A High-Throughput Microtiter-Based Fungicide Sensitivity Assay for Oomycetes Using Z'-Factor Statistic

Current methods to quantitatively assess fungicide sensitivity for a diverse range of oomycetes are slow and labor intensive. Microtiter-based assays can be used to increase throughput. However, many factors can affect their quality and reproducibility. Therefore, efficient and reliable methods for detection of assay quality are desirable. The objective of this study was to develop and validate a robust high-throughput fungicide phenotyping assay based on spectrophotometric quantification of mycelial growth in liquid culture and implementation of quality control with Z' factor and growth curves. Z' factor was used to ensure that each isolate grew enough in the absence of fungicides compared with the negative control, and growth curves were used to ensure active growth at the time of concentration of a fungicide that reduces growth by 50% (EC₅₀) estimation. EC₅₀ and relative growth values were correlated in a side-by-side comparison with values obtained using the amended medium (gold standard) assay. Concordance correlation indicated that the high-throughput assay is accurate but may not be as precise as the amended medium assay. To demonstrate the utility of the high-throughput assay, the sensitivity of 216 oomycete isolates representing four genera and 81 species to mefenoxam and ethaboxam was tested. The assay developed herein will enable high-throughput fungicide phenotyping at a population or community level.