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

Yan, G. P., Smiley, R. W., Okubara, P. A., Skantar, A. M., and Reardon, C. L. 2013. Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Dis. 97:757-764. 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 largescale 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. Pratylenchus neglectus is one of the most important root-lesion nematodes that invades roots of plants and restricts productivity of wheat in the Pacific Northwest (PNW) (28,30,32,34). This nematode has been detected in 95% of dryland wheat-producing fields surveyed in Idaho, Oregon, and Washington (32,35) and in 40% of fields in Montana (14). P. neglectus was more widely distributed than P. thornei, another damaging root-lesion nematode species in the PNW, but mixed populations also occurred (32). Population density of P. neglectus at preplanting has often been inversely correlated with wheat grain yield (32,33) and yield reductions were generally 8 to 36% for susceptible spring wheat cultivars in Oregon (34). P. neglectus together with P. thornei were estimated to reduce farm profits by about $51 million annually in the three PNW states (28,29). Multiple factors such as variable climate and weather, host genotype, nematode virulence, and cropping system make it difficult to determine economic damage thresholds for Pratylenchus spp. (33). However, an initial population of 2,000 P. neglectus per kilogram of soil reduced wheat grain yield in one dryland and one irrigated field experiment in Oregon (34). Potentially damaging high populations have been detected in up to 60% of the wheat fields sampled in the PNW (29). Management of lesion nematodes at high population densities is best approached by integrating crop rotation and use of resistant and tolerant wheat cultivars (29,33,37). To some extent, crop rotation alone is limited because P. neglectus is polyphagous and has a broad range of hosts such as legumes, cereals, biofuel crops, and many other genera of broadleaf and grass weeds (5,29,33,39). Minimizing damage by crop rotation is possible only if resistant or tolerant rotation crops are available and profitable for growers. However, many crops differed in susceptibility to each Pratylenchus spp. (33,39), and these differences were also observed at the cultivar level. For instance, a wheat cultivar with resistance or tolerance to P. neglectus was not necessarily resistant or tolerant to P. thornei, and vice versa (28). Therefore, nematode species identification and quantification are essential for selecting the bestperforming rotation crops and wheat cultivars for optimal disease management. Distinguishing P. neglectus from P. thornei and other closely related Pratylenchus spp. using traditional microscopic methods is based on subtle morphological differences in lip annule number, tail shape, and vulva position (6,10). This process is time-consuming and it is difficult to count and identify these species from large numbers of field soil samples, especially when other nematodes are present. Commercial laboratories provide nematode diagnostic services and routinely report total numbers of lesion nematodes at the genus level but do not identify and quantify them to the species level. DNA-based technologies provide a rapid alternative to microscopic methods. A commercial laboratory, South Australia Research and Development Institute (SARDI, Adelaide, Australia), provides a comprehensive DNA-based testing service for quantification of P. neglectus along with two other nematode pathogens and six fungal pathogens of wheat (12,20,23). However, their protocols are proprietary. There is a demand for a publically available quantitative polymerase chain reaction (qPCR) procedure for quantitative testing of P. neglectus in soil, combined with identification to species level. At least one PNW commercial laboratory has an expert nematode diagnostician, and possesses qPCR capabilities for fungal crop pathogens but lacks the option for qPCR diagnosis of nematodes such as P. neglectus in soil samples (http://www. westernlaboratories.com). A number of PCR-based methods have been developed for differentiating species of plant-parasitic nematodes (19). PCR in combination with restriction fragment length polymorphism was used to differentiate Pratylenchus spp. (21,36,40). One-step species-specific PCR followed by gel electrophoresis was performed for discrimination of six Pratylenchus spp., including P. neglectus Corresponding author: G. P. Yan, E-mail: [email protected] Accepted for publication 30 December 2012. http://dx.doi.org/10.1094 / PDIS-08-12-0729-RE © 2013 The American Phytopathological Society