Developing a Real-Time PCR Assay for Direct Identification and Quantification of Pratylenchus penetrans in Soil

Abstract

The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato worldwide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are essential for developing effective integrated pest control measures. However, distinction between P. penetrans and other Pratylenchus spp. based on morphology is a tedious task. A SYBR Green I-based qPCR assay was developed to discriminate, identify, and quantify P. penetrans in field soil. P. penetrans-specific qPCR primers were designed from the D2-D3 region of the 28S rDNA. The specificity of the assay was evaluated using eight isolates of P. penetrans populations and 31 isolates of other nematode species. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. There was a high correlation between the P. penetrans numbers artificially added to soil or estimated from naturally infested field soils by conventional methods, and the numbers quantified using the qPCR assay. Grinding the field soil prior to DNA extraction improved P. penetrans detection from soil. The qPCR assay will not only be useful for differentiating P. penetrans from mixed populations of Pratylenchus spp., but also for efficient detection and quantification of P. penetrans from field soil. The assay requires no expertise in nematode taxonomy and morphology, and may serve as a useful diagnostic tool in research, diagnostic labs, and extension services for pest management.

Root‐lesion nematodes of potato: Current status of diagnostics, pathogenicity and management

“…Similar results were obtained for the detection and quantification of P. thornei (Mokrini et al, 2014). In both studies, there were no cases of underestimation or overestimation compared to enumeration under the microscope, as previously reported by other qPCR methods (Sato et al, 2007(Sato et al, , 2010Goto et al, 2011;Baidoo et al, 2017;Oliveira et al, 2017;Dauphinais et al, 2018). Nevertheless, further tests must be done to check their reproducibility in other laboratories worldwide.…”

“…However, these methods showed an overestimation of nematode numbers compared to conventional enumeration under the microscope (Baidoo et al, 2017;Dauphinais et al, 2018).…”

“…Both qPCR methods allowed the detection of a single individual of P. penetrans without evidence of cross‐reaction with other root‐lesion nematode species or plant‐parasitic nematodes. However, these methods showed an overestimation of nematode numbers compared to conventional enumeration under the microscope (Baidoo et al , ; Dauphinais et al , ).…”

“…Attention has also been given to the D2‐D3 expansion segment of 28S rDNA as a potential target for qPCR methods for detection and quantification of P. penetrans (Baidoo et al , ; Dauphinais et al , ). Both qPCR methods allowed the detection of a single individual of P. penetrans without evidence of cross‐reaction with other root‐lesion nematode species or plant‐parasitic nematodes.…”

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

“…Since then, numerous studies have identified and differentiated Pratylenchus spp. to the species level using DNA-based molecular diagnostic methods such as PCR restriction fragment length polymorphism (RFLP), species-specific PCR, and DNA sequencing [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. The RFLP potion of the RFLP–PCR is an additional time-consuming step that is not amenable to commercial high-throughput applications [ 22 ].…”