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Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes

December 2012 , Volume 96 , Number  12
Pages  1,757 - 1,762

Ronald J. Sayler, Department of Plant Pathology, University of Arkansas, Fayetteville 72701; Courtney Walker, University of Central Arkansas, Conway 72035; Fiona Goggin, Department of Entomology, University of Arkansas, Fayetteville 72701; Paula Agudelo, Clemson University, School of Agricultural, Forest, and Environmental Sciences, Clemson, SC 29634; and Terrence Kirkpatrick, Department of Plant Pathology, University of Arkansas, Fayetteville 72701

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Accepted for publication 17 June 2012.

Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

© 2012 The American Phytopathological Society