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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Mycology and Nematology Genetic Diversity and Biology Laboratory » Research » Publications at this Location » Publication #337834

Title: Developing a real-time PCR assay for direct identification and quantification of Pratylenchus penetrans in soil

item BAIDOO, RICHARD - North Dakota State University
item YAN, GUIPING - North Dakota State University
item NAGACHANDRABOSE, SEENIVASAN - North Dakota State University
item Skantar, Andrea

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2017
Publication Date: 8/1/2017
Citation: Baidoo, R., Yan, G., Nagachandrabose, S., Skantar, A.M. 2017. Developing a real-time PCR assay for direct identification and quantification of Pratylenchus penetrans in soil. Plant Disease. 101(8):1432-1441.

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. The root-lesion nematode Pratylenchus penetrans is one of the most widespread and economically important nematodes that invade plant roots and restrict the productivity of many crops throughout the U.S., including potato. This nematode can be difficult to distinguish from other lesion nematodes by microscopic methods. In the present study, scientists from North Dakota State University and an ARS scientist from Beltsville, MD developed a highly sensitive molecular diagnostic assay to detect P. penetrans using DNA extracted directly from soil samples. The results are significant because the assay can be used to quantify P. penetrans in soil and accurately distinguish this species from other lesion nematodes present in mixed nematode populations. Commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research.

Technical Abstract: The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato world-wide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the Potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are essential for developing effective integrated pest control measures. However, distinction between P. penetrans and other Pratylenchus spp. based on morphology is a tedious task. A SYBR Green I-based qPCR assay was developed to discriminate, identify and quantify P. penetrans in field soil. P. penetrans-specific qPCR primers were designed from the D2-D3 region of the 28S rDNA. The specificity of the assay was evaluated using eight isolates of P. penetrans populations and 31 isolates of other nematode species. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. There was a high correlation between the P. penetrans numbers artificially added to soil or estimated from naturally infested field soils by conventional methods, and the numbers quantified using the qPCR assay. Grinding the field soil prior to DNA extraction improved P. penetrans detection from soil. The qPCR assay will not only be useful for differentiating P. penetrans from mixed populations of Pratylenchus spp., but also for efficient detection and quantification of P. penetrans from field soil. The assay requires no expertise in nematode taxonomy and morphology, and may serve as a useful diagnostic tool in research, diagnostic labs and extension services for pest management.