Developing a Real-Time PCR Assay for Direct Identification and Quantification of Soybean Cyst Nematode, Heterodera glycines, in Soil and Its Discrimination from Sugar Beet Cyst Nematode, Heterodera schachtii.

Abstract

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the Heterodera schachtii sensu stricto group is not only difficult and time-consuming but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes and to quantify SCN directly from DNA extracts of field soils. SCN- and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated on the basis of target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and they may serve as useful diagnostic tools in research, diagnostic laboratories, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.