Quantitative Polymerase Chain Reaction (qPCR) By Matthew Tucker, School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA
L
ance nematodes have become increasingly problematic on golf course putting greens across Virginia. Turfgrass professionals are monitoring plant parasitic nematode populations more frequently by sending routine samples into nematode assay labs for quantification. Previous research has demonstrated variability between selected state labs in population estimates due to the use of different diagnostic methods, highlighting the need for a standard objective quantification method. The use of quantitative polymerase chain reaction (qPCR) for quantitative estimates of various organisms is widely adopted for many diagnostics approaches but is understudied for turfgrass nematode estimations. In general, qPCR works using reaction additives to target a select region of DNA that is unique to an organism through a process of heating and cooling, referred to as a cycle (x40). Each cycle will multiply that specific region of DNA and activates a fluorescent dye proportional to the amount of DNA present. The number of cycles required to see the fluorescence is known as the cycle threshold (Ct) value. When the Ct value is low, it means there is a lot of DNA present that is causing fluorescence to be seen quickly and when the Ct value is high, it means that more cycles are required to identify the presence of any DNA. The objective of this research is to refine a qPCR technique for identification and quantification of lance nematodes from golf course putting greens. Five samples of DNA extracted from 100 handpicked lance nematode using a Qiagen Powersoil DNA kit produced a Ct range of 28.9-34.7. This range is wide and suggests inconsistency. Lance nematode populations that were hand counted from 28 plots collected at Belmont Golf Course (BGC) in Richmond, VA resulted in a cycle threshold range of 20.949-25.686. Based on information previously stated, one might assume higher levels of lance nematodes from the BGC samples, but some things were not adding up upon closer inspection. Some samples should have fallen around the range of the handpicked nematodes, but all reactions were lower in Ct values. This suggests a degree of error across reactions.
We then handpicked samples of 250, 100, 50, and 20 lance nematodes to validate our range for 100 handpicked lance nematodes by addressing multiple avenues of human error. Results from the new handpicked samples produced Ct values that made more logical sense with the sample of 250 resulting in the lowest Ct value and the sample of 20 the highest. These results suggest that qPCR is a valid and objective way to both identify and quantify lance nematode presence; however, future research will continue to refine this process of reducing human error to maximize quantification consistency.
Interlab variability
Nematode assay extraction methods vary by lab for quantifying plant-parasitic nematodes (PPN) from turfgrass samples. The objective of this research was to compare the relative extraction efficiencies of homogenous turfgrass samples by various state nematode labs. Random samples were collected from three locations across the Mid-Atlantic United States, homogenized, and divided into thirty 500 cc subsamples. Three subsamples from each location were sent to ten state nematode assay labs (n=90). Semi-automatic elutriation (n=27) was used to extract PPN by three labs; Louisiana, North Carolina, and Virginia. Nematodes were extracted by hand sieving (n=63) in the remaining seven labs; Alabama, Florida, Georgia, Massachusetts, Michigan, Mississippi, and South Carolina. Six PPN genera identified across all labs included lance, ring, stunt, root-knot, spiral, and stubby root. Five or six genera of PPN were different by state where spiral was similar across states due to low or no populations present. Estimated populations of PPN varied among states with the same extraction methods. Extractions using semi-automatic elutriation were similar among the three tested labs for all genera except lance, where estimated populations were higher from the Virginia lab than from Louisiana or North Carolina labs. Estimated populations were significantly different for four of six genera when evaluated using the handsieving method. Analysis by method suggests that a component of automation may reduce variability among PPN counts. Ultimately, this research demonstrates the need to use the same lab over time for the most consistent PPN information.
Table 1. Comparison of nematode extraction efficiencies of six genera of plant parasitic nematode by state and by method used. Highlighted sections denote significance. This table suggests that semi-automatic elutriation is a more consistent method of extracting nematodes compared with hand sieving. By State
By Semi-Automatic Elutriation
By Hand Sieving
P-value
P-value
P-value
Lance
0.0001
0.0021
0.0027
Ring
0.0385
0.1487
0.1030
Stunt
0.0051
0.1001
0.0344
Root-knot
0.0329
0.0639
0.0802
Spiral
0.443
0.3678
0.0066
Stubby root
0.0191
0.5025
0.0462
16 | VIRGINIA TURFGRASS JOURNAL January/February 2024 www.vaturf.org
