Improved deferred antagonism technique for detecting antibiosis

Authors: KLEIN, JEANNIE - University Of Florida; Stockwell, Virginia; MINSAVAGE, GERALD - University Of Florida; VALLAD, GARY - University Of Florida; GOSS, ERICA - University Of Florida; JONES, JEFFREY - University Of Florida

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2020
Publication Date: 6/7/2020
Citation: Klein, J.M., Stockwell, V.O., Minsavage, G.V., Vallad, G.E., Goss, E.M., Jones, J.B. 2020. Improved deferred antagonism technique for detecting antibiosis. Letters in Applied Microbiology. https://doi.org/10.1111/lam.13339.
DOI: https://doi.org/10.1111/lam.13339

Interpretive Summary: We are interested in biological control of plant diseases caused by bacteria and/or fungi. Biocontrol is the introduction of a harmless microorganism to a plant that can slow or stop the growth of plant pathogens and reduce the incidence of disease. A common feature of effective biocontrol microorganisms is the production of antimicrobial compounds that inhibit growth of the pathogen. When screening microorganisms for their ability to control pathogens via production of antimicrobial compounds, we use the 'deferred antagonism assay.' This assay has been used by numerous scientists since the 1960s to detect antibiosis or the production of antimicrobial compounds. For the deferred antagonism assay, test bacteria are grown on culture media in glass petri dishes for two days, allowing time for antimicrobial compounds to collect in the agar. Then, the test bacteria are killed with chloroform vapors. Afterwards, we overlay the medium with a thin layer of agar containing the pathogen. Over time, we look for zones of inhibition of the pathogen, which would indicate that the test bacterium had produced antimicrobial compounds. The results of the assay are very useful, but it is a tedious process which involves a lot of handling and time when screening hundreds of microorganisms. The manuscript describes a modified deferred antagonism assay. Instead of killing the microorganism with hazardous chloroform vapors, we add an antibiotic to the overlay medium that inhibits the growth of test bacteria, but not the growth of the pathogen. We demonstrate that the modified method is a rapid, consistent, and cost-effective method to screen microorganisms for the production of antimicrobial compounds.

Technical Abstract: The deferred antagonism technique has been utilized for several decades for detecting antibiotic activity. Most protocols require the elimination of antibiotic-producing cells by exposing cells to chloroform vapor, UV radiation, or filter-sterilizing the filtrate; steps that require additional time and expense to complete. We provide a modified approach to current soft agar overlay practices, which involves addition of antibiotics, which inhibit growth of the producer but not the indicator strain, to the soft agar overlay. This technique can be used to reproducibly and efficiently screen for antibiotic production with ease. We demonstrate the effectiveness of this technique with three bacterial systems: inhibition of the fire blight pathogen, Erwinia amylovora, by Pantoea vagans C9-1 or Pseudomonas fluorescens A506; and inhibition of the bacterial spot of tomato pathogen, Xanthomonas euvesicatoria, by its pathogenic competitor Xanthomonas perforans.