Research Update

Authors: Aaron Tucker, Caleb Henderson, David McCall, Ph.D., Jon Eisenback, and David Haak

Standard methods of lance nematode identification and quantification involve hand counting nematodes. These standard methods require time and a specific level of expertise to hand count both accurately and precisely. This is a subjective process that is time exhaustive when many samples need attention. The goal of this research is to evaluate if using a qPCR approach to identify and quantify lance nematodes can be equivalent to the standard methods of nematode counting and more time efficient with bulk sampling. A qPCR reaction shows amplification of targeted DNA in real time measured by the number of cycles required to meet a threshold of amplification (Ct). Twenty-four samples were collected in September 2022 from Foundry Golf Club in Powhatan, VA. Each sample was saved in 25ml vials after traditional methods of nematode extraction and hand counting for qPCR methods. Hand counts showed 12.5% of samples were at or above the current lance damage threshold of 100 nematodes per 100cc of soil. The average Ct value for the current economic threshold was determined to be 32.15 and any qPCR samples that are below that Ct value are considered problematic for golf course greens. Primers were selected based on former gene selection research for lance nematodes (Bae et al., 2008) and specificity was tested against ring, spiral, and sheath nematodes within the 24 samples. Primer specificity assays of lance alone exhibited a positive identification where assays of ring, spiral, and sheath alone were negative. Combinations of lance with each selected plant parasitic nematodes above resulted in positive identifications. Overall, results show a level of success in amplification; however, these methods are inconsistent and currently would not be a viable alternative to traditional methods of nematode extraction. Future work will look at diluting DNA in qPCR reactions to achieve more specific amplification.