Development of a droplet digital PCR assay for detection and quantification of stubby root nematode, Paratrichodorus allius, in soil

Authors: LAWAJU, BISHO RAM - North Dakota State University; YAN, GUIPING - North Dakota State University; Whitworth, Jonathan

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2023
Publication Date: 11/6/2023
Citation: Lawaju, B., Yan, G., Whitworth, J.L. 2023. Development of a droplet digital PCR assay for detection and quantification of stubby root nematode, Paratrichodorus allius, in soil. Plant Disease. (2023) 107:3344-3353. https://doi.org/10.1094/PDIS-03-23-0439-SR.
DOI: https://doi.org/10.1094/PDIS-03-23-0439-SR

Interpretive Summary: Droplet digital PCR (ddPCR) is a novel PCR technique which can be used to detect stubby root nematode (Paratrichodorus allius) DNA from a soil sample. This work shows that this technique can specifically detect DNA from stubby root nematodes down to a detection limit of just 0.02 portion of a single nematode. The specificity was tested against species from eight different genera of nematodes, including two other species of stubby root nematode (P. minor and P. porosus). From all of these samples, only P. allius was detected by the assay. Correlation between traditional microscope counts and the use of ddPCR is high (R2=0.7963), allowing this method to be used to detect stubby root nematode in soil samples without the need to manually extract nematodes, identify them under the microscope and quantify them using real-time PCR. This ddPCR technique has the potential for a new diagnostic tool for plant-parasitic nematodes.

Technical Abstract: The stubby root nematode, Paratrichodorus allius is an important plant-parasitic nematode species within Trichodoridae family. It can directly harm the plants by feeding on the roots or indirectly by transmitting Tobacco rattle virus. These nematodes are mostly diagnosed either by traditional microscopic method or polymerase chain reaction (PCR)-based method. Droplet digital PCR (ddPCR) is a novel PCR technique which is sensitive and precise in quantifying DNA templates of the test samples. In this study, we developed a ddPCR assay to detect and quantify P. allius in soil. The specificity and sensitivity of the assay was first determined using P. allius nematode DNA or DNA from sterilized soil artificially inoculated with P. allius and then the assay was used to quantify P. allius populations in field soils. The assay did not detect nematodes other than P. allius, showing high specificity. It was able to detect P. allius equivalent to a 0.02 portion of a single nematode. Highly linear relationships between DNA copy numbers from ddPCR and serial dilutions of known concentrations were observed with DNA from P allius nematodes (R2 = 0.9842) and from artificially infested soil (R2 = 0.9464). Moreover, the P. allius populations from field soils determined by ddPCR were highly correlated with traditional microscopic counts (R2 = 0.7963). To our knowledge, this is the first report of applying ddPCR assay to detect and quantifying stubby root nematode in soil. The results of this study support the potential of ddPCR assay as a new research tool in diagnostics of plant-parasitic nematodes.