4.1. Soil Sampling and Nematode Extraction from Soil

Soil samples were collected from a soybean field in Walcott, Richland County, ND, which was previously reported to have been infested with P. dakotaensis [28]. Nematodes were then extracted from the soil samples using the standard hand sieving, decanting, and sugar centrifugal–floatation method [58]. Briefly, this method relies principally on agitating the soil in tap water (200 mL) through stirring and pouring the mixture through a stack of sieves (250 µm and 20 µm apertures) to separate larger organic and inorganic soil particles from nematodes and smaller soil particles. Then, the smaller soil particles and nematodes that were collected in the 20-µm-aperture sieve were separated from each other by suspending them in a 1.3 M sucrose solution (American Crystal Sugar Company, Moorhead, MN, USA). Once suspended, the solution was centrifuged at 4000 RPM for 30 s. The supernatant containing the vermiform nematodes was poured through 20-µm-aperture sieve to remove the sugar solution and the nematodes were then rinsed with tap water and collected in 15 mL tap water for quantification, and the pellet containing the soil particles was discarded. Subsequently, root-lesion nematodes were identified and enumerated at the genus level based on their key morphological features [32] under a dissecting microscope (Zeiss Stemi 305; Zeiss, Thornwood, NY, USA) at 80× magnification.