A method for quantifying Phytophthora oospore viability using fluorescent dyes and automated image analysis.

Abstract

Phytophthora are eukaryotic microbes that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via sexual reproduction. These resting spores can survive in the soil for several years in the absence of a host plant, thus providing a long-term inoculum for disease. The ability to quantitatively evaluate oospore viability is an important part of many phytopathology studies. Here, we tested six fluorescent viability dyes for their ability to differentially stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate (FDA), 5-carboxyfluorescein diacetate (CFDA), propidium iodide, and TOTO-3 iodide. Each dye was first tested individually with untreated or heat-treated oospores as proxies for viable and non-viable oospores, respectively. SYTO9, FUN-1, CFDA and propidium iodide stained untreated and heat-treated oospores indiscriminately. In contrast, FDA (a green-fluorescent viable cell stain) and TOTO-3 (a red-fluorescent non-viable cell stain) differentially stained untreated or heat-treated oospores with no cross-fluorescence. We then tested the efficacy of dual viability staining and in conjunction with a pipeline for automated image analysis. To validate the method, untreated and heat-treated oospores were mixed at specific ratios, dual-stained, and analyzed using the pipeline. Linear regression of the resulting data showed a clear correlation between the expected and measured oospore ratios (dy/dx=0.95, R2=0.88). Overall, the combination of dual-fluorescence staining and automated image analysis provides a high-throughput method for quantitatively assessing oospore viability and therefore can facilitate further studies on this key part of the Phytophthora disease cycle.