Development of Real-Time and Conventional PCR Assays for Identifying a Newly Named Species of Root-Lesion Nematode (Pratylenchus dakotaensis) on Soybean.

Intiaz Amin Chowdhury,Guiping Yan

doi:10.3390/ijms22115872

Intiaz Amin Chowdhury, Guiping Yan

Open Access

https://doi.org/10.3390/ijms22115872

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Abstract

A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode (Pratylenchus dakotaensis) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of P. dakotaensis. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only P. dakotaensis DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R2 value of 0.95 between quantification cycle number and log number of P. dakotaensis. To validate the assays to distinguish P. dakotaensis from other Pratylenchus spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of P. dakotaensis and discriminated it from other Pratylenchus spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of P. dakotaensis.

Highlights

Root-lesion nematodes, Pratylenchus spp., are one of the most economically important nematode pests of crops worldwide [1]
The objectives of this research were to develop conventional and real-time polymerase chain reaction (PCR) assays with species-specific primers for rapid, reliable, and sensitive detection of P. dakotaensis, and to evaluate the capability of the assays to discriminate between P. dakotaensis and other commonly found Pratylenchus spp. in North Dakota soybean fields
The multiple sequence alignment of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) from Pratylenchus spp. revealed a unique region in the genome of P. dakotaensis that is polymorphic between different species

Summary

Introduction

Root-lesion nematodes, Pratylenchus spp., are one of the most economically important nematode pests of crops worldwide [1] These plant-parasitic nematodes have a migratory endoparasitic nature, ability to reproduce sexually and/or asexually, excellent adaptability to diverse environmental conditions, and broad host range [2]. They are ranked as the third most important group of plant-parasitic nematodes after root-knot nematodes and cyst nematodes [3]. Numerous studies have reported high yield losses caused by root-lesion nematodes in infested crop fields. In Australia, P. thornei was reported to cause yield losses in wheat as high as 85% [5].

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