Assessment of factors associated with real-time PCR quantification of Paratrichodorus allius from field soil DNA

Authors: HUANG, D - North Dakota State University; YAN, G - North Dakota State University; GUDMESTAD, N - North Dakota State University; Whitworth, Jonathan

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/18/2019
Publication Date: 6/22/2019
Citation: Huang, D., Yan, G., Gudmestad, N.C., Whitworth, J.L. 2019. Assessment of factors associated with real-time PCR quantification of Paratrichodorus allius from field soil DNA. Plant Disease. 103(12):3265-3273. https://doi.org/10.1094/PDIS-12-18-2240-RE.
DOI: https://doi.org/10.1094/PDIS-12-18-2240-RE

Interpretive Summary: Stubby root nematode (Paratrichodorus allius) is one of the vectors of Tobacco rattle virus in potato. Symptoms caused by this virus can make potato tubers unmarketable. Identification of stubby root nematode based on physical characteristics using a microscope is difficult and time-consuming. DNA-based molecular identification of this nematode species can be done using a soil sample from a field in question. Molecular probes specific for the nematode are used in a real-time PCR test that can quantify stubby root nematodes in the sample. Inhibitory factors were studied, and results showed that pre-treating the soil sample by autoclaving reduced the amount and quality of DNA detected. Air or oven drying of the soil sample also yielded less DNA, but the quality was similar to samples from natural field soil. The effect of PCR inhibitors was decreased by the addition of bovine serum albumin in the test. Results also showed more nematode DNA was detected when larger nematodes were present compared to smaller nematodes. These results will help when testing is done for the detection and quantification of stubby root nematodes in a field soil sample.

Technical Abstract: Factors relating to SYBR Green quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sands from potato fields in North Dakota and Idaho. Results showed that the largest individuals (body length > 720 µm) produced significantly lower Cq values than smallest individuals (< 359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatment studies showed that autoclaved field soil significantly reduced the final DNA amount and quality, and air or oven dried soil yielded less amount of DNA with a similar purity compared to natural field soil. PCR inhibitors were detected in soil DNA substrate without additional purification targeting pBluescript II SK(+)- plasmid DNA, and Al(NH4)(SO4)2 treatment during DNA extraction significantly reduced the inhibitors compared to post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin used as a PCR enhancer factor. The quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA with known nematode numbers, produced similar quantification correlation and amplification efficiency. The targets in soil DNA quantified by qPCR using either of the standard curves correlated well with the microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.