Evaluation of Fluorescent Stains for Viability Assessment of the Potato Cyst Nematodes Globodera pallida and G. ellingtonae

Abstract

Potato cyst nematodes (PCNs), Globodera pallida and Globodera rostochiensis, are quarantine pests of potato which cause significant damage to production and farm gate revenue worldwide. Accurately assessing viability of PCN eggs is important for eradication and management programs. The goal of this study was to develop a quick and reliable fluorescent staining method to evaluate viability of G. pallida and Globodera ellingtonae eggs. The staining efficiency of eight fluorescent stains was evaluated using G. pallida eggs compared with the conventional Meldola’s Blue (MB) staining method. The staining efficiency of the fluorescent stains ranged from 80.33 ± 2.99 (Sytox Green) to 100% (Acridine Orange) for non-viable eggs. Two stains were further evaluated for their efficiency in assessing viability of encysted eggs from five different greenhouse-reared G. pallida cyst sources which contained both viable and non-viable eggs. For the G. pallida cyst sources, viability ofencysted eggs were estimated to be 41.02 ± 3.81 to 62.66% ± 3.12% when stained with Acridine Orange (AO) and 79.52% ± 1.54% viability for G. ellingtonae. Both staining time and stain concentration were significant for staining efficiency of released and encysted eggs. Staining time and concentration were optimized for released eggs at 4 h at 10 µg/ml and for encysted eggs at 16 h at 25 µg/ml respectively for AO. Fluorescent stains accurately and rapidly assessed percent egg viability and were determined to be as sensitive as a seven-day incubation with the Meldola’s Blue staining method.