Compatibility analysis of Trichoderma koningii with selected systemic fungicides under laboratory conditions

Introduction

Biological control has become a cornerstone of sustainable agriculture, providing an environmentally safe alternative to synthetic pesticides. Among beneficial microorganisms, fungi of the genus Trichoderma are particularly notable due to their diverse antagonistic strategies, including mycoparasitism, nutrient and space competition, production of antimicrobial compounds and induction of plant defense mechanisms (Mukherjee et al., 2022; Contreras-Cornejo et al., 2016) [24,7] . These filamentous fungi are widely distributed across agricultural soils, decomposing plant residues, forest litter and organic waste environments. Their ecological success is attributed to rapid growth, efficient substrate utilization, opportunistic behavior and prolific production of spores and bioactive metabolites, enabling effective rhizosphere colonization and pathogen suppression (Alexander, 1961; Singh et al., 2009) [2,35] . Research over the past decades has demonstrated that Trichoderma not only suppresses pathogens but also enhances plant growth, nutrient uptake and soil health (Harman et al., 2004; Regragui & Lahlou, 2012) [15,28] . Different species exhibit multiple antagonistic mechanisms, including enzymatic degradation of pathogen cell walls, antibiosis, nutrient competition and environmental modification, contributing to improved plant physiology and the establishment of a favorable rhizosphere microbial community. Among Trichoderma species, Trichoderma koningii is recognized as an effective biocontrol agent for managing fungal diseases and is often suggested as an alternative to chemical fungicides (Samuels et al., 2006; Oyesola et al., 2024). It exerts its biocontrol potential through mycoparasitism, competition, antibiosis via secondary metabolites and induction of systemic resistance in plants. In addition, T. koningii promotes plant growth and nutrient availability by solubilizing phosphorus and iron through organic acid and siderophore production (Gutiérrez-Moreno et al., 2025) [12] and synthesizes antibiotics such as gliotoxin and harzianic acid, which inhibit the growth of soilborne pathogens like Gaeumannomyces graminis var. tritici (Simon et al., 1988) [33] . Despite promising results under laboratory and greenhouse conditions, Trichoderma-based biocontrol agents often fail to achieve consistent efficacy in field settings. A major factor is the incompatibility between fungal bioagents and commonly applied synthetic fungicides. For successful integration into disease management programs, a biocontrol agent must remain physiologically active and competitive in the presence of these chemicals (Desai et al., 2002; Khandelwal et al., 2012) [8,17] . Fungicides vary in their toxicity depending on their active ingredients, formulations, dosages and the sensitivity of specific Trichoderma isolates (Abeysinghe et al., 2019) [1] . Therefore, assessing fungicide compatibility for individual isolates is critical before recommending them for field use (Mukherjee et al., 2020) [23] . While compatibility studies are available for species such as T. harzianum and T. asperellum, there is limited information on the response of T. koningii to systemic fungicides. Given its strong antagonistic activity, production of cell-walldegrading enzymes and antifungal secondary metabolites, T. koningii is a promising candidate for managing soil-borne and foliar pathogens (Zhang et al., 2018) [39] . However, its practical field application requires a careful evaluation of fungicide interactions to avoid potential inhibition. Therefore, the present study, entitled Compatibility Analysis of Trichoderma koningii with Selected Systemic Fungicides Under Laboratory Conditions was undertaken to evaluate the tolerance and compatibility of T. koningii with selected systemic fungicides. The findings are expected to guide the selection of compatible fungicide-bioagent combinations, support integrated disease management strategies and enhance the bioefficacy and commercial use of T. koningii under field conditions.

Materials and Methods

Source of Trichoderma culture

Rhizospheric soil was collected from various crops at the College of Agriculture, Baramati farm to isolate Trichoderma koningii. The isolation of Trichoderma koningii was performed using the serial dilution plate technique as described by Johnson and Curl (1972). Soil samples were cultured on Potato Dextrose Agar (PDA) and Trichoderma Selective Medium (TSM) to promote fungal growth while minimizing contamination. Emerging fungal colonies were initially screened based on characteristic Trichoderma morphology, including rapid growth, colony texture and pigmentation. Species-level identification of T. koningii was carried out by examining both macroscopic and microscopic features, focusing on colony morphology, growth patterns and the structure of conidiophores, phialides and conidia, following the taxonomic keys of Samuels et al. (2012) [30] and Kubicek et al. (2008) [18] . To ensure the purity of cultures, each isolate was purified using the hyphal tip method (Tuite, 1969) [37] . Confirmed T. koningii isolates were preserved throughout the study by periodic sub-culturing on PDA and TSM slants under aseptic conditions, ensuring their viability, morphological stability and consistent biocontrol potential.

Fungicides

Seven systemic fungicides-Fosetyl-Al 80% WP, Tebuconazole 25.9% EC, Fluxapyroxad 333 g/L FS, Difenoconazole 25% EC, Kresoxim-methyl 44.3% SC, Propiconazole 25% EC and Hexaconazole 5% EC-were procured from the Plant Pathology Department, Dr. Sharadchandra Pawar College of Agriculture, Baramati and used in the study.

In vitro compatibility of Trichoderma koningii with fungicides

The compatibility of Trichoderma koningii with selected systemic fungicides was evaluated using the Standard Poisoned Food Technique as described by Nene and Thapliyal (1993) [25] . Fungicide concentrations were calculated based on their active ingredient content and incorporated into sterilized, cooled Potato Dextrose Agar (PDA). About 20 ml of the amended medium was poured into 90 mm sterile Petri plates and allowed to solidify. Three replicates were maintained for each fungicide and concentration. A 5 mm mycelial disc from a 7-day-old actively growing T. koningii culture was placed at the center of each plate under aseptic conditions. Plates were incubated at 28 ± 1 °C, while uninoculated PDA plates without fungicide served as controls. Radial mycelial growth was measured once the control plates reached the edge of the Petri dish. Percentage inhibition of growth for each treatment was calculated relative to the control using Vincent's formula (1927) [38] .

Where, I = Percent growth inhibition C = Radial growth of fungus in control plate T = Radial growth of fungus in treated plate

The compatibility categories were determined based on the percentage of mycelial growth inhibition-Saha et al. (2023) [29]

Inhibition

Nature of compatibility 0-30% Highly compatible 31-60%

Moderately compatible 61-90% Slightly compatible 91-100% Non-compatible

Experimental Design and Statistical Analyses

The experiment was conducted in Completely Randomized Design (CRD) with 8 treatments and three replications.

Technique used for conducting this experiment was Poison Food technique (Nene & Thapliyal, 1993) [25] The data gathered in this study was analyzed by using the appropriate statistical methods as explained by Panse and Sukhatme (1967) [27] . The standard error (SE) and critical difference (CD) at the 5% significance level (P = 0.05) were calculated to compare treatment means.

Results and Discussion

As presented in Table These findings align with previous reports. Haritha (2003) [14] observed the incompatibility of Hexaconazole 5% EC with Trichoderma species, which was corroborated by Manandhar et al. (2020) [21] . Haque et al. (2023) [13] reported non-compatibility of Propiconazole 25% EC with several Trichoderma species, while Bagwan (2010) [4] found T. viride and T. harzianum highly sensitive to Tebuconazole and Propiconazole. Saha et al. (2023) [29] identified Kresoxim-methyl 44.3% as safe for T. asperelloides, and Dinkwar et al. (2023) [9] reported high compatibility of Fluxapyroxad 333 g/L FS with various Trichoderma isolates. Arain et al. (2022) [3] found no growth of T. harzianum with Difenoconazole 25% EC and Propiconazole 25% EC. Similarly, Bharadwaz et al. (2023) [6] reported total incompatibility of Tebuconazole, Propiconazole, and Hexaconazole with T. viride. Comparable results have also been documented by Sharma et al. (2016) [32] , Sonavane and Venkataravanappa (2017) [36] , Dutta et al. (2017) [10] , Gunda et al. (2018) [11] , Meena Ravindra et al. (2018) [22] , Kumar et al. (2019) [19] , Maheshwary et al. (2020) [20] , Singh et al.

(2021) [34] and Bai et al. (2022) [5] .

Tables Table 1: In-vitro effect of systemic fungicides on the growth of Trichoderma koningii.

Conclusion

Compatibility analysis of Trichoderma koningii with selected systemic fungicides under laboratory conditions revealed considerable variation in fungal tolerance. At half of the recommended concentrations, Fluxapyroxad 333 g/L FS and Kresoxim-methyl 44.3% SC were highly compatible with T. koningii, showing minimal inhibition of mycelial growth i.e., 4.81 and 23.89% respectively. When applied at the recommended concentrations, Kresoxim-methyl 44.3% SC exhibited moderate compatibility, indicating a slight increase in inhibitory effect (33.70%) with higher doses. In contrast, Fosetyl-Al 80% WP was slightly compatible at both half and full recommended concentrations with 63.33 and 68.89 % growth inhibition, demonstrating moderate suppression of fungal growth while still allowing T. koningii to survive and retain its activity. These results suggest that the degree of compatibility of systemic fungicides with T. koningii is both fungicide-specific and concentrationdependent, highlighting the importance of selecting appropriate fungicides when integrating chemical and biological disease management strategies.

Acknowledgement

AcknowledgmentsI sincerely acknowledge Dr. Sharadchandra Pawar College of Agriculture, Baramati, for providing the essential facilities that enabled the successful completion of this research.

References

1. Abeysinghe S, Bhattacharyya D, Ghag SB, Savaliya SD. Effect of systemic fungicides on growth and sporulation of Trichoderma spp. under in vitro conditions. J Mycol Plant Pathol. 2019;49(3):305-312.
2. Alexander M. Introduction to soil microbiology. New York: John Wiley and Sons; 1961.
3. Arain U, Dars MJ, Ujjan AA, Bozdar HB, Rajput AQ, Shahzad S. Compatibility of myco-fungicide isolate (Trichoderma harzianum Rifai) with fungicides and their in-vitro synergism assessment. Pak J Phytopathol. 2022;34(2):140-147.
4. Bagwan NB. Evaluation of Trichoderma compatibility with fungicides, pesticides, organic cakes and botanicals for integrated management of soil borne diseases of soybean (Glycine max (L.) Merril). Int J Plant Prot. 2010;3(2):206-209.
5. Bai AT, Vibha JB, Nair R, Upadhyay A. Compatibility of Trichoderma viride with commonly used fungicides in management of early blight pathogen of tomato. Pharma Innov J. 2022;11:1734-1736.
6. Bharadwaz P, Nath BC, Chetia R, Saikia S, Bora P, Bhattacharyya PN. in vitro studies on compatibility of Trichoderma viride with commonly used agrochemicals in vegetable cropping system. Pest Manag Hortic Ecosyst. 2023;29(1):136-143.
7. Contreras-Cornejo HA, Macías-Rodríguez L, AlfaroCuevas R, López-Bucio J. Trichoderma spp. improve growth of Arabidopsis seedlings under salt stress through enhanced root development and auxin production. Microbiol Res. 2016;183:75-83.
8. Desai S, Raut JG, Ingle ST. Compatibility of biocontrol agents with fungicides used in plant disease management. Indian Phytopathol. 2002;55(4):473-475.
9. Dinkwar GT, Yadav VK, Kumar A, Nema S, Mishra S. Compatibility of fungicides with potent Trichoderma isolates. Int J Plant Soil Sci. 2023;35(18):1934-1948.
10. Dutta P, Kakati N, Das A, Kaushik H, Boruah S, Bhowmick P, Hazarika GN. Trichoderma pseudokoningii showed compatibility with certain commonly used inorganic pesticides, fertilizers and sticker-cum-spreaders. Int J Curr Microbiol Appl Sci. 2017;6(2):140-146.
11. Gunda VNS, Kiran M, Thara SS, Jyothi KR. Studies on compatibility of biocontrol agents with chemical fungicides for integrated management of Alternaria leaf spot of cabbage. J Pharmacogn Phytochem. 2018;7(5):2974-2977.
12. Gutiérrez-Moreno K, Olguín-Martínez AI, MontoyaMartínez AC, de los Santos-Villalobos S. Trichoderma in sustainable agriculture and challenges related to its effectiveness. Diversity. 2025;17(10):734.
13. Haque Z, Gupta N, Rajana RN. Compatibility of multifacial isolates of Trichoderma species with six common fungicides used against soil-borne fungal pathogens. J Mycopathol Res. 2023;61(4):545-552.
14. Haritha V. Studies on the management of damping-off disease incited by Pythium aphanidermatum (Edson) Fitzp. in tobacco nurseries [thesis]. Hyderabad: Acharya N.G. Ranga Agricultural University; 2003.
15. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichoderma species-opportunistic, avirulent plant symbionts. Nat Rev Microbiol. 2004;2(1):43-56.
16. Johnson LF, Curl EA. Methods for research on the ecology of soil-borne plant pathogens. Minneapolis: Burgess Publishing Co.; 1972.
17. Khandelwal AK, Singh A, Yadav RN. Compatibility of Trichoderma spp. with commercial fungicides for enhanced disease management. Int J Plant Prot. 2012;5(2):308-313.
18. Kubicek CP, Komon-Zelazowska M, Druzhinina IS. Fungal genus Trichoderma: from food spoilage to enzyme production and biocontrol. Curr Opin Biotechnol. 2008;19(4):428-435.
19. Kumar A, Bansal RD, Chelak YK. Compatibility of Trichoderma viride with fungicides for plant disease management. Int J Pure Appl Biosci. 2019;7(3):44-51.
20. Maheshwary N, Gangadhara Naik B, Amoghavarsha Chittaragi M, Naik SK, Nandish M. Compatibility of Trichoderma asperellum with fungicides. Pharma Innov J. 2020;9(8):136-140.
21. Manandhar S, Timila R, Karkee A, Gupt S, Baidya S. Compatibility study of Trichoderma isolates with chemical fungicides. J Agric Environ. 2020;21:9-18.
22. Meena Ravindra, Arsia SK, Jain YK, Dongre M. Compatibility of fungicides with Trichoderma viridae against wilt caused by Fusarium udum. Int J Agric Sci. 2018;10(5):5268-5271.
23. Mukherjee PK, Horwitz BA, Kenerley CM, Schmoll M. Trichoderma research in the genome era. Annu Rev Phytopathol. 2020;58:105-123.
24. Mukherjee PK, Mehetre ST, Sherkhane PD, Verma M. Advances in Trichoderma research in the post-genomic era. Fungal Biol Rev. 2022;38:100-110.
25. Nene YL, Thapliyal PN. Fungicides in plant disease control. 3rd ed. New Delhi: Oxford & IBH Publishing; 1993. p. 531-532.
26. Oyesola OL, Tonjock RK, Bello AO, Taiwo OS, Obembe OO. Trichoderma: A review of its mechanisms of action in plant disease control. Preprints. 2024. doi:10.20944/preprints202405.1378.v1
27. Panse VG, Sukhatme PV. Statistical analysis for agricultural workers. New Delhi: ICAR; 1967.
28. Regragui A, Lahlou H. Effect of Trichoderma isolates on growth and nutrient uptake of plants. Afr J Agric Res. 2012;7(32):4532-4539.
29. Saha S, Pharate S, Thosar RU, Chavan V. Compatibility of Trichoderma asperelloides with fungicides controlling downy mildew and powdery mildew in grapes. Grape Insight. 2023;1(1):32-36.
30. Samuels GJ, Dodd SL, Lu BS, Petrini O. The Trichoderma koningii aggregate species. Stud Mycol. 2012;75:131-159.
31. Samuels GJ, Dodd SL, Lu BS, Petrini O, Schroers HJ, Druzhinina IS. The Trichoderma koningii aggregate species. Stud Mycol. 2006;56:67-133.
32. Sharma D, Sharma R, Puri S. Compatibility of biocontrol agents with fungicides. Int Q J Life Sci. 2016;11(4):2863-2866.
33. Simon A, Dunlop RW, Ghisalberti EL, Sivasithamparam K. Trichoderma koningii produces a pyrone compound with antibiotic properties. Soil Biol Biochem. 1988;20(4):263-264.
34. Singh M, Singh R, Mishra P, Sengar RS, Shahi UP. in vitro compatibility of Trichoderma harzianum with systemic fungicides. Int J Chem Stud. 2021;9(1):28842888.
35. Singh N, Sharma P, Singh AK. Biocontrol potential of Trichoderma species and their growth characteristics. Ann Plant Prot Sci. 2009;17(2):389-392.
36. Sonavane P, Venkataravanappa. Compatibility studies of Trichoderma harzianum isolate with fungicides used against soil-borne disease in Coorg mandarin-peppercoffee plantations. Int J Curr Microbiol Appl Sci. 2017;6(8):346-354.
37. Tuite J. Plant pathological methods: fungus and bacteria. Vol. 1. Minneapolis: Burgess Publishing Co.; 1969. p. 238.
38. Vincent JM. Distortion of fungal hyphae in the presence of certain inhibitors. Nature. 1927;159-180.
39. Zhang F, Ge H, Zhang W, Zhang X. Antifungal activity and mechanisms of Trichoderma koningii against soilborne phytopathogens. Microbiol Res. 2018;215:1-9.