Development of a spore-based confrontation assay to screen for biocontrol organisms with antagonistic activity against oomycete and fungal pathogens

Abstract

Aims Screenings for biocontrol organisms against fungal and oomycete pathogens are typically performed on mycelium. While this allows for high-throughput screenings, it omits a major actor in pathogenicity, i.e. the spores. This study aims to improve the screening strategy using a spore-based confrontation assay (SBCA), as well as comparing its performance to the traditional mycelium-based confrontation assay (MBCA) and microscopy analyses of spore germination. Methods and Results The SBCA was used to screen for 38 candidate biocontrol bacteria against two relevant broad-spectrum phytopathogens, Botrytis cinerea and Phytophthora cactorum. The performance of the SBCA was benchmarked to the traditional mycelium-based confrontation assay and microscopy observations for spore germination inhibition. The SBCA demonstrated a higher hit rate and reproducibility than its counterparts. The bacteria tested exhibited diverse traits in vitro such as production of lytic enzymes, biosurfactant, bioactive volatile organic compounds, and cell-free extracts. These characteristics suggest potential biocontrol modes of action, such as antibiosis (via diffusible metabolites and enzymes) or competition for nutrients and space. For two Pseudomonas strains, the biocontrol activity against P. cactorum was confirmed in planta in a detached leaf assay. Conclusions This study showcases a versatile and robust spore-based screening that outperforms conventional screening methods. Through the use of the SBCA, two promising biocontrol strains with antagonistic activity against P. cactorum in strawberry were identified.