Developing a one-step multiplex PCR assay for rapid detection of four Stubby-Root Nematode species, Paratrichodorus allius, P. minor, P. porosus and Trichodorus obtusus

Authors: HUANG, DANQIONG - North Dakota State University; YAN, GUIPING - North Dakota State University; GUDMESTAD, NEIL - North Dakota State University; Whitworth, Jonathan; FROST, KENNETH - Oregon State University; CROW, WILLIAM - University Of Florida; HAJIHASSANI, ABOLFAZL - University Of Georgia

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2018
Publication Date: 1/9/2019
Citation: Huang, D., Yan, G., Gudmestad, N., Whitworth, J.L., Frost, K., Crow, W. 2019. Developing a one-step multiplex PCR assay for rapid detection of four Stubby-Root Nematode species, Paratrichodorus allius, P. minor, P. porosus and Trichodorus obtusus. Plant Disease. 103(3):404-410. https://doi.org/10.1094/PDIS-06-18-0983-RE.
DOI: https://doi.org/10.1094/PDIS-06-18-0983-RE

Interpretive Summary: Stubby-root nematodes in the Trichodoridae family are found in multiple states in the United States. Diagnosis of four species in this family based on morphological characteristics requires the time and the skills of an experienced taxonomist. This study developed a molecular test using a one-step multiplex PCR that can simultaneously diagnose the four species (Paratrichodorus allius, P. minor, P. porosus and Trichodorus obtusus) based on sequences for 18S ribosomal DNA and the internal transcribed spacer 1 region. The test was verified by using 10 other trichodorid species, 20 other plant-parasitic nematodes and three non-plant-parasitic nematodes and results confirmed the test specificity for the four trichodorid species. The trichocorid species confirmed were collected from multiple geographic regions. This new multiplex PCR test provides a simple, rapid, and cost-friends assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

Technical Abstract: Four trichodorid species Paratrichodorus allius, P. minor, P. porosus and Trichodorus obtusus were found in multiple states in the United States. The traditional diagnosis based on morphometrics is laborious and requires an experienced taxonomist. Unfortunately, rapid and low-cost end-point diagnosis using PCR was only available for P. allius. To increase the diagnostic efficiency and reduce the cost, a one-step multiplex PCR assay was developed to simultaneously diagnose these four species through one PCR reaction. Available sequences of the 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer located in ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental PCR tests with these four species, 20 other plant-parasitic nematodes, and three non-plant-parasitic nematodes confirmed the primer specificity. The multiplex PCR successfully amplified desirable fragments using a set of artificially mixed templates containing one, two, three or four targets. The reliability of multiplex PCR diagnostic results was demonstrated by using nematode populations isolated from infested soils from multiple geographic regions. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.