Potato Cyst Nematode Egg Viability Assessment and Preparasitic Juvenile Screening Using a Large Particle Flow Cytometer and Sorter

Abstract

Potato cyst nematode (PCN) cysts consist of heterogenous populations of eggs, juveniles, and eggshells that make manual sorting of individual life stages cumbersome. The number of viable PCN eggs is a major determinant of crop damage. An accurate high-throughput PCN egg viability assay is useful for developing effective management and eradication plans. In this study, we present a method for rapid and precise enumeration and sorting of PCN eggs and juveniles, along with an egg viability assessment by staining eggs with the fluorescent stain, acridine orange, and sorting with the Complex Object Parametric Analyzer and Sorter (COPAS) system, a large particle flow cytometer. Both size sorting and fluorescent sorting capabilities of the COPAS were explored. By using the COPAS, sorting efficiency for eggs and preparasitic second-stage juveniles (J2s) was 97.6 and 97.2%, respectively, with 99% recovery at a flow rate of 15 events/s. Purity of sorted live and dead eggs was 95.5 and 94.1%, respectively. Sorting of J2s by size indicated that 15 to 16.4% of Globodera ellingtonae or G. pallida had an average body length of 436.1 ± 3.4 µm compared with an average size of 512.9 ± 4.4 µm for the majority of the J2 population for both species. A red autofluorescing J2 population was also identified through sorting. Sorting of eggs by flow cytometry did not significantly affect hatching (55.1 ± 1.2 and 53.9 ± 1.6%, respectively, for sorted or nonsorted eggs) or juvenile motility (91.3 ± 1.0 or 90.1 ± 1.1%, respectively), thus confirming that the method does not impair the biological activity of the nematode.