Development of a redox dye-based rapid colorimetric assay for the quantitation of viability/mortality of pine wilt nematode

Abstract

Control of pine wilt disease, which is caused by pine wilt nematode Bursaphelenchus xylophilus, is heavily dependent on the use of chemicals such as abamectin. Although such chemicals are highly effective, demands for alternatives that are derived preferentially from natural sources, are increasing out of environmental concerns. One of the challenges to discovery of alternative control agents is lack of fast and efficient screening method that can be used in a high-throughput manner. Here we described the development of colorimetric assay for the rapid and accurate screening of candidate nematicidal compounds/biologics targeting B. xylophilus. Contrary to the conventional method, which relies on laborious visual inspection and counting of nematode population under microscope, our method utilizes a redox dye that changes its color in response to metabolic activity of nematode population in a given sample. In this work, we optimized parameters of our colorimetric assay including number of nematodes and amount of redox dye, and tested applicability of our assay for screening of chemicals and biologics. We demonstrated that our colorimetric assay can be applied to rapid and accurate quantification of nematode viability/mortality in a nematode population treated with candidate chemicals/biologics. Application of our method would facilitate high-throughput endeavors aiming at finding environment-friendly control agents for deadly disease of pine trees.