Title: Development of a species-specific PCR assay for differentiation of Heterodera filipjevi and H. avenae Authors
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 24, 2013
Publication Date: December 20, 2013
Citation: Yan, G., Smiley, R.W., Okubara, P.A., Skantar, A.M. 2013. Development of a species-specific PCR assay for differentiation of Heterodera filipjevi and H. avenae. Plant Disease. 97(12):241-246. Interpretive Summary: The cereal cyst nematodes are a widespread and important group of microscopic, plant parasitic soil worms that limit production of cereal crops throughout the world. High populations of the nematode Heterodera avenae are estimated to reduce profits from wheat production by at least $3.4 million annually in the Pacific Northwest (PNW). A second wheat-damaging cyst nematode, Heterodera filipjevi, was discovered in the PNW in 2008, and its distribution there is currently unknown. Distinguishing these two nematodes from each other by microscopic methods is laborious and sometimes inconclusive. In the present study, scientists from Oregon State University and ARS scientists from Beltsville, MD and Pullman, WA developed highly specific molecular diagnostic assay to detect and distinguish H. avenae and H. filipjevi. The results are significant because the assay can be used to identify these nematodes and distinguish them from other nematode species, thus providing accurate identifications needed for selection of appropriate wheat cultivars and management practices. Commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research.
Technical Abstract: Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest (PNW) USA and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) region of Heterodera rDNA. The primers were highly specific when tested on 15 isolates from 10 Heterodera spp. and 23 isolates from Globodera spp., Meloidogyne spp., Pratylenchus spp., and other plant-parasitic nematode species. Optimal PCR reaction and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 bp and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using typical worm lysis buffer, and DNA from 0.5 egg or 0.5 J2 using a commercial kit. This PCR assay was successfully employed for differentiation of H. filipjevi and H. avenae populations collected from various locations in three PNW states. This is the first report for developing species-specific PCR to rapidly and reliably detect and identify H. filipjevi. This assay will enhance detection and identification efficiencies for cereal cyst nematodes in infested fields.