Sep 11, 2025
Dual Culture Antagonism Assay
- Md Sahadat Ali1,
- Fatima Tuz Zohora Mony1,
- Jonathan D. Eisenback1
- 1Virginia Tech
Protocol Citation: Md Sahadat Ali, Fatima Tuz Zohora Mony, Jonathan D. Eisenback 2025. Dual Culture Antagonism Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrwk12lmk/v1
Manuscript citation:
Ali MS, Mony FTZ, Evans M, Rideout S, Haak D, Eisenback JD (2025)
Unveiling the antagonistic activity of ‘Candidatus Pseudomonas auctus’ JDE115 against Agroathelia rolfsii: a soybean nodule endophyte with biocontrol potential. Manuscript under review in BioControl (Springer Nature). (In preparation)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 10, 2025
Last Modified: September 11, 2025
Protocol Integer ID: 226934
Keywords: Dual culture assay, antagonism, Pseudomonas auctus JDE115, Agroathelia rolfsii, biocontrol, soil-borne pathogen, plant growth-promoting rhizobacteria (PGPR), fungal suppression, sclerotia, hyphal inhibition, fungal interaction, fungal pathogen, phytopathogenic fungus, borne fungal pathogen, candidatus pseudomonas auctus, jde115 against the phytopathogenic fungus, bacterial biocontrol agent, antagonistic activity of bacterial biocontrol agent, fungus, dual culture antagonism assay, dual culture antagonism assay this protocol, hyphal plugs of the fungus, potato dextrose agar, agroathelia rolfsii, dextrose agar, biocontrol potential
Disclaimer
This protocol is intended for research purposes only. Results may vary depending on laboratory conditions, microbial strains, and experimental design. Users are responsible for ensuring compliance with relevant biosafety and institutional guidelines before implementation.
Abstract
This protocol describes the dual culture antagonism assay used to evaluate the biocontrol potential of ‘Candidatus Pseudomonas auctus’ JDE115 against the phytopathogenic fungus Agroathelia rolfsii (syn. Sclerotium rolfsii). The assay was performed on potato dextrose agar (PDA) under both direct contact and non-contact conditions using sclerotia and hyphal plugs of the fungus. Multiple experimental setups were tested to assess bacterial–fungal interactions, with replicates conducted to ensure reproducibility. Antagonistic effects were evaluated through visual observation, photographic documentation, and radial growth measurements, followed by quantification of growth suppression. This standardized protocol provides a reproducible framework for assessing the antagonistic activity of bacterial biocontrol agents against soil-borne fungal pathogens.
Attachments
Guidelines
The antagonistic potential of ‘Candidatus P. auctus’ JDE115 against A. rolfsii was evaluated using a dual culture assay on potato dextrose agar (PDA). PDA was prepared by dissolving 24 g of potato dextrose broth and 15 g of agar in 1 L of deionized water, sterilization at 121°C for 15 minutes. The media was then poured into sterile petri dishes and allowed to solidify.
Seven parameters were tested: (a) JDE115 placed directly on A. rolfsii sclerotia, (b) JDE115 alone, (c) JDE115 and sclerotia placed distantly, (d) sclerotia alone, (e) JDE115 and fungal hyphal plug placed distantly, (f) hyphal plug alone, and (g) JDE115 placed directly on hyphae.
Fresh cultures of JDE115 (OD600 ≈ 0.6) and A. rolfsii (sclerotia or 6–8 mm hyphal plugs) were used in all treatments (Fig. 1). Plates were incubated at 28°C for 7 days. Each treatment included five biological replicates and was repeated independently five times. Fungal growth inhibition was assessed visually and documented photographically to evaluate antagonistic effects under both contact and non-contact conditions (Khan et al. 2018).
In a complimentary dual plate assay, the radial growth of A. rolfsii was measured in centimeters after 7 days of incubation at 28°C. The experiment consisted of two treatments: (1) A. rolfsii control, where sterile water was applied instead of JDE115, (2) JDE115 treatment, in which the bacterium was streaked 4 cm away from the fungal plug (Ali et al. 2020). Each treatment included 10 biological replicates, and the experiment was independently repeated three times under identical conditions.
Fungal inhibition was quantified by calculating the percentage suppression of radial growth using the following formula (Montealegre et al. 2003):
Suppression (%) = (1 – Mean growth in treatment / Mean growth in control) × 100
Materials
- Potato dextrose broth: 24 g per 1 L
- Agar: 15 g per 1 L
- Deionized water: 1 L
- Potato dextrose agar (PDA) plates / sterile petri dishes
- Autoclave (sterilization at 121°C for 15 minutes)
- Fresh culture of JDE115 (OD600 ≈ 0.6)
- A. rolfsii sclerotia or 6–8 mm hyphal plugs
- Sterile water
- Incubator capable of 28°C
Troubleshooting
Safety warnings
- Handle Agroathelia rolfsii (syn. Sclerotium rolfsii) with care, as it is a plant pathogen that can spread through contaminated surfaces or soil.
- Dispose of fungal and bacterial cultures according to institutional biosafety guidelines.
- Work under Biosafety Level 2 (BSL-2) practices when handling live cultures.
- Avoid direct contact with fungal structures and bacterial suspensions; wear gloves, a lab coat, and eye protection.
Ethics statement
This study did not involve human participants or vertebrate animals. All experiments were conducted with microbial cultures under the approval and biosafety regulations of Virginia Tech’s Institutional Biosafety Committee. No additional ethical approvals were required.
Before start
Ensure all microbial cultures are fresh and prepared under aseptic conditions. Confirm that potato dextrose agar (PDA) and all other media are sterilized and properly solidified before inoculation. Calibrate spectrophotometer for accurate OD600 measurements of bacterial suspensions. Maintain sterile workspace and equipment to prevent contamination that may affect results.
Dual Culture Antagonism Assay
Prepare potato dextrose agar (PDA) by dissolving 24 g potato dextrose broth and 15 g agar in 1 L deionized water; sterilize at 121°C for 15 minutes. Pour the sterilized media into sterile petri dishes and allow to solidify.
Set up the seven treatment parameters on solidified PDA: (a) JDE115 placed directly on A. rolfsii sclerotia; (b) JDE115 alone; (c) JDE115 and sclerotia placed distantly; (d) sclerotia alone; (e) JDE115 and fungal hyphal plug placed distantly; (f) hyphal plug alone; (g) JDE115 placed directly on hyphae.
Use fresh cultures of JDE115 (OD600 ≈ 0.6) and A. rolfsii (sclerotia or 6–8 mm hyphal plugs) for all treatments (Fig. 1).
Incubate the inoculated plates at 28°C for 7 days.
For the dual culture assay include five biological replicates per treatment and repeat the entire experiment independently five times. Assess fungal growth inhibition visually and document photographically to evaluate antagonistic effects under both contact and non-contact conditions.
Perform the complementary dual plate (radial growth) assay: after 7 days incubation at 28°C measure radial growth of A. rolfsii in centimeters. Use two treatments: (1) A. rolfsii control — apply sterile water instead of JDE115; (2) JDE115 treatment — streak JDE115 4 cm away from the fungal plug. Use 10 biological replicates per treatment and repeat the experiment independently three times under identical conditions.
Quantify fungal inhibition by calculating percentage suppression of radial growth using the formula: Suppression (%) = (1 – Mean growth in treatment / Mean growth in control) × 100.
Protocol references
Khan N, Martínez-Hidalgo P, Ice TA, et al (2018) Antifungal Activity of Bacillus Species Against Fusarium and Analysis of the Potential Mechanisms Used in Biocontrol. Front Microbiol 9:2363. https://doi.org/10.3389/fmicb.2018.02363
Ali S, Hameed S, Shahid M, et al (2020) Functional characterization of potential PGPR exhibiting broad-spectrum antifungal activity. Microbiol Res 232:126389. https://doi.org/10.1016/j.micres.2019.126389
Montealegre JR, Reyes R, Pérez LM, et al (2003) Selection of bioantagonistic bacteria to be used in biological control of Rhizoctonia solani in tomato. Electron J Biotechnol 6:0–0. https://doi.org/10.2225/vol6-issue2-fulltext-8
Acknowledgements
We are grateful to the members of the Plant Pathology, Physiology, and Weed Science Department for their technical assistance and helpful discussions. We also acknowledge the Nematode Diagnostic Lab at Virginia Tech for providing facilities and resources essential for conducting this work.