Catherine L. Reardon

Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil

Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

Rapid Quantification of Soilborne Pathogen Communities in Wheat-Based Long-Term Field Experiments

Rainfed experiments operated continuously for up to 84 years in semiarid eastern Oregon are among the oldest agronomic trials in North America. Disease incidence and severity had been quantified visually but quantification of inoculum density had not been attempted. Natural inoculum of 17 fungal and nematode pathogens were quantified for each of 2 years on eight trials using DNA extracts from soil. Crop type, tillage, rotation, soil fertility, year, and their interactions had large effects on the pathogens. Fusarium culmorum and Pratylenchus thornei were more dominant than F. pseudograminearum and P. neglectus where spring crops were grown, and the opposite species dominances occurred where winter wheat was the only crop. Bipolaris sorokiniana and Phoma pinodella were restricted to the presence of spring cereals and pulse crops, respectively. Helgardia spp. occurred in winter wheat-fallow rotations but not in annual winter wheat. Gaeumannomyces graminis var. tritici was more prevalent in cultivated than noncultivated soils and the opposite generally occurred for Rhizoctonia solani AG-8. Densities of Pythium spp. clade F were high but were also influenced by treatments. Significant treatment effects and interactions were more prevalent in two long-standing (>50-year) annually cropped experiments (29%) than two long-standing 2-year wheat-fallow rotations (14%). Associations among pathogens occurred mostly in an 84-year-old annual cereals experiment. This survey provided guidance for research on dynamics of root-infecting pathogens of rainfed field crops and identified two pathogens (Drechslera tritici-repentis and P. pinodella) not previously identified at the location.