In vitro suppression of major soil-borne pathogens

Introduction

The agricultural productivity across the globe is threatened by the incidence of various pests and diseases with estimated yield losses of 20-40% each year (Savary et al., 2019) [10] . Plant diseases reduce yields by 21-30% in several important crops worldwide (El-Saadony et al., 2022) [2] . Plant diseases caused by various seed and soil-borne pathogens determine the dynamics of plant populations in both natural and agricultural environments. Soil-borne fungal pathogens are some of the most persistent and destructive constraints to global agricultural productivity. These pathogens survive for extended periods in soil as sclerotia, microsclerotia or mycelial fragments and initiate infection at the root, crown or collar regions of susceptible hosts, often leading to severe seedling mortality and substantial yield losses. Necrotrophic soil inhabitants such as Rhizoctonia solani, Macrophomina phaseolina and Sclerotium rolfsii are among the most damaging causal agents of sheath blight of rice, charcoal rot of maize, and stem rot of groundnut, respectively. Their wide host range, ability to survive under diverse agro-ecological conditions, and capacity to persist in soil for many years make them particularly challenging to manage (Panth et al., 2020) [9] . Yield losses caused by soil-borne pathogens can reach 50-75% in several major crops, especially under environmental conditions that favour pathogen growth, survival, and infection processes (Savary et al., 2019; El-Saadony et al., 2022) [10,2] . The resilience of these pathogens arises from their hardy survival structures-sclerotia and microsclerotia-which enable them to withstand unfavourable conditions and serve as primary inoculum across seasons. Once established, these pathogens cause root rot, collar rot, wilt, damping-off, and stem blight symptoms that severely impair plant growth and productivity. Host plant resistance is an ideal and eco-effective strategy for managing soil-borne diseases. However, frequent pathogenic variability and the necrotrophic nature of these fungi often limit the [8] . Hence, integrated disease management approaches that combine host resistance with cultural, biological and chemical strategies are essential for achieving reliable disease suppression. Among these, chemical fungicides remain a key component due to their rapid and broad-spectrum action, ease of use, and effectiveness in reducing earlyseason disease establishment (Machado et al., 2017) [6] . Seed treatment and soil application with fungicides are widely adopted to protect emerging seedlings from soil borne inoculum and to reduce primary infection foci. The efficacy of fungicides, however, varies with active ingredient, formulation, concentration and the specific pathogen targeted (Lamichhane et al., 2020) [5] . Therefore, laboratory-based evaluations of fungicide performance under controlled conditions are critical before recommending field-level applications (Maitlo et al., 2014) [7] .

In this context, the present study was undertaken to evaluate the in vitro efficacy of commonly used fungicides against three major soil-borne fungal pathogens-R. solani, M. phaseolina and S. rolfsii-to identify the most effective chemistries for their management.

Materials and Methods

Evaluation of fungicides against Fusarium spp. under in vitro conditions

Six fungicides (Table 2), commonly used for seed treatment were selected from the pesticides recommended as per the Central Insecticides Board & Registration Committee, India (CIB & RC, 2024) [1] were tested for their efficacy against five pathogens (Table 1) at six concentrations viz., 100 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL, 1500 µg/mL and 2000 µg/mL using the poisoned food technique (Nene and Thapliyal, 1993) under in vitro conditions. Stock solution of 100000 ppm concentration was prepared using 10 ml of sterilized distilled water. Desired concentration of fungicide was obtained by diluting the stock solution using the following formula

Where, C1 = concentration of the stock solution (mg/L), V1 = volume of the stock solution to be added (mg/L), C2 = desired concentration (mg/L) and V2 = volume of Potato Dextrose Agar (PDA) in which fungicide is to be amended (ml).

The required quantity of each fungicide was added separately to the sterilized PDA medium, mixed thoroughly and poured in sterilized 90 mm diameter glass Petri plates and allowed to solidify. Three replications were maintained.

A control plate was also maintained, where medium was not supplemented with any fungicides. Each plate was inoculated with 5 mm discs of 5 days old pathogen cultures taken with the help of sterilized cork borer and incubated at 25 ± 2 °C for 7 days. Mycelial growth of the pathogen was recorded daily in the control plate starting from the initiation of the pathogen growth in correspondence to treatment plates till the fungal growth was full in control. Based on the observations recorded, the per cent growth inhibition will be calculated by the formula as mentioned (Vincent, 1947)-I = (C-T) ×100/C where I = Per cent inhibition of mycelial growth over control C = Mycelial growth of the pathogen in control (mm) T = Mycelial growth of the pathogen in treatment (mm)

Statistical Analysis

The data collected were analysed by analysis of variance (ANOVA) using the Statistical Package for Agricultural Scientists (OPSTAT). Duncan Multiple Range Test (DMRT) was used to analyse the data.

Results and Discussion

The six fungicides showed significant variation in their efficacy against the five pathogens with distinct inhibition pattern noted for each pathogen.

Evaluation of fungicides on mycelial growth of Rhizoctonia solani under in vitro conditions

All the fungicides tested in this study significantly (p≤0.05) inhibited the mycelial growth of R. solani at varying concentrations when compared to the untreated control (Figure 1). Notably, carboxin 37.5% + thiram 37.5% DS and prochloraz 24.4% + tebuconazole 12.1% EW showed complete inhibition (100%) of mycelial growth at all concentrations ranging from 100 to 2000 µg/mL, indicating their broad-spectrum and potent fungitoxic action. Fungicides captan 50 WP and thiophanate methyl 450 g/L + pyraclostrobin 50 g/L FS also showed considerable suppression. Thiram 75% DS was the least effective with o% inhibition at 100-500 µg/mL. However, once the fungicide concentration was at 1000 µg/mL it was able to completely inhibit the mycelial growth. On the other hand, the effectiveness of Tebuconazole 5.36% FS was gradual across the concentrations with 81% inhibition at 1000 µg/mL followed by 85% and 100% inhibition at higher concentrations. Recently, Vittal et al. (2025) [14] also demonstrated the broad-spectrum effectiveness of carboxin 37.5% + thiram 37.5% DS and prochloraz 24.4% + tebuconazole 12.1% EW against three Fusarium species (Vittal et al., 2025) [14] .

Evaluation of fungicides on mycelial growth of Macrophomina phaseolina under in vitro conditions.

In case of M. phaseolina, significant differences in the antifungal activity of the tested fungicides were observed with carboxin 37.5% + thiram 37.5% DS being the most effective fungicide achieving complete (100%) inhibition of mycelial growth at concentrations ranging from 250 to 2000 µg/mL (Figure 2). This was followed by thiophanate methyl 450 g/L + pyraclostrobin 50 g/L FS exhibiting comparable inhibition at concentrations above 500 µg/mL. Except for thiram 75% DS, all the fungicides were effective against M. phaseolina at 1000 µg/mL and above concentrations. The mycelia showed 0% inhibition at 100-500 µg/mL concentrations and 11% inhibition at 1000 µg/mL concentrations indicating that thiram was ineffective against M. phaseolina. This clearly indicated the superior efficacy of thiram when used in combination with carboxin and also suggested the use of integrated management practices against soil-borne pathogens such as M. phaseolina (Vittal et al., 2025) [14] . Recent studies by Khamari and Patra (2018) [4] and Karibasappa et al. (2020) [3] also confirmed the efficacy of the combination fungicide carboxin + thiram against M. phaseolina under in vitro conditions

Evaluation of fungicides on mycelial growth of Sclerotium rolfsii under in vitro conditions

All the tested fungicides significantly (p≤0.05) suppressed the mycelial growth of S. rolfsii across the concentration gradient when compared to the control (Figure 3). Among them, tebuconazole 5.36% FS, carboxin 37.5% + thiram 37.5% DS, prochloraz 24.4% + tebuconazole 12.1% EW and thiophanate methyl 450 g/L + pyraclostrobin 50 g/L FS completely inhibited mycelial growth (100%) at concentrations ranging from 250-2000 µg/mL. Interestingly, tebuconazole performed better against S. rolfsii compared to R. solani and M. phaseolina even at lower concentrations of 100-500 µg/mL. This might be because S. rolfsii grows fast with soft, actively expanding hyphae, hat depend on continuous membrane (sterol) synthesis. Tebuconazole, being a DMI inhibits ergosterol synthesis, might be blocking this thus quickly slowing the fungal growth. In contrast, M. phaseolina and R. solani produce thicker and denser mycelia and early survival structures compared to S. rolfsii, reducing their sensitivity to tebuconazole in PDA. Thiram 75% DS followed by Captan 50 WP however showed comparatively lower inhibition, indicating variability in fungitoxicity among the formulations. These results align with Shirsole et al. (2019) [11] , who reported that Carboxin + Thiram completely inhibited S. rolfsii under in vitro conditions. Similarly, Vamshi et al. (2019) [12] observed 100% inhibition of S. rolfsii growth with Thiram and Tebuconazole, supporting the current findings. The six fungicides showed clear differences in their sensitivity patterns against the three pathogens, which might be attributing to their contrasting biology and different fungitoxic potential of the fungicide molecules. Among them, carboxin 37.5% + thiram 37.5% DS and prochloraz 24.4% + tebuconazole 12.1% EW consistently produced complete inhibition across a wide concentration range, reflecting their strong systemic action and multi-site interference which agrees with earlier reports on other necrotrophs (Khamari and Patra, 2018; Vittal et al., 2025) [18, 14] .

Conclusion

The study revealed that fungicides varied significantly in their efficacy against major soil-borne pathogens under in vitro conditions. Among the tested fungicides, Carboxin 37.5% + Thiram 37.5% DS and Prochloraz 24.4% + Tebuconazole 12.1% EW showed complete inhibition of mycelial growth across all concentrations, indicating their superior and broad-spectrum antifungal activity. These formulations can be effectively recommended to manage soil-borne fungal pathogens, ensuring healthy crop establishment and improved productivity.

Acknowledgement

AcknowledgementThe authors are thankful to the Microbial Culture Collection Centre, Department of Plant Pathology, College of Agriculture, Rajendranagar and Institute of Biotechnology, Professor Jayashankar Telangana Agricultural University, Rajendranagar, Hyderabad, Telangana, India for providing the microbial cultures and facilities for conducting the experiments.

References

1. CIB & RC. Major uses of pesticides. Central Insecticide Board & Registration Committee, Faridabad (Haryana), India; 2024. p. 1. Available from: https://ppqs.gov.in/sites/default/files/fungicides_31.03.2 024.pdf (accessed June 2025).
2. El-Saadony MT, Saad AM, Soliman SM, Salem HM, Ahmed AI, Mahmood M, et al. Plant growth-promoting microorganisms as biocontrol agents of plant diseases: mechanisms, challenges and future perspectives. Front Plant Sci. 2022;13:923880-923880.
3. Karibasappa CS, Bhat BN, Rao SC. Evaluation of potential fungicides, botanicals, biocontrol agents and their combination against Macrophomina phaseolina inciting root rot of sesame. Int J Chem Stud. 2020;8(2):1765-1771.
4. Khamari B, Patra C. Evaluation of potentiality of different fungitoxicants against Macrophomina phaseolina causing stem and root rot disease of sesame. Int J Chem Stud. 2018;6(6):424-426.
5. Lamichhane JR, You MP, Laudinot V, Barbetti MJ, Aubertot JN. Revisiting sustainability of fungicide seed treatments for field crops. Plant Dis. 2020;104(3):610623.
6. Machado FJ, Santana FM, Lau D, Del Ponte EM. Quantitative review of the effects of triazole and benzimidazole fungicides on Fusarium head blight and wheat yield in Brazil. Plant Dis. 2017;101(9):16331641.
7. Maitlo SA, Syed RN, Rustamani MA, Khuhro RD, Lodhi AM. Comparative efficacy of different fungicides against Fusarium wilt of chickpea (Cicer arietinum L.). Pak J Bot. 2014;46(6):2305-2312.
8. Monga D, Sain SK, Nakkeeran S, Bhattiprolu SL, Rajani VV, Kulkarni V, et al. Effectiveness of seed treatment with recommended fungicides on seed-and soil-borne diseases and productivity of cotton. J Mycol Plant Pathol. 2018;48(3):311-323.
9. Panth M, Hassler SC, Baysal-Gurel F. Methods for management of soilborne diseases in crop production. Agriculture. 2020;10(1):16-16.
10. Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A. The global burden of pathogens and pests on major food crops. Nat Ecol Evol. 2019;3(3):430-439.
11. Shirsole SS, Khare N, Lakpale N, Kotasthane AS. Evaluation of fungicides against Sclerotium rolfsii Sacc., incitant of collar rot of chickpea. Pharma Innov J. 2019;8(12):310-316.
12. Vamshi J, Uma Devi G, Uma Maheswari T, Supriya K. Evaluation of fungicides on Sclerotium rolfsii, incitant of stem rot disease in groundnut (Arachis hypogaea L.). Statistics. 2019;6:1.
13. Vincent JM. Distortion of fungal hyphae in the presence of certain inhibitors. Nature. 1947;159(4051):850-850.
14. Vittal R, Kampa R, Basha SA, Karni A, Bokka V. in vitro suppression of Fusarium spp. infecting major crops. J Adv Biol Biotechnol. 2025;28(8):1759-1770.