Morphological and Molecular Characterization of Pratylenchus dakotaensis n. sp. (Nematoda: Pratylenchidae), a New Root-Lesion Nematode Species on Soybean in North Dakota, USA

Abstract

Root-lesion nematodes (Pratylenchus spp.) of the genus Pratylenchus Filipjev, 1936, are among the most important nematode pests on soybean (Glycine max (L.) Merr.), along with soybean cyst and root-knot nematodes. In May 2015 and 2016, a total of six soil samples were collected from a soybean field in Walcott, Richland County, ND and submitted to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), USDA, ARS, MD for analysis. Later, in 2019, additional nematodes recovered from a greenhouse culture on soybean originally from the same field were submitted for further analysis. Males, females, and juveniles of Pratylenchus sp. were recovered from soil and root samples and were examined morphologically and molecularly. DNA from single nematodes were extracted, and the nucleotides feature of three genomic regions targeting on the D2–D3 region of 28S rDNA and ITS rDNA and mitochondrial cytochrome oxidase subunit I (COX1) gene were characterized. Phylogeny trees were constructed to ascertain the relationships with other Pratylenchus spp., and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to provide a rapid and reliable differentiation from other common Pratylenchus spp. Molecular features indicated that it is a new, unnamed Pratylenchus sp. that is different from morphologically closely related Pratylenchus spp., including P. convallariae, P. pratensis, P. fallax, and P. flakkensis. In conclusion, both morphological and molecular observations indicate that the North Dakota isolate on soybean represents a new root-lesion nematode species which is named and described herein as Pratylenchus dakotaensis n. sp.

Pratylenchus smoliki, a new nematode species (Pratylenchidae: Tylenchomorpha) from the Great Plains region of North America

“…as a member of a group consisting of four related described species (P. scribneri, P. hexincisus, P. pseudocoffeae, P. alleni) and a putative undescribed species labeled as haplotype groups 9 and 10 in Figure 7. This grouping of species is consistently supported by 28S, 18S, and ITS trees in the literature (Inserra et al, 2007;Subbotin et al, 2008;Araya et al, 2016;Troccoli et al, 2016;Janssen et al, 2017;Singh et al, 2018;Nguyen et al, 2019;Ozbayrak et al, 2019;Handoo et al, 2021) and is highly congruent with grouping based on lip patterns. A maximum likelihood ITS1 tree also illustrates the distinctiveness of P. smoliki n. sp.…”

“…DNA barcoding with the COI mitochondrial gene has been an accurate approach for species identification in Pratylenchus (Troccoli et al, 2016;Singh et al, 2018;Nguyen et al, 2019;Ozbayrak et al, 2019;Handoo et al, 2021). Since COI provides diffe rentiation at the population level as well as species-level discrimination, it is necessary to generate an estimate of withinspecies genetic variation for the mitochondrial marker.…”

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

“…While conventional PCR is more inexpensive, easy to use, and less affected by PCR inhibitors [ 35 ], real-time PCR is less time consuming, more sensitive, allows continuous monitoring of data, and provides quantitative data [ 20 ]. Thus, we designed and developed a conventional PCR assay, and a SYBR Green-I based real-time PCR assay, for identification and quantitative detection of P. dakotaensis , which is a new species of Pratylenchus recently discovered in a soybean field in North Dakota [ 28 , 29 ]. The IC-ITS1F/IC-ITS1R primer set designed was able to specifically and sensitively amplify the DNA of P. dakotaensis in both conventional and real-time PCR assays.…”

“…To confirm the species identity of the root-lesion nematodes extracted from samples that were collected from the field where P. dakotaensis was first discovered [ 28 , 29 ], two genomic regions were sequenced. The universal primers D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) were used to amplify the D2-D3 expansion region of 28S rDNA, following the PCR protocol described by Subbotin et al [ 59 ].…”

“…Greenhouse experiments proved that this Pratylenchus species reproduced well on soybeans with a reproductive factor (final population/initial population) as high as 5.02, thus, allowing us to conclude that this is a new species of Pratylenchus that had never been reported in the literature prior to 2017. Recently, Handoo et al [ 29 ] named this new species of root-lesion nematodes as Pratylenchus dakotaensis , paying homage to the state in which it was discovered. However, no species-specific PCR diagnostic method exists to identify this nematode species prior to this study.…”