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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 #332137

Research Project: Enhanced alfalfa and forage productivity through molecular detection and characterization of plant nematodes

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

Title: Use of chemical flocculation and nested PCR for Heterodera glycines detection in DNA extracts from field soils with low population densities

Author
item Baidoo, Richard - North Dakota State University
item Yan, Guiping - North Dakota State University
item Nelson, Berlin - North Dakota State University
item Skantar, Andrea
item Chen, Senyu - University Of Minnesota

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/24/2017
Publication Date: 4/10/2017
Citation: Baidoo, R., Yan, G., Nelson, B., Skantar, A.M., Chen, S. 2017. Use of chemical flocculation and nested PCR for Heterodera glycines detection in DNA extracts from field soils with low population densities. Plant Disease. doi: 10.1094/PDIS-08-16-1163-RE.

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 soybean cyst nematode (SCN) is a major pathogen of soybean throughout the world. It is difficult to distinguish SCN from closely related nematodes based solely on the anatomical features of juveniles, many of which appear very similar among species. This report describes how a team of scientists from ARS, North Dakota State University, and the University of Minnesota developed a molecular assay to detect and identify low densities of SCN from field soils. This research is significant because it describes a unique method for removing contaminants and inhibitors from soil-extracted DNA. It also improves the sensitivity and reliability of this type of diagnostic test, and does not require nematodes to be isolated from soil samples. This research will be used by scientists and diagnostic laboratories to accurately detect and quantify SCN from infested fields and for directing management decisions in soybean cultivation.

Technical Abstract: The soybean cyst nematode (SCN) Heterodera glycines is a major pathogen of soybean world-wide. Distinction between SCN and other members of H. schachtii sensu stricto group based on morphology is a tedious task. A molecular assay was developed to detect SCN in field soils with low population densities and to differentiate SCN from other species. Different numbers of SCN eggs or juveniles were inoculated into 10 g of sterilized soil from which soil DNA was extracted using the PowerSoil DNA Isolation Kit. A specific amplicon was amplified using published SCN-specific primers SCNF1/SCNR1. This primer set was evaluated for the first time to detect SCN directly in soil DNA extracts. The specificity of the primers was confirmed by testing 36 isolates of other nematode species. The PCR assay detected one SCN egg or juvenile added to 10 g of soil. The assay was validated using 36 field soil samples. Grinding the field soil coupled with PCR inhibitors removal by AlNH4(SO4)2 and PVPP treatment of soil DNA extracts followed by nested PCR enabled SCN detection as low as 12 SCN eggs/200 g soil. The PCR assay not only provides a sensitive method for SCN detection at low densities but also provides a discrimination method for SCN from other closely related nematodes.