2019 ; Vol.29-7: 1117~1123
|Author||Kyeongmin Han, Jaejoon Lee, Gnanendra Shanmugam, Sun Keun Lee, Junhyun Jeon|
|Place of duty||Department of Biotechnology, College of Applied and Life Sciences, Yeungnam University, Republic of Korea|
|Title||Development of a redox dye-based rapid colorimetric assay for the quantitation of viability/mortality of pine wilt nematode|
J. Microbiol. Biotechnol.2019 ;
|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 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 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.|
|Key_word||Pine wilt disease, pine wilt nematode, Bursaphelenchus xylophilus, colorimetric assay, redox dye|
Mota MM, Vieira P. 2008. Pine wilt disease: a worldwide threat to forest ecosystems, pp. 1-14. Springer, Heidelberg, Germany.
Yano S. 1913. Investigation on pine death in Nagasaki prefecture. Sanrin-Kouhou. 4: 1-14.
Steiner G, Buhrer EM. 1932. Aphelenchoides xylophilus, a nematode associated with blue-stain and other fungi in timber. J. Agric. Res. 48: 949-951.
Evans HF, McNamara DG, Braasch H, Chadoeuf J, Magnusson C. 1996. Pest risk analysis (PRA) for the territories of the European Union (as PRA area) on Bursaphelenchus xylophilus and tis vector in the genus Monochamus. EPPO Bulletin. 26: 199-249.
Jikumaru S, Togashi K. 2001. Transmission of Bursaphelenchus mucronatus (Nematoda : Aphelenchoididae) through feeding wounds by Monochamus saltuarius (Coleoptera : Cerambycidae). Nematology 3: 325-333.
Mota MM, Braasch H, Bravo MA, Penas AC, Burgermeister W, Metge K, et al. 1999. First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1: 727-734.
Mamiya Y. 1988. History of pine wilt disease in Japan. J. Nematol. 20: 219-226.
Mamiya Y. 1983. Pathology of the pine wilt disease caused by Bursaphelenchus xylophilus. Annu. Rev. Phytopathol. 21:201-220.
Futai K. 2013. Pine wood nematode, Bursaphelenchus xylophilus. Annu. Rev. Phytopathol. 51: 61-83.
Kwon TS, Song MY, Shin SC, Park YS. 2005. Effects of aerial insecticide sprays on ant communities to control pine wilt disease in Korean pine forests. Appl. Entomol. Zool. 40: 563-574.
Rajasekharan SK, Lee JH, Ravichandran V, Lee J. 2017. Assessments of iodoindoles and abamectin as inducers of methuosis in pinewood nematode, Bursaphelenchus xylophilus. Sci. Rep. 7: 6803
Kim J, Lee SM, Park CG. 2016. Bursaphelenchus xylophilus is killed by homologues of 2-(1-undecyloxy)-1-ethanol. Sci. Rep. 6: 29300
Shanmugam G, Lee SK, Jeon J. 2018. Identification of potential nematicidal compounds against the pine wood nematode, Bursaphelenchus xylophilus through an In Silico Approach. Molecules 23: 1828
Lee HR, Lee SC, Lee JE, Seo SM, Jeong YC, Jung CS, et al. 2017. Nematicidal activity of 3-acyltetramic acid analogues against pine wood nematode, Bursaphelenchus xylophilus. Molecules 22: 1568
Seo SM, Kim J, Koh SH, Ahn YJ, Park IK. 2014. Nematicidal activity of natural ester compounds and their analogues against pine wood nematode, Bursaphelenchus xylophilus. J. Agr. Food Chem. 62: 9103-9108.
Liu MJ, Hwang BS, Jin CZ, Li WJ, Park DJ, Seo ST, et al. 2018. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest Manag. Sci. 75:1585-1593.
Ferreira SR, Mendes TA, Bueno LL, de Araujo JV, Bartholomeu DC, Fujiwara RT. 2015. A new methodology for evaluation of nematode viability. Biomed. Res. Int. 2015:879263.
Shanmugam G, Dubey A, Ponpandian LN, Rim SO, Seo ST, Bae H, et al. 2018. Genomic insights into nematicidal activity of a bacterial endophyte, Raoultella ornithinolytica MG against pine wilt nematode. Plant Pathol. J. 34: 250-255.