Comparison of Meldola’s Blue staining and hatching assay with potato root diffusate for assessment of Globodera sp. egg viability

Authors: KROESE, DUNCAN - Oregon State University; Zasada, Inga; INGHAM, R - Oregon State University

Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2011
Publication Date: 9/1/2011
Citation: Kroese, D., Zasada, I.A., Ingham, R.E. 2011. Comparison of Meldola’s Blue staining and hatching assay with potato root diffusate for assessment of Globodera sp. egg viability. Journal of Nematology. 43(3/4):182-186.

Interpretive Summary: The potato cyst nematodes (Globodera rostochiensis and Globodera pallida) are of regulatory concern to the United States because of their potential impact on potato production and on the ability of U.S. growers to export potatoes to other countries. Because of these concerns, surveys have been conducted to determine the occurrence and distribution of these nematodes in the United States. When Globodera cysts are detected in a sample, it would be useful to have a rapid and robust means to assess nematode viability. This research was conducted to compare two nematode viability methods: one which uses a stain (Meldola's Blue) to distinguish living vs. dead Globodera eggs and another which uses potato root diffusates (PRD; chemical compounds released by roots and known to be important for the nematodes to complete their life cycles) to stimulate Globodera egg hatch. Results indicated that staining with Meldola's Blue provides a conservative assessment of egg viability compared to the hatch method with PRD. These results are significant because it supports the use of a rapid and robust nematode viability method (Meldola's Blue) over the more time-consuming PRD hatch method. This research will be used by scientists and regulatory agencies involved in the management and possible regulation of these nematode in the United States.

Technical Abstract: Laboratory-based methods to test egg viability include staining with Meldola’s Blue and/or juvenile (J2) hatching assays using potato root diffusate (PRD). These two methods have not been tested under identical conditions to directly compare their assessments of Globodera egg viability. Using two bioassay strategies, cysts from a Globodera sp. population found in Oregon were subjected to both viability assessment methods. In strategy one, intact cysts were first stained with Meldola’s Blue for one week (primary staining) and the numbers of stained and unstained eggs were determined. Eggs were then transferred to PRD (secondary hatching), and one week later the numbers of J2 and remaining eggs were determined. In the second strategy, intact cysts were exposed to PRD for one week (primary hatching), the numbers of J2 and remaining eggs were determined per cyst, then the eggs were transferred to Meldola’s Blue (secondary staining). After one week, the numbers of stained and unstained eggs were determined. Two different cohorts of cysts were evaluated using these experimental strategies: one contained eggs that exhibited low hatch when exposed to PRD (greenhouse) and the other whose eggs yielded significant hatch when exposed to PRD. With field-produced cysts, primary staining with Meldola’s Blue and primary hatching with PRD produced similar viability estimates, with averages of 74.9% and 76.3%, respectively. In contrast, with greenhouse-produced cysts the two methods yielded much lower and unequal estimates 32.4% to 2.2%, respectively for primary hatching and staining methods. In addition, J2 hatch from unstained (viable) greenhouse-produced eggs was 13.7% after secondary exposure to PRD compared to 61.5% for field-produced eggs. The majority of eggs remaining unhatched after primary exposure to PRD (> 87%) stained with Meldola’s Blue regardless of cyst cohort. Staining with Meldola’s Blue provided a conservative assessment of egg viability compared to hatch assay with PRD regardless of diapause.