003cem\u003eIn Vitro Evaluation of Antagonistic Potential of\u003c/em\u003e \u003cb\u003eTrichoderma\u003c/b\u003e \u003cem\u003espp\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eSix isolates of \u003cem\u003eTrichoderma\u003c/em\u003e spp. were evaluated for their antagonistic activity against five phyto pathogenic fungi: \u003cem\u003eS. rolfsii\u003c/em\u003e, \u003cem\u003eM. phaseolina\u003c/em\u003e, \u003cem\u003eA. alternata\u003c/em\u003e, \u003cem\u003eF. oxysporum\u003c/em\u003e, and \u003cem\u003eColletotrichum\u003c/em\u003e spp. The dual culture technique was employed, where a 5 mm disc of the test pathogen and the respective \u003cem\u003eTrichoderma\u003c/em\u003e isolate were inoculated on opposite sides of a PDA (Potato Dextrose Agar) plate. The plates were incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and radial growth inhibition of the pathogens was recorded after seven days. The percentage inhibition of mycelial growth was calculated using the formula:\u003c/p\u003e\u003cp\u003ePercent Inhibition (I%) = [(C - T)/C] \u0026times; 100\u003c/p\u003e\u003cp\u003ewhere,\u003c/p\u003e\u003cp\u003e\u003cem\u003eC\u003c/em\u003e\u0026thinsp;=\u0026thinsp;radial growth of pathogen in control plate\u003c/p\u003e\u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u0026thinsp;=\u0026thinsp;radial growth of pathogen in dual culture plate\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Morphological and Cultural Variability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological and cultural characteristics of six \u003cem\u003eTrichoderma\u003c/em\u003e isolates were studied using monoconidial cultures grown on \u003cem\u003eTrichoderma Selective Medium\u003c/em\u003e (TSM). Observations were recorded on colony colour, morphology, concentric ring formation, pigmentation on lower side of culture plates, and spore size (Table 1 \u0026amp; Fig. 1). Results indicated noticeable variation among isolates, highlighting intraspecific diversity within the genus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetailed analysis of morphological traits revealed distinct characteristics among the isolates:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony colour:\u0026nbsp;\u003c/strong\u003eThe\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eisolates exhibited the various shades of green, a characteristic traits of \u003cem\u003eTrichoderma\u003c/em\u003e sps. \u003cem\u003eTrichoderma\u003c/em\u003e 1 \u0026amp; 3 showed dark green colonies, while \u003cem\u003eTrichoderma\u003c/em\u003e 2, 4, and 6 displayed a transition from light to dark green. \u003cem\u003eTrichoderma\u003c/em\u003e 5 was characterized by a uniform light green coloration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony morphology:\u003c/strong\u003e Most isolates, including \u003cem\u003eTrichoderma\u003c/em\u003e 1, 2, 4, Tr 5, showed a rough, spongy, and raised morphology, suggesting active mycelial growth and aeration. In contrast, \u003cem\u003eTrichoderma\u003c/em\u003e 3 and 6 presented smooth, flat colonies with dense sporulation localized at the border, indicating potential differences in sporulation patterns and radial growth behaviour.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcentric ring formation:\u003c/strong\u003e Concentric rings, indicative of the distinct sporulation phases or mycelial growth cycles observed in \u003cem\u003eTrichoderma\u003c/em\u003e 1 \u0026amp; 2. Their presence may be associated with periodic nutrient availability or intrinsic sporulation rhythms. The remaining isolates (\u003cem\u003eTrichoderma\u003c/em\u003e 3, 4, 5 and 6) did not exhibit ring formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePigmentation (lower side of plate):\u003c/strong\u003e Significant differences in pigmentation were observed on reverse side of culture plates. \u003cem\u003eTrichoderma\u003c/em\u003e 1, 2, and 4 exhibited dark brown pigmentation, potentially indicating robust secondary metabolite production. \u003cem\u003eTrichoderma\u003c/em\u003e 3 and 5 showed whitish-creamy pigmentation, whereas \u003cem\u003eTrichoderma\u003c/em\u003e 6 demonstrated a distinctive pinkish hue, suggesting variability in metabolic activity among isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpore size:\u0026nbsp;\u003c/strong\u003eSpore size analysis revealed minor yet distinct differences across isolates. The largest spores were recorded in \u003cem\u003eTrichoderma\u003c/em\u003e 3 (3.74\u0026times;3.46 \u0026micro;m), while smallest were observed in \u003cem\u003eTrichoderma\u003c/em\u003e 6 (3.06 \u0026times; 2.92 \u0026micro;m). Such variations could affect spore germination, dispersal efficiency, and potential biocontrol performance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Molecular Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCR amplification of ITS region yielded a 600 bp fragment in all six isolates, indicating genetic similarity within \u003cem\u003eTrichoderma\u003c/em\u003e genus. No inter-or intra-species ITS length diversity was detected, suggesting conservation of ITS region among isolates (Fig. 2). This result implies a high degree of genetic homogeneity among isolates, reinforcing their classification within \u003cem\u003eTrichoderma g\u003c/em\u003eenus. Use of ITS-PCR as a molecular identification tool confirmed that these isolates belong to same genetic cluster, highlighting reliability of molecular methods in fungal taxonomy and genetic differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Evaluation of \u003cem\u003eTrichoderma\u003c/em\u003e isolates for pH and temperature tolerance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth and development of \u003cem\u003eTrichoderma\u003c/em\u003e isolates are significantly influenced by pH and temperature, as these factors regulate metabolic and enzymatic activities essential for biomass accumulation and sporulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of pH and temperature on mycelial growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mycelial growth was assessed across six \u003cem\u003eTrichoderma\u003c/em\u003e isolates at pH levels ranging from 4.0 to 8.0 (in 0.5-unit increments) and at six temperature levels (15\u0026deg;C, 20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C and 40\u0026deg;C). Results revealed that the significant variability among isolates, emphasizing influence of these factors on fungal physiology (Tables 2 and 3; Figure 3 and 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Optimal growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHighest biomass production was recorded at pH levels between 4.5 \u0026amp; 6.5, with \u003cem\u003eTrichoderma\u003c/em\u003e 2 achieving highest mycelial weight (1.09 g) at pH 6.5, followed by \u003cem\u003eTrichoderma\u003c/em\u003e 6 (1.08 g). A progressive increase in biomass was observed from pH 4.0 to 6.5, but growth declined significantly at extreme pH levels (4.0 \u0026amp; 8.0). While mycelial growth remained satisfactory at these extremes, sporulation notably poor, indicating that suboptimal pH conditions might inhibit the reproductive processes in \u003cem\u003eTrichoderma\u003c/em\u003e isolates (Fig. 5). Temperature played a crucial role in colony expansion, with 30\u0026deg;C identified as optimal temperature. \u003cem\u003eTrichoderma\u003c/em\u003e 3 exhibited largest colony diameter (88.00 mm), followed by Tr2 (87.00 mm) and \u003cem\u003eTrichoderma\u003c/em\u003e 6 (86.33 mm). Growth declined at temperatures below 30\u0026deg;C, with \u003cem\u003eTrichoderma\u003c/em\u003e 3 maintaining superior growth at 25\u0026deg;C (84.67 mm). At 20\u0026deg;C and 15\u0026deg;C, colony diameters were significantly reduced, with \u003cem\u003eTrichoderma\u003c/em\u003e 4 consistently showing lowest growth across all temperatures. At elevated temperatures (35\u0026deg;C and 40\u0026deg;C), growth progressively declined, with severe inhibition at 40\u0026deg;C, where \u003cem\u003eTrichoderma\u003c/em\u003e 3 was recorded the highest colony diameter (21.67 mm). These results indicate that \u003cem\u003eTrichoderma\u003c/em\u003e isolates thrive best within moderate temperature conditions and that extreme temperatures adversely affect fungal proliferation and survival.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e3.5 Effects of\u003c/strong\u003e \u003cstrong\u003eAntagonistic Potential of\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;spp. Against Major Plant Pathogenic Fungi\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll six isolates of\u0026nbsp;\u003cem\u003eTrichoderma\u003c/em\u003e spp. showed significant inhibitory effects against the tested pathogens. However, the degree of antagonism varied among isolates and pathogens(Table 4 and Figure 6).\u003c/p\u003e\n\u003ch5\u003e\u003cstrong\u003eAntagonism Against\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAlternaria alternata\u003c/strong\u003e\u003c/em\u003e\u003c/h5\u003e\n\u003cp\u003e\u003cem\u003eAlternaria alternata\u003c/em\u003e is a foliar pathogen responsible for causing leaf spots and blights in various crops. The inhibition percentage ranged between\u0026nbsp;\u003cstrong\u003e17.37% and 60.86%\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Among the isolates,\u0026nbsp;\u003cstrong\u003eTr 2\u003c/strong\u003e demonstrated the highest inhibition (\u003cstrong\u003e60.86%\u003c/strong\u003e\u003cstrong\u003e),\u003c/strong\u003e indicating its strong antagonistic potential, followed by\u0026nbsp;\u003cstrong\u003eTr 1 (58.69%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 3 (56.51%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 6 (52.16%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 5 (47.81%)\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 4\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erecorded the lowest inhibition (\u003cstrong\u003e17.37%\u003c/strong\u003e\u003cstrong\u003e),\u003c/strong\u003e suggesting its limited efficacy against\u0026nbsp;\u003cem\u003eA. alternata\u003c/em\u003e.\u003c/p\u003e\n\u003ch5\u003e\u003cstrong\u003eAntagonism Against\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eColletotrichum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;spp.\u003c/strong\u003e\u003c/h5\u003e\n\u003cp\u003e\u003cem\u003eColletotrichum\u003c/em\u003e spp. cause anthracnose disease, which affects fruits, flowers, and stems. All six\u0026nbsp;\u003cem\u003eTrichoderma\u003c/em\u003e isolates effectively inhibited\u0026nbsp;\u003cem\u003eColletotrichum\u003c/em\u003e spp. growth. The maximum inhibition was observed with\u0026nbsp;\u003cstrong\u003eTr 1 (79.39%)\u003c/strong\u003e, followed by\u0026nbsp;\u003cstrong\u003eTr 6 (76.35%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 2 (71.62%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 5 (64.19%)\u003c/strong\u003e, and\u0026nbsp;\u003cstrong\u003eTr 3 (61.48%)\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 4\u003c/strong\u003e again showed the least inhibition \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e47.97%\u003c/strong\u003e\u003cstrong\u003e).\u003c/strong\u003e The strong performance of Tr 1 and Tr 6 suggests their potential utility in managing anthracnose in crops.\u003c/p\u003e\n\u003ch5\u003e\u003cstrong\u003eAntagonism Against\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFusarium oxysporum\u003c/strong\u003e\u003c/em\u003e\u003c/h5\u003e\n\u003cp\u003e\u003cem\u003eF. oxysporum\u003c/em\u003e is a notorious soil-borne pathogen responsible for vascular wilt in numerous crops. All\u0026nbsp;\u003cem\u003eTrichoderma\u003c/em\u003e isolates significantly suppressed its growth, with inhibition ranging from\u0026nbsp;\u003cstrong\u003e28.70% to 65.74%\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The highest inhibition was recorded with\u0026nbsp;\u003cstrong\u003eTr 1 (65.74%)\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e followed by\u0026nbsp;\u003cstrong\u003eTr 3 (60.19%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 5 (49.07%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 2 (44.44%)\u003c/strong\u003e, and\u0026nbsp;\u003cstrong\u003eTr 6 (38.89%)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The lowest inhibition was again observed with\u0026nbsp;\u003cstrong\u003eTr 4 (28.70%)\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e suggesting that isolates Tr 1 and Tr 3 hold promise as biocontrol agents against\u0026nbsp;\u003cem\u003eFusarium\u003c/em\u003e wilt.\u003c/p\u003e\n\u003ch5\u003e\u003cstrong\u003eAntagonism Against\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMacrophomina phaseolina\u003c/strong\u003e\u003c/em\u003e\u003c/h5\u003e\n\u003cp\u003e\u003cem\u003eM. phaseolina\u003c/em\u003e, the causal organism of charcoal rot, was effectively suppressed by all isolates of\u0026nbsp;\u003cem\u003eTrichoderma\u003c/em\u003e. The percent inhibition ranged from\u0026nbsp;\u003cstrong\u003e47.10% to 73.55%\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e with\u0026nbsp;\u003cstrong\u003eTr 5\u003c/strong\u003e demonstrating the highest inhibition \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e73.55%\u003c/strong\u003e\u003cstrong\u003e),\u003c/strong\u003e followed by\u0026nbsp;\u003cstrong\u003eTr 3 (71.61%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 1 (64.52%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 4 (60.65%)\u003c/strong\u003e, and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 2 (56.78%)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The minimum inhibition was observed with\u0026nbsp;\u003cstrong\u003eTr 6 (47.10%)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e This suggests that Tr 5 and Tr 3 may be potent candidates for managing\u0026nbsp;\u003cem\u003eM. phaseolina\u003c/em\u003e in crops prone to dry root rot and stem rot.\u003c/p\u003e\n\u003ch5\u003e\u003cstrong\u003eAntagonism Against\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSclerotium rolfsii\u003c/strong\u003e\u003c/em\u003e\u003c/h5\u003e\n\u003cp\u003e\u003cem\u003eSclerotium rolfsii\u003c/em\u003e causes collar rot, particularly in legume crops and vegetables. All\u0026nbsp;\u003cem\u003eTrichoderma\u003c/em\u003e isolates significantly reduced its growth. The maximum inhibition was shown by\u0026nbsp;\u003cstrong\u003eTr 3 (69.33%)\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e followed by\u0026nbsp;\u003cstrong\u003eTr 6 (64.00%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 2 (62.67%)\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 5 (60.00%)\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e and\u0026nbsp;\u003cstrong\u003eTr 1 (57.33%)\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTr 4\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erecorded the minimum inhibition (\u003cstrong\u003e36.00%\u003c/strong\u003e), suggesting its comparatively lower antagonistic potential against\u0026nbsp;\u003cem\u003eS. rolfsii\u003c/em\u003e.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eResults align with previous studies by \u003csup\u003e\u003cb\u003e16\u003c/b\u003e\u003c/sup\u003e, who observed similar colony growth rates and colour variations under different temperature conditions. Singh et al. (2014) also reported that \u003cem\u003eTrichoderma\u003c/em\u003e sps. exhibit the optimal growth between 25\u0026deg;C and 30\u0026deg;C, which was consistent with the present observations. \u003csup\u003e\u003cb\u003e21\u003c/b\u003e\u003c/sup\u003e confirmed the importance of molecular tools in species identification, supporting our findings that ITS-PCR is an effective method for genetic differentiation. Similar distinctions among \u003cem\u003eTrichoderma\u003c/em\u003e species based on cultural and morphological characteristics were also observed by \u003csup\u003e\u003cb\u003e22\u003c/b\u003e\u003c/sup\u003e, who identified difference between \u003cem\u003eT. asperellum\u003c/em\u003e, \u003cem\u003eT. harzianum, T. reesei, T. hamatum\u003c/em\u003e based on the radial growth, colony morphology, pigmentation and conidial shape. These morphological differences indicate that native \u003cem\u003eTrichoderma\u003c/em\u003e isolates exhibit species-specific traits, which could influence their biocontrol potential. Presence of concentric rings in some isolates suggests that sporulation efficiency may vary based on environmental adaptation, as also observed by research findings of \u003csup\u003e\u003cb\u003e23\u003c/b\u003e\u003c/sup\u003e in their genotypic and phenotypic classification of \u003cem\u003eTrichoderma\u003c/em\u003e sps. The observed colony colour variations could be attributed to metabolic adaptations to the different soil conditions, as previously discussed by \u003csup\u003e\u003cb\u003e24\u003c/b\u003e\u003c/sup\u003e, who reported pH \u0026amp; temperature significantly influence colony pigmentation and growth.\u003c/p\u003e\u003cp\u003eThe PCR amplification of ITS region yielded a 600 bp fragment in all six isolates, indicating genetic similarity within \u003cem\u003eTrichoderma\u003c/em\u003e genus. These findings are in agreement with \u003csup\u003e\u003cb\u003e25\u003c/b\u003e\u003c/sup\u003e, who reported 98.6\u0026ndash;100% DNA similarity among \u003cem\u003eTrichoderma\u003c/em\u003e isolates based on ITS barcode analysis. \u003csup\u003e\u003cb\u003e26\u003c/b\u003e\u003c/sup\u003e also noted the high similarity among \u003cem\u003eTrichoderma\u003c/em\u003e species using ITS sequencing, reinforcing genetic homogeneity observed in the present investigations. Molecular techniques had become reliable \u0026amp; highly suitable tools for identifying microbial sps \u0026amp; for assessing genetic variation within collections \u0026amp; population \u003csup\u003e\u003cb\u003e27\u003c/b\u003e\u003c/sup\u003e. \u003csup\u003e\u003cb\u003e28\u003c/b\u003e\u003c/sup\u003e emphasized impact of environmental factors on \u003cem\u003eTrichoderma\u003c/em\u003e growth, which aligns with present observations that some isolates exhibited the morphological changes depending on culture conditions\u003c/p\u003e\u003cp\u003eDespite variation in the colony colour and sporulation, ITS-PCR results indicated that genetic uniformity. This suggests that while phenotypic plasticity exists among isolates, their genetic identity remains conserved, which aligns with findings by \u003csup\u003e\u003cb\u003e29\u003c/b\u003e\u003c/sup\u003e, who demonstrated that environmental conditions influence morphological traits while genetic markers remain stable. \u003csup\u003e\u003cb\u003e30\u003c/b\u003e\u003c/sup\u003e analysed the genetic relatedness of these isolates using RAPD (Random Amplified Polymorphic DNA) profiling with six-random primers. RAPD profiles revealed genetic diversity among the isolates, forming two distinct clusters corresponding to \u003cem\u003eTrichoderma viride\u003c/em\u003e \u0026amp; \u003cem\u003eTrichoderma harzianum\u003c/em\u003e, each further divided into five subgroups. \u003csup\u003e\u003cb\u003e15\u003c/b\u003e\u003c/sup\u003e identified \u003cem\u003eTrichoderma\u003c/em\u003e strain morphologically, microscopically, and biochemically, after which it was further analysed and confirmed using ITS sequencing molecular technique.\u003c/p\u003e\u003cp\u003eThe study confirmed that growth was entirely inhibited at 10\u0026deg;C and 35\u0026deg;C, regardless of pH \u003csup\u003e\u003cb\u003e29\u003c/b\u003e\u003c/sup\u003e. Similar findings were reported in previous studies by \u003csup\u003e\u003cb\u003e31\u003c/b\u003e\u003c/sup\u003e,\u003csup\u003e\u003cb\u003e32\u003c/b\u003e\u003c/sup\u003e where the optimal temperature range for \u003cem\u003eTrichoderma\u003c/em\u003e growth was identified as 25\u0026deg;C to 30\u0026deg;C. Notably, a temperature of 25\u0026deg;C and a pH of 5.5 were not limiting factors for \u003cem\u003eR. solani\u003c/em\u003e growth. According to \u003csup\u003e\u003cb\u003e33\u003c/b\u003e\u003c/sup\u003e these conditions actually favoured its mycelial growth \u0026amp; survival, highlighting the potential competition between \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eR. solani\u003c/em\u003e under such conditions. These results reinforce the necessity of maintaining moderate temperature and pH conditions for the optimal fungal performance. According to our results, \u003cem\u003eTrichoderma\u003c/em\u003e isolates demonstrated optimal growth at 30\u0026deg;C and a pH range of 4.5 to 6.5. Growth progressively declined beyond these condition, underscoring sensitivity of \u003cem\u003eTrichoderma\u003c/em\u003e isolates to extreme environmental variations. These findings emphasize importance of maintaining optimal temperature and pH conditions to enhance the biocontrol potential of \u003cem\u003eTrichoderma\u003c/em\u003e species in agricultural applications.\u003c/p\u003e\u003cp\u003eThe efficacy of \u003cem\u003eTrichoderma\u003c/em\u003e spp. as biocontrol agents has been well-documented across various crops and pathogens. In the present study, native \u003cem\u003eTrichoderma\u003c/em\u003e isolates exhibited significant in vitro antagonism against \u003cem\u003eSclerotium rolfsii\u003c/em\u003e, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e, \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eAlternaria alternata\u003c/em\u003e, and \u003cem\u003eColletotrichum\u003c/em\u003e spp., with isolates Tr 1, Tr 2, and Tr 3 showing superior inhibition rates. These findings are consistent with those of \u003csup\u003e\u003cb\u003e34\u003c/b\u003e\u003c/sup\u003e, who tested sixteen \u003cem\u003eTrichoderma\u003c/em\u003e strains against \u003cem\u003eRhizoctonia solani\u003c/em\u003e, \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, and \u003cem\u003eS. rolfsii\u003c/em\u003e, observing average inhibition rates of 60% for \u003cem\u003eR. solani\u003c/em\u003e and \u003cem\u003eS. sclerotiorum\u003c/em\u003e, and up to 70% for \u003cem\u003eS. rolfsii\u003c/em\u003e. The study also highlighted considerable variation among strains, with certain isolates demonstrating more aggressive antagonism. Similarly, \u003csup\u003e\u003cb\u003e35\u003c/b\u003e\u003c/sup\u003e reported that \u003cem\u003eT. asperellum\u003c/em\u003e restricted the growth of multiple fungal phytopathogens by 65\u0026ndash;74% and inhibited spore germination by 30\u0026ndash;75%. Further supporting these outcomes, \u003csup\u003e\u003cb\u003e36\u003c/b\u003e\u003c/sup\u003e demonstrated the efficacy of \u003cem\u003eT. asperellum\u003c/em\u003e against a spectrum of phytopathogens in dual culture assays, with the highest inhibition observed against \u003cem\u003eF. oxysporum\u003c/em\u003e (53.24%).\u003c/p\u003e\u003cp\u003eCollectively, these studies reinforce the relevance of native \u003cem\u003eTrichoderma\u003c/em\u003e isolates as promising agents for biological disease management. The observed variability in antagonistic potential underscores the need for precise selection and characterization of strains tailored to specific pathogens and agro ecological condition.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis present study highlights the diverse morphological \u0026amp; molecular characteristics of native \u003cem\u003eTrichoderma\u003c/em\u003e isolates, reinforcing their potential as an effective biocontrol agent. Variations in colony traits, pigmentation, and sporulation suggest adaptations to different environments, while ITS-PCR analysis confirmed that the genetic stability within genus. Despite their morphological differences, isolates share a conserved genetic identity, making them reliable candidates for agricultural applications. Given that \u003cem\u003eTrichoderma\u003c/em\u003e ability to suppress the plant pathogens and promote plant growth, its role in sustainable farming production systems is undeniable. Future research should focus on large-scale field trials, exploring its interactions with other beneficial microbes, and enhancing its biocontrol potential through genetic and biochemical studies. Additionally, developing the stable bio-formulations and stress-tolerant strains will be key to maximizing its practical applications. \u003cem\u003eTrichoderma\u003c/em\u003e thrives the best at a temperature of 30\u0026deg;C and within a pH range of 4.5 to 6.5. Growth was significantly reduced at extreme conditions, highlighting importance of maintaining optimal environmental factors for its application in modern crop production. All six \u003cem\u003eTrichoderma\u003c/em\u003e isolates possessed significant antagonistic activity against the five tested plant pathogenic fungi under in vitro conditions. Among them, Tr 1, Tr 2, Tr 3, and Tr 5 consistently showed higher efficacy across most pathogens, indicating their potential for further evaluation under greenhouse and field conditions. In contrast, Tr 4 exhibited the least inhibition in all pathogen interactions and may not be ideal for biocontrol purposes.\u003c/p\u003e\u003cp\u003eUltimately, \u003cem\u003eTrichoderma\u003c/em\u003e offers a promising, eco-friendly alternative to agro-chemical pesticides, contributing to the resilient and sustainable agricultural production systems. By further refining its use through integrated research, it can play a crucial role in improving crop health and soil fertility while minimizing environmental impacts.\u003c/p\u003e"},{"header":"6. Future scope of study","content":"\u003cp\u003eFuture of \u003cem\u003eTrichoderma\u003c/em\u003e research lies in advancing its applications for biocontrol, plant growth promotion, and sustainable agricultural production systems in broad blew of climate change scenarios. Large-scale field trials will validate its efficacy across diverse environments, while molecular studies will elucidate its antifungal mechanisms and role in systemic resistance. Genetic improvements through selective breeding and CRISPR could enhance its adaptability and efficiency. Investigating its interactions within soil microbiomes will further its role in nutrient cycling and ecosystem balance. Additionally, developing stable bio-formulations and innovative application methods will improve its practicality for farmers. With the growing emphasis on climate resilience, \u003cem\u003eTrichoderma\u003c/em\u003e also holds promise in mitigating abiotic stresses, ultimately supporting environmentally friendly and sustainable crop production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSanjeev Kumar, Sardar Sunil Singh and Chandra Shekhar Azad conceptualized the study, guided the research framework, and supervised the manuscript preparation.\u003cbr\u003e\u0026nbsp;Devendra Mandal and Mohammad Shamim were involved in data collection, field experimentation, and initial analysis. Rakesh Kumar, Pravin Kumar Upadhyay, Niru Kumari, Mahesh Kumar, Malkhan Singh Gurjar, Erayya, Satendra Singh and Subrat Keshori Behera contributed to data curation, statistical analysis, and interpretation of results. Achin Kumar, Rajeev Padbhushan, and Yalamareddy Kiranmai supported literature review, drafting of methodology, and technical validation. Brajendra Parmar contributed to reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Bihar Agricultural University, Sabour, for providing comprehensive institutional support and research assistance for the successful conduct of this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePermission for Soil Samples Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We hereby confirm that permission has been obtained from the respective farmers for collecting soil samples from their fields for the isolation of \u003cem\u003eTrichoderma\u003c/em\u003e spp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSingh, A. \u003cem\u003eet al. \u003c/em\u003eReview on plant-Trichoderma-pathogen interaction. \u003cem\u003eInt. 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Ind. https://doi.org/10.1007/978-81-322-1764-4 (2014).\u003c/li\u003e\n\u003cli\u003eSingh, S., Singh, A. K., Pradhan, B., Tripathi, S., Kumar, K. S., Chand, S., Rout, P. R., and Shahid, M. K. Harnessing Trichoderma mycoparasitism as a tool in the management of soil dwelling plant pathogens. \u003cem\u003eMicrob. Ecol.\u003c/em\u003e 87, 158. https://doi.org/10.1007/s00248-024-02472-2 (2024).\u003c/li\u003e\n\u003cli\u003eNgegba, P. M. \u003cem\u003eet al.\u003c/em\u003e Use of botanical pesticides in agriculture as an alternative to synthetic pesticides. \u003cem\u003eAgri.\u003c/em\u003e 12(5), 600. https://doi.org/10.3390/agriculture12050600 (2022).\u003c/li\u003e\n\u003cli\u003eMuthukumar, A. \u003cem\u003eet al.\u003c/em\u003e Eco-friendly management of damping-off of solanaceous crops caused by \u003cem\u003ePythium\u003c/em\u003e species. In: \u003cem\u003eCurrent Trends in Plant Disease Diagnostics and Management Practices\u003c/em\u003e. Sprin., Cham, pp. 49\u0026ndash;90. https://doi.org/10.1007/978-3-319-32396-2_4 (2016).\u003c/li\u003e\n\u003cli\u003eSingh, R. \u003cem\u003eet al.\u003c/em\u003e Biodiversity of \u003cem\u003eTrichoderma\u003c/em\u003e species in different agro-ecological habitats. In: Manoharachary, C., Singh, H. B., and Varma, A. (eds) \u003cem\u003eTrichoderma: Agricultural Applications and Beyond\u003c/em\u003e. \u003cem\u003eSoil Bio.\u003c/em\u003e 61, 21\u0026ndash;40. Springer, Cham. https://doi.org/10.1007/978-3-030-54758-5_2 (2020).\u003c/li\u003e\n\u003cli\u003eGhorbanpour, M. \u003cem\u003eet al.\u003c/em\u003e Mechanisms underlying the protective effects of beneficial fungi against plant diseases. \u003cem\u003eBiol. Cont.\u003c/em\u003e 117, 147\u0026ndash;157. https://doi.org/10.1016/j.biocontrol.2017.11.006 (2018).\u003c/li\u003e\n\u003cli\u003eBłaszczyk, L., Basińska-Barczak, A., Ćwiek-Kupczyńska, H., Gromadzka, K., Popiel, D., and Stępień, Ł. Suppressive effect of \u003cem\u003eTrichoderma\u003c/em\u003e spp. on toxigenic \u003cem\u003eFusarium\u003c/em\u003e species. \u003cem\u003ePol. J. Microbiol.\u003c/em\u003e 66, 85\u0026ndash;100. https://doi.org/10.5604/17331331.1234996 (2017).\u003c/li\u003e\n\u003cli\u003eContreras-Cornejo, H. A. \u003cem\u003eet al.\u003c/em\u003e Ecological functions of \u003cem\u003eTrichoderma\u003c/em\u003e spp. and their secondary metabolites in the rhizosphere: interactions with plants. \u003cem\u003eFEMS Micro.. Ecol.\u003c/em\u003e 92(4), 36. https://doi.org/10.1093/femsec/fiw036 (2016).\u003c/li\u003e\n\u003cli\u003eSarrocco, S. \u003cem\u003eet al.\u003c/em\u003e Genes involved in the secondary metabolism of \u003cem\u003eTrichoderma\u003c/em\u003e and the biochemistry of these compounds. \u003cem\u003eAdv. in Tricho. Bio. for Agri. Appli.\u003c/em\u003e, 113\u0026ndash;135. https://doi.org/10.1007/978-3-030-91650-3_4 (2022). \u003c/li\u003e\n\u003cli\u003eYao, X., Guo, H., Zhang, K., Zhao, M., Ruan, J., and Chen, J. \u003cem\u003eTrichoderma\u003c/em\u003e and its role in biological control of plant fungal and nematode disease. \u003cem\u003eFront. Microbiol.\u003c/em\u003e 14, 1160551. https://doi.org/10.3389/fmicb.2023.1160551 (2023). \u003c/li\u003e\n\u003cli\u003eManzar, N., Kashyap, A. S., Goutam, R. S., Rajawat, M. V. S., Sharma, P. K., Sharma, S. K., and Singh, H. V. \u003cem\u003eTrichoderma\u003c/em\u003e: advent of versatile biocontrol agent, its secrets and insights into mechanism of biocontrol potential. \u003cem\u003eSustain.\u003c/em\u003e 14(19), 12786. https://doi.org/10.3390/su141912786 (2022).\u003c/li\u003e\n\u003cli\u003eHaque, Z. \u003cem\u003eet al.\u003c/em\u003e Molecular characterization of \u003cem\u003eTrichoderma\u003c/em\u003e spp. isolates by internal transcribed spacer (ITS) region sequencing technique and its use as a biocontrol agent. \u003cem\u003eOpen Biotech.. J.\u003c/em\u003e 14(1), 70\u0026ndash;77. https://doi.org/10.2174/1874070702014010070 (2020).\u003c/li\u003e\n\u003cli\u003eGupta, V., \u003cem\u003eet al.\u003c/em\u003e Assessment of optimum temperature of \u003cem\u003eTrichoderma harzianum\u003c/em\u003e by monitoring radial growth and population dynamics in different compost manures under different temperature. \u003cem\u003eOcta J. Biosci.\u003c/em\u003e, \u003cstrong\u003e1\u003c/strong\u003e(2), 151\u0026ndash;157 (2013).\u003c/li\u003e\n\u003cli\u003eSingh, A., Shahid, M., Srivastava, M., Pandey, S., Sharma, A., Kumar, V. Optimal physical parameters for growth of \u003cem\u003eTrichoderma\u003c/em\u003e species at varying pH, temperature and agitation. \u003cem\u003eVirol. Mycol.\u003c/em\u003e, \u003cstrong\u003e3\u003c/strong\u003e, 127. https://doi.org/10.4172/2161-0517.1000127 (2014).\u003c/li\u003e\n\u003cli\u003eSamuels, G. J. Trichoderma: A review of biology and systematic of the genus. \u003cem\u003eMycol. Res.,\u003c/em\u003e 100, 923\u0026ndash;935. (1996).\u003c/li\u003e\n\u003cli\u003eDoyle, J. J. \u003cem\u003eet al. \u003c/em\u003eCATB isolation: The true story. \u003cem\u003ePlant Sci. Bull.,\u003c/em\u003e 65, 15\u0026ndash;18 (2019).\u003c/li\u003e\n\u003cli\u003eChaverri, P. \u003cem\u003eet al. \u003c/em\u003eHypocrea virens, the teleomorph of \u003cem\u003eTrichoderma virens\u003c/em\u003e. \u003cem\u003eMycologia,\u003c/em\u003e 93, 1113\u0026ndash;1124 (2001).\u003c/li\u003e\n\u003cli\u003eSavitha, M. J. \u003cem\u003eet al.\u003c/em\u003e Morphological and molecular identification of \u003cem\u003eTrichoderma \u003c/em\u003eisolates with biocontrol potential against Phytophthora blight in red pepper. \u003cem\u003ePest Manag. Hortic. Ecosyst.\u003c/em\u003e, 21, 194\u0026ndash;202 (2015).\u003c/li\u003e\n\u003cli\u003ePayal, V. K. \u003cem\u003eet al.\u003c/em\u003e Cultural and morphological characteristics of Trichoderma spp. and soil borne plant pathogens. \u003cem\u003eInt. J. Adv. Biochem. Res.,\u003c/em\u003e 8, 244\u0026ndash;249 (2024).\u003c/li\u003e\n\u003cli\u003eKumar, J. \u003cem\u003eet al.\u003c/em\u003e Morphological and molecular characterization of Trichoderma spp. from rhizosphere soil and their antagonistic activity against \u003cem\u003eFusarium\u003c/em\u003e spp. Int. J. Plant Soil Sci., 33, 100\u0026ndash;112 (2021).\u003c/li\u003e\n\u003cli\u003eMishra, P. K. \u003cem\u003eet al.\u003c/em\u003e Effect of different growth media and physical factors on biomass production of \u003cem\u003eTrichoderma viride\u003c/em\u003e. \u003cem\u003ePeople\u0026rsquo;s J. Sci. Res.,\u003c/em\u003e 8, 11\u0026ndash;16 (2015).\u003c/li\u003e\n\u003cli\u003eIsmaiel, M. H. \u003cem\u003eet al.\u003c/em\u003e Molecular and morphological identification of Trichoderma isolates from Egyptian agriculture wastes-rich soil. \u003cem\u003eSABRAO J. Breed. Genet.,\u003c/em\u003e 54, 598\u0026ndash;607 (2022).\u003c/li\u003e\n\u003cli\u003eJaisani, P. \u003cem\u003eet al.\u003c/em\u003e Morphological and molecular characterization for identification of isolates of Trichoderma spp. from rhizospheric soils of crops in middle Gujarat. \u003cem\u003eInd.Phytopath.,\u003c/em\u003e 70, 238\u0026ndash;245 (2017).\u003c/li\u003e\n\u003cli\u003eSundravadana, S. \u003cem\u003eet al.\u003c/em\u003e Exploration of molecular variability in Rhizoctonia bataticola, the incitant of root rot disease of pulse crops. \u003cem\u003eJ. Plant Prot. Res.,\u003c/em\u003e 51, 184\u0026ndash;189 (2011).\u003c/li\u003e\n\u003cli\u003eZehra, A. \u003cem\u003eet al.\u003c/em\u003e Effect of different environmental conditions on growth and sporulation of some \u003cem\u003eTrichoderma\u003c/em\u003e species. \u003cem\u003eJ. Environ. Biol.\u003c/em\u003e, 38, 197\u0026ndash;203 (2017).\u003c/li\u003e\n\u003cli\u003eAndres, P. A. \u003cem\u003eet al.\u003c/em\u003e Comparative study of different strains of Trichoderma under different conditions of temperature and pH for the control of \u003cem\u003eRhizoctonia solani.\u003c/em\u003e\u003cem\u003eAgric. Sci\u003c/em\u003e., 13, 702\u0026ndash;714 (2022).\u003c/li\u003e\n\u003cli\u003eChakraborty, B. N. \u003cem\u003eet al.\u003c/em\u003e Morphological and molecular characterization of Trichoderma isolates of North Bengal. \u003cem\u003eJ. Mycol. Plant Pathol.,\u003c/em\u003e 41, 207\u0026ndash;214 (2011).\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez, A. A. \u003cem\u003eet al.\u003c/em\u003e Selecci\u0026oacute;n de aislamientos de \u003cem\u003eTrichoderma\u003c/em\u003e spp. in vitro como potenciales biofungicidas para el control de Rhizoctonia solani K\u0026uuml;hn en papa. \u003cem\u003eAgri. Scientia\u003c/em\u003e, 37, 21\u0026ndash;33 (2020).\u003c/li\u003e\n\u003cli\u003eZin, N. A. \u003cem\u003eet al. \u003c/em\u003eBiological functions of Trichoderma spp. for agriculture applications. \u003cem\u003eAnn. Agric. Sci.\u003c/em\u003e, 65, 168\u0026ndash;178 (2020). \u003c/li\u003e\n\u003cli\u003eRaza, W. \u003cem\u003eet al.\u003c/em\u003e Volatile and non-volatile antifungal compounds produced by Trichoderma harzianum SQR-T037 suppressed the growth of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. niveum. \u003cem\u003eSci. Lett.\u003c/em\u003e, 1, 21\u0026ndash;24 (2013).\u003c/li\u003e\n\u003cli\u003eManganiello G., Nicastro N., Caputo M., Zaccardelli M., Cardi T., and Pane C. Functional hyperspectral imaging by high-related vegetation indices to track the wide-spectrum Trichoderma biocontrol activity against soil-borne diseases of baby-leaf vegetables. Front. in Plant Sci., 12, 630059. https://doi.org/10.3389/fpls.2021.630059 (2021).\u003c/li\u003e\n\u003cli\u003eWin, T.T. \u003cem\u003eet al.\u003c/em\u003e Newly isolated strain of Trichoderma asperellum from disease suppressive soil is a potential bio-control agent to suppress Fusarium soil borne fungal phytopathogens. \u003cem\u003eJ. of Plant Path\u003c/em\u003e., 103, 549-561. http://dx.doi.org/10.1007/s42161-021-00780-x (2021).\u003c/li\u003e\n\u003cli\u003eSehim, A.E., Hewedy, O.A., Altammar, K.A., Alhumaidi, M.S., Abd Elghaffar RY. Trichoderma asperellum empowers tomato plants and suppresses Fusarium oxysporum through priming responses. \u003cem\u003eFront. in Microbio.,\u003c/em\u003e 14, 1140378. https://doi.org/10.3389/fmicb.2023.1140378 (2023).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Morphological characterization of Trichoderma isolates\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eS.N.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eIsolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eColony Colour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eConcentric Rings\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eMorphology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003ePigmentation (Lower Side)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTr 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eDark green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eRough, spongy and raised\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eDark brown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTr 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eLight to dark green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eRough, spongy and raised\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eDark brown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTr 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eDark green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eSmooth, flat and dense at border\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eWhitish creamy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTr 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eLight to dark green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eRough, spongy and raised\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eDark brown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTr 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eLight green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eRough, spongy and raised\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eWhitish creamy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eTr 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eLight to dark green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eSmooth, flat and dense at border\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003ePinkish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. Mycelial weight (g) of \u003cem\u003eTrichoderma\u003c/em\u003e isolates at different pH levels\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"102%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTrichoderma\u0026nbsp;isolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003epH\u0026nbsp;4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003epH\u0026nbsp;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003epH\u0026nbsp;5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003epH\u0026nbsp;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003epH\u0026nbsp;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003epH\u0026nbsp;6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003epH 7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003epH\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eC.V.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e3.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e3.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e4.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3. Colony diameter (in mm) of \u003cem\u003eTrichoderma\u003c/em\u003e isolates at different temperature range\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eisolates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e15\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e20\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e30\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e35\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e40\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTr 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e45.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e59.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e82.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e85.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e83.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e18.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTr 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e48.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e61.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e82.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e87.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e85.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e17.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTr 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e40.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e63.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e84.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e88.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e85.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e21.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTr 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e12.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e29.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e35.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e54.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e47.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e17.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTr 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e44.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e60.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e83.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e85.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e84.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e18.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eTr 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e47.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e58.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e80.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e86.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e83.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e18.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eC.V.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2.585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e1.954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e4.772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3.517\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eTable 4.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eevaluation of antagonism of \u003cem\u003eTrichoderma\u0026nbsp;\u003c/em\u003eisolates against major plant pathogenic fungi.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"963\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eisolates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cem\u003eA.\u0026nbsp;alternata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cem\u003eColletotrichum\u0026nbsp;spp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cem\u003eF.\u0026nbsp;oxysporium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cem\u003eM.\u0026nbsp;phaseolina\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cem\u003eS.\u0026nbsp;rolfsi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eRadial\u0026nbsp;growth\u0026nbsp;(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003ePer cent\u0026nbsp;inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eRadial\u0026nbsp;growth\u0026nbsp;(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003ePer cent\u0026nbsp;inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eRadial\u0026nbsp;growth\u0026nbsp;(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003ePer cent inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eRadial\u0026nbsp;growth\u0026nbsp;(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003ePer\u0026nbsp;cent inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eRadial\u0026nbsp;growth\u0026nbsp;(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003ePer cent\u0026nbsp;inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e58.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e79.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e11.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e38.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e18.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e64.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e57.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTr2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e60.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e14.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e71.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e22.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e56.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e9.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e62.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTr3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e56.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e19.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e61.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e7.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e60.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e14.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e71.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e7.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e69.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e12.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e17.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e25.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e47.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e12.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e28.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e20.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e60.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e16.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e36.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTr\u0026nbsp;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e8.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e47.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e17.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e64.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e9.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e49.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e13.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e73.55\u003c/p\u003e\n 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76px;\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e18.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e51.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e25.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eC\u0026nbsp;V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colony colour, Morphology, Pigmentation, Concentric ring formation, spore size","lastPublishedDoi":"10.21203/rs.3.rs-6989372/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6989372/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e species are widely recognized for their biocontrol potential and ability to promote plant growth. This study aimed to evaluate the morphological, cultural, molecular, and antagonistic variability of native \u003cem\u003eTrichoderma\u003c/em\u003e isolates under laboratory conditions. Six isolates were collected from different crops and agro ecological zones, and their growth characteristics were assessed using Trichoderma Selective Medium (TSM). Morphological characterization was conducted based on colony colour, morphology, pigmentation, concentric ring formation, and spore size, while molecular characterization using ITS marker analysis confirmed the genetic identity of the isolates. The results revealed considerable morphological and cultural variability among the isolates. Colony colours ranged from dark green to light green, with most showing rough, spongy, and raised morphology; two isolates (Tr 1 and Tr 2) exhibited prominent concentric ring formation, indicating higher sporulation. Reverse side pigmentation varied from dark brown to whitish creamy and pinkish, reflecting metabolic diversity. Spore sizes ranged from 3.06 \u0026times; 2.92 \u0026micro;m (Tr 6) to 3.74 \u0026times; 3.46 \u0026micro;m (Tr 3), showing moderate intraspecific variation. Despite these differences, PCR amplification of the ITS region consistently yielded a\u0026thinsp;~\u0026thinsp;600 bp product across all isolates, confirming genetic uniformity within the \u003cem\u003eTrichoderma\u003c/em\u003e genus. Environmental adaptability studies indicated that \u003cem\u003eTrichoderma\u003c/em\u003e isolates thrived best at 30\u0026deg;C and in a pH range of 4.5\u0026ndash;6.5, with significantly reduced growth under extreme conditions. In vitro antagonistic assays using the dual culture technique demonstrated that all isolates significantly inhibited the growth of major soil-borne pathogens including \u003cem\u003eSclerotium rolfsii\u003c/em\u003e, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e, \u003cem\u003eAlternaria alternata\u003c/em\u003e, \u003cem\u003eFusarium oxysporum\u003c/em\u003e, and \u003cem\u003eColletotrichum\u003c/em\u003e spp. The highest inhibition percentages were recorded with isolates Tr 1, Tr 2, and Tr 3, indicating their strong biocontrol efficacy. These findings underscore the potential of morphologically and genetically diverse native \u003cem\u003eTrichoderma\u003c/em\u003e isolates as promising biocontrol agents under variable agro-climatic conditions, and support their integration into sustainable plant disease management strategies.\u003c/p\u003e","manuscriptTitle":"Assessment of the morphological, molecular and environmental adaptability of the native Trichoderma isolates for biocontrol applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 10:55:01","doi":"10.21203/rs.3.rs-6989372/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad5d6a6e-7218-4593-ad6c-82d591053cd6","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51448122,"name":"Biological sciences/Biotechnology"},{"id":51448123,"name":"Biological sciences/Microbiology"},{"id":51448124,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-07-29T04:38:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 10:55:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6989372","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6989372","identity":"rs-6989372","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}