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Title: Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

Author
item NAKHLA, MARK - Animal And Plant Health Inspection Service (APHIS)
item OWENS, KRISTINA - Animal And Plant Health Inspection Service (APHIS)
item LI, WENBIN - Animal And Plant Health Inspection Service (APHIS)
item WEI, GANG - Animal And Plant Health Inspection Service (APHIS)
item Skantar, Andrea
item LEVY, LAURENE - Animal And Plant Health Inspection Service (APHIS)

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2010
Publication Date: 8/1/2010
Citation: Nakhla, M.K., Owens, K.J., Li, W., Wei, G., Skantar, A.M., Levy, L. 2010. Multiplex real-time PCR Assays for the identification of the potato cyst and tobacco cyst nematodes. Plant Disease. 94(8):959-965.

Interpretive Summary: Plant-parasitic nematodes are microscopic worms that attack plant roots and cause an estimated ten billion dollars of crop loss each year in the United States and 100 billion dollars globally. Two potato cyst nematodes (PCN), known as the pale cyst nematode and the golden nematode, are regulated as quarantine pests by many countries and cause economic damage to potato worldwide. Growers and regulatory officials face an enormous problem in that the existing molecular methods for distinguishing these PCN species are slow and occasionally inaccurate. In the present study, scientists from APHIS and ARS designed a new molecular diagnostic test that can rapidly detect and distinguish the pale cyst nematode from the golden cyst nematode, and from the anatomically similar tobacco cyst nematode (TCN). This research is significant because the new assay is species-specific, highly sensitive, and much faster than existing molecular methods for PCN detection. This test was specifically designed for PCN detection by federal and state diagnostic labs, but will also be useful to domestic and international research scientists, regulatory personnel, or extension agencies for identifying and preventing further damage by potato cyst nematodes.

Technical Abstract: TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another primer-probe set specific for Globodera rostochiensis (golden nematode). A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for Globodera tabacum (the morphologically similar tobacco cyst nematode). This ITS rDNA-based system was specific for the Globodera species of interest and successfully identified several populations of PCN. This rapid, sensitive and specific TaqMan PCR assay has been successfully used in the confirmation of PCN found during voluntary surveys, and presents a useful tool for PCN regulatory response and management programs.