Environmental conditions and ecological interactions among microorganisms isolated from diseased samples shape their biocontrol efficacy against Erwinia amylovora

Introduction Fire blight, caused by Erwinia amylovora , is a devastating disease of apples and pears. Limitations in current control strategies using antibiotics and copper demand for more sustainable alternatives . This study aimed to evaluate the potential of 13 microorganisms (12 bacteria, 1 yeast) co-isolated with E. amylovora from symptomatic apple tissues as biocontrol agents (BCAs) and determine how environmental conditions and ecological interactions influence their efficacy against the pathogen. Methods We optimized an in vitro agar plug assay to determine antagonistic activities of microorganisms under different environmental conditions. We utilized ex vivo assays on detached fruitlets to determine the efficacies of BACs. Bliss independence and best single agent frameworks were used to identify BAC helpers and hinderers. Results In vitro agar plug assays revealed that antagonistic activity of these microbes was enhanced by environmental factors, including minimal media, acidic pH (6.0), lower temperature (22°C), and specific carbon sources (fructose and glucose), which significantly enhanced their pathogen inhibition abilities. Ex vivo assays on detached fruitlets demonstrated that preventive application of BCAs was more effective at reducing disease symptoms than a co-inoculation (curative) regime. Most combinations of antagonists reduced infection rates, with combinations containing Erwinia sp. 2186 achieving the highest disease suppression. Net interaction analysis using Bliss independence and best single agent frameworks identified biocontrol helper and hinderer organisms. As an example, Rahnella sp. EL51 and Pseudomonas sp. 2180 acted to synergistically enhance or antagonistically decrease disease suppression in paired treatments, respectively. Population dynamics of the top-performing pair ( Erwinia sp. 2186 plus Rahnella sp. EL51) indicated a link between disease suppression, sustained BCA presence, bacteriostatic effects on E. amylovora populations and medium acidification. Conclusion These findings underscore the importance of ecological interactions and environmental effects in developing biocontrol strategies against E. amylovora .