Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the New Root-lesion Nematode Pratylenchus dakotaensis on Soybean.

Root-lesion nematodes, Pratylenchus spp. are reported to cause serious yield losses in various crops including soybean. A new root-lesion nematode species was recently detected in a soybean field in North Dakota (ND) and named Pratylenchus dakotaensis. Nematode detection and differentiation from other species are critical in management strategies. Thus, a recombinase polymerase amplification (RPA) assay was developed for rapid detection of this nematode from field soils under isothermal conditions. New primers and probes were designed from ITS-rDNA region of the nematode genome and tested for both specificity and sensitivity. The RPA assay was able to detect DNA from a single adult nematode at 39.5°C in 20 minutes using both Basic and Exo kits. The specificity of the primers was initially confirmed through in silico analyses, followed by laboratory tests. The assay successfully amplified DNA from the target species, while no amplification occurred for other Pratylenchus spp. and non-Pratylenchus control species. Sensitivity testing with real-time RPA revealed its ability to detect DNA in dilutions equivalent to 1/32 of a single nematode from DNA extracted from inoculated sterile soil. To further validate the assay, it was tested with 19 field soil samples collected in ND. This assay amplified soil DNA extracts of all P. dakotaensis-infested field samples confirmed through conventional PCR. It did not amplify DNA from 13 other field soils infested with other Pratylenchus spp. This is the first report of RPA develoment for detecting a root-lesion nematode species. The RPA assay developed can help in the rapid detection of this nematode species for effective nematode management.

Rapid and Direct Detection of the Stubby Root Nematode, Paratrichodorus allius, from Soil DNA Extracts Using Recombinase Polymerase Amplification Assay.

The stubby root nematode, Paratrichodorus allius, is one of the most important plant-parasitic nematodes. Besides root feeding, P. allius also transmits the Tobacco rattle virus in potatoes, which causes corky ringspot disease. Rapid detection of P. allius is key for efficient management. This study was conducted to develop a real-time recombinase polymerase amplification (RPA) assay that is capable of detecting P. allius directly in DNA extracts from soil using a simple portable device in real time. A fluorophore-attached probe was designed to target the internal transcribed spacer (ITS)-rDNA of P. allius and was used along with primers designed previously. The real-time RPA assay had the ability to detect P. allius DNA extracted directly from infested soil with a sensitivity of one-sixteenth portion of a single nematode. This RPA assay was specific, as it did not produce positive signals from non-target nematodes tested. The real-time RPA was found to be rapid as it could even detect P. allius in as little as 7 min. Testing with 15 field soil samples validated the RPA assay developed in this study. This is the first report of P. allius detection directly from soil DNA using real-time RPA and is the fastest method for P. allius detection in soil to date.

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil.

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0-3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 µg/µL or 0.4 µg/µL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR's capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA.

Host Suitability of Cover Crops to the Root-Lesion Nematode Pratylenchus penetrans Associated with Potato.

Nonhost or poor host cover crops can provide an alternative method for nematode management. A total of 25 cover crop species/cultivars, along with three controls were evaluated in greenhouse experiments for their host suitability to the root-lesion nematode Pratylenchus penetrans. Trials were conducted in a completely randomized design using nematode-infested soil and terminated 3 months after planting. Nematodes were extracted from the roots and soil of each crop to determine their final population densities, reproductive factor (Rf = final population density/initial population density), and distributions in the soil and root habitats. Reproductive factor was used to categorize the host suitability of crops. Faba bean cv. Petite produced the greatest nematode population density in all trials, whereas only alfalfa cv. Bullseye constantly demonstrated the poor host ability to P. penetrans. Annual ryegrass, winter rye cv. ND Dylan, and white proso millet also showed poor hosts in most trials. Five cover crops consistently maintained the population throughout the experiments, with Rf values less than 2, and the remaining tested cover crops were suitable hosts for P. penetrans. The majority of the tested cover crops had less than or equal to 30% of the final population residing in the roots after three months of growth in all the trials. This research helps us gain the knowledge on cover crops and P. penetrans interaction and will be useful for potato growers to select better cover crops and avoid susceptible hosts to manage P. penetrans in infested fields to minimize potato yield losses.

Development of a Droplet Digital PCR Assay for Detection and Quantification of Stubby Root Nematode, Paratrichodorus allius, in Soil.

The stubby root nematode Paratrichodorus allius is an important plant-parasitic nematode species within the 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 methods or a 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 the assay was used to quantify P. allius populations in field soils. The assay did not detect nematodes other than P. allius, thus showing high specificity. It was able to detect P. allius equivalent to a 0.01 and 0.02 portion of a single nematode in soil DNA and nematode DNA extracts, respectively. 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). 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 to detect and quantify stubby root nematode in soil. The results of this study support the potentiality of a ddPCR assay as a new research tool in diagnostics of plant-parasitic nematodes.

Evaluation of Wheat Cultivars and Germplasm Lines for Resistance to Pratylenchus neglectus Populations Collected in North Dakota.

Root-lesion nematode (RLN; Pratylenchus neglectus) is a migratory endoparasite and a major soilborne pathogen that affects wheat (Triticum spp.) production worldwide. Genetic resistance is one of the most economical and effective ways to manage P. neglectus in wheat. This study evaluated 37 local cultivars and germplasm lines in seven greenhouse experiments, including 26 hexaploid wheat, six durum wheat, two synthetic hexaploid wheat, one emmer wheat, and two triticale for P. neglectus resistance from 2016 to 2020. North Dakota field soils infested with two RLN populations (350 to 1,125 nematodes per kilogram of soil) were used for resistance screening under controlled greenhouse conditions. The final nematode population density for each cultivar and line was counted under the microscope to categorize the resistance ranking of these entries as resistant, moderately resistant, moderately susceptible, and susceptible. Out of the 37 cultivars and lines, one was classified as resistant (Brennan); 18 were moderately resistant (Divide, Carpio, Prosper, Advance, Alkabo, SY Soren, Barlow, Bolles, Select, Faller, Briggs, WB Mayville, SY Ingmar, W7984, PI 626573, Ben, Grandin, and Villax St. Jose); 11 were moderately susceptible; and seven were susceptible to P. neglectus. The resistant to moderately resistant lines identified in this study could be used in breeding programs after the resistance genes or loci are further elucidated. This research provides valuable information about P. neglectus resistance among wheat and triticale cultivars used in the Upper Midwest region of the United States.

First Draft Genome Assembly of Root-Lesion Nematode Pratylenchus scribneri Generated Using Long-Read Sequencing.

Root-lesion nematodes (genus Pratylenchus) belong to a diverse group of plant-parasitic nematodes (PPN) with a worldwide distribution. Despite being an economically important PPN group of more than 100 species, genome information related to Pratylenchus genus is scarcely available. Here, we report the draft genome assembly of Pratylenchus scribneri generated on the PacBio Sequel IIe System using the ultra-low DNA input HiFi sequencing workflow. The final assembly created using 500 nematodes consisted of 276 decontaminated contigs, with an average contig N50 of 1.72 Mb and an assembled draft genome size of 227.24 Mb consisting of 51,146 predicted protein sequences. The benchmarking universal single-copy ortholog (BUSCO) analysis with 3131 nematode BUSCO groups indicated that 65.4% of the BUSCOs were complete, whereas 24.0%, 41.4%, and 1.8% were single-copy, duplicated, and fragmented, respectively, and 32.8% were missing. The outputs from GenomeScope2 and Smudgeplots converged towards a diploid genome for P. scribneri. The data provided here will facilitate future studies on host plant-nematode interactions and crop protection at the molecular level.

Early Detection and Temporal Dynamics of Pratylenchus scribneri Infection in Potato Roots Determined Using Quantitative PCR and Root Staining.

The root-lesion nematode, Pratylenchus scribneri, is a migratory endo-parasitic nematode that impacts potato production on a large scale. Effective management of this nematode requires an understanding of its population dynamics alongside early detection. Typically, the nematode population estimates are made from infested soil; however, considering the endo-migratory lifestyle of this nematode, it also is crucial to determine the nematode population residing inside the host roots. In this study, a SYBR green-based quantitative real-time PCR (qPCR) assay was developed for detection and quantification of P. scribneri in potato roots. The assay used a previously reported primer pair (ITS-2F/ITS-2R), and it proved to be specific and sensitive, detecting as low as 1/128th equivalents of a P. scribneri individual per 0.2 g of potato roots. The robustness of the assay was reflected in high correlation observed between the P. scribneri densities determined microscopically and the densities detected by qPCR in artificially inoculated (R2 = 0.93) and naturally infected (R2 = 0.73) root samples. A time-course experiment conducted in the greenhouse using qPCR detected P. scribneri in potato roots as early as 5 days after planting. The results correlated well with the microscopic observations (R2 = 0.80) and were complemented further with root staining. Additionally, three P. scribneri reproduction peaks were observed during one 3-month growth cycle of potato. Overall, the assay developed in this study is specific to P. scribneri in DNA extracts of root tissue and allows early detection and understandings of reproduction timings of this important nematode of potato.

Population Development of the Root-Lesion Nematode Pratylenchus dakotaensis on Soybean Cultivars.

Two greenhouse experiments were conducted with the new species of root-lesion nematode, Pratylenchus dakotaensis, discovered in North Dakota, USA in 2017 and named in 2021. Experiment 1 was conducted to ascertain resistance levels of soybean (Glycine max [L.] Merr.) cultivars to the new species P. dakotaensis and Experiment 2 was conducted to evaluate the population levels of P. dakotaensis in roots versus soil. A total of 20 soybean cultivars, and the positive control, cultivar Barnes, accompanying a nonplanted control, were evaluated in both experiments; they each had five replicates and the experiments were repeated. Among the 20 soybean cultivars evaluated, a cultivar with the ID# 13 consistently produced the greatest population densities of P. dakotaensis across all the experiments and therefore it was selected as the susceptible check. The ratio of the final nematode population density of a test cultivar relative to the final population density of the susceptible check was used to scale resistance rating. Combined results of all the trials indicated that seven of the cultivars were moderately resistant, nine of the cultivars plus 'Barnes' were moderately susceptible, and four of the cultivars were susceptible. However, none of the cultivars tested were classified as resistant. Analysis of P. dakotaensis population levels revealed that ≥50% of the population resided in the root habitat and the remaining nematodes were recovered from soil at 9 weeks after planting for a majority of the cultivars tested. These results provide an insight into the virulence of P. dakotaensis on commercial soybean cultivars.

Characterization of Virulence Phenotypes of Soybean Cyst Nematode (Heterodera glycines) Populations in North Dakota.

Soybean cyst nematode (SCN; Heterodera glycines) continues to be the greatest threat to soybean production in the United States. Because host resistance is the primary strategy used to control SCN, knowledge of SCN virulence phenotypes (HG types) is necessary for choosing sources of resistance for SCN management. To characterize SCN virulence phenotypes in North Dakota, a total of 419 soybean fields across 22 counties were sampled during 2015, 2016, and 2017. SCN was detected in 42% of the fields sampled, and population densities in these samples ranged from 30 to 92,800 eggs and juveniles per 100 cm3 of soil. The SCN populations from some of the infested fields were virulence-phenotyped with seven soybean indicator lines and a susceptible check ('Barnes') using the HG type tests. Overall, 73 SCN field populations were successfully virulence-phenotyped. The HG types detected in North Dakota were HG types 0 (frequency rate: 36%), 7 (27%), 2.5.7 (19%), 5.7 (11%), 1.2.5.7 (4%), and 2.7 (2%). However, before this study only HG type 0 was detected in North Dakota. The designation of each of these HG types detected was also validated by repeating the HG type tests for 33 arbitrarily selected samples. This research for the first time reports several new HG types detected in North Dakota and confirms that the virulence of SCN populations is shifting and overcoming resistance, highlighting the necessity of using different resistance sources, rotating resistance sources, and identifying novel resistance sources for SCN management in North Dakota.

Developing a Real-Time PCR Assay for Direct Identification and Quantification of Soybean Cyst Nematode, Heterodera glycines, in Soil and Its Discrimination from Sugar Beet Cyst Nematode, Heterodera schachtii.

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the Heterodera schachtii sensu stricto group is not only difficult and time-consuming but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes and to quantify SCN directly from DNA extracts of field soils. SCN- and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated on the basis of target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and they may serve as useful diagnostic tools in research, diagnostic laboratories, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.

Effects of Cover Crops on Population Reduction of Soybean Cyst Nematode (Heterodera glycines).

Microplot experiments were conducted to evaluate the effects of cover crops on population reduction of a major soybean pest, soybean cyst nematode (SCN; Heterodera glycines Ichinohe) in 2016 and 2017. Ten crop species, including annual ryegrass (Lolium multiflorum L.), Austrian winter pea (Pisum sativum L. subsp. arvense), carinata (Brassica carinata A. Braun), faba bean (Vicia faba Roth), foxtail millet (Setaria italica (L.) P. Beauvois), daikon radish (Raphanus sativus L.), red clover (Trifolium pratense L.), sweetclover (Melilotus officinalis L.), turnip (Brassica rapa subsp. rapa L.), and winter rye (Secale cereale L.), were planted along with susceptible soybean (Glycine max (L.) Merr. 'Barnes') in soil naturally infested with each of two SCN populations (SCN103 and SCN2W) from two North Dakota soybean fields. Crops were grown in large plastic pots for 75 days in an outdoor environment (microplot). Soil samples were collected from each pot for nematode extraction and SCN eggs were counted to determine the final SCN egg density. The population reduction was determined for each crop and nonplanted natural soil (fallow). All of the tested crops and nonplanted natural soil had significantly (P < 0.0001) lower final population densities compared with susceptible soybean (Barnes). Also, a significant difference (P < 0.0001) was observed between the SCN population suppressions caused by cover crops versus the fallow treatment. All cover crops except Austrian winter pea, carinata, faba bean, and foxtail millet had consistently lower SCN egg numbers than in fallow in both years of the experiments. The average population reductions of SCN by the cover crops ranged from 44 to 67% in comparison with the initial population density, while the fallow had natural reductions from 4 to 24%. Annual ryegrass and daikon radish reduced SCN egg numbers to a greater extent than the other cover crops, with an average of 65 and 67% reduction of initial population density, respectively, from 2 years. The results suggested that cover crops reduced the SCN populations in external microplot conditions, and their use has great potential for improving SCN management in infested fields.

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

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.

On the molecular identity of Paratylenchus nanus (Nematoda: Tylenchida).

In this study, molecular characterization of Paratylenchus nanus collected from the type locality in Four Mile Run, Fall Church, Virginia using COI, D2-D3 of 28 S rRNA and ITS rRNA gene sequences was provided. We molecularly also characterized, Paratylenchus specimens collected from grasses in Devils Lake, Ramsey County, North Dakota indicated as the type locality in the original description of P. nanus by Cobb (1923). These nematodes were identified as representatives of the species P. projectus. Populations of P. nanus belonging to the molecular types A and B, and previously designated by Van den Berg et al. (2014) should be now identified as P. nanus and P. projectus, respectively.

Reproduction Ability and Growth Effect of Pin Nematode, Paratylenchus nanus, With Selected Field Pea Cultivars.

Greenhouse experiments were conducted to determine the reproductive ability and effect of the pin nematode Paratylenchus nanus from North Dakota on field pea cultivars. Reproduction of P. nanus was determined on seven field pea cultivars using naturally infested field soils at low (1,500/kg of soil) and high (4,500/kg soil) initial pin nematode densities. Nematode effect on plant growth and seed yield was evaluated at 4,500 P. nanus per 1 kg of soil by artificially inoculating P. nanus on six field pea cultivars. Reproductive factor (RF) of P. nanus was observed to be greater at the low density than the high density of the nematode. At the low population density, RF values ranged from 1.10 to 11.20, whereas at the high density, RF ranged from 1.20 to 2.50. In experiments evaluating P. nanus effects on cultivar growth, the nematode (4,500 P. nanus per 1 kg soil) caused reduction (P < 0.05) of plant height in most cultivars tested, and it also significantly impacted dry shoot weight and dry seed weight in some experiments. Plant height and shoot weight reductions were the highest in the cultivar Arcadia (up to 37 and 53%, respectively), with a dry seed weight reduction up to 32%. This research demonstrated for the first time the negative impact of P. nanus on field peas in controlled greenhouse conditions, which is an important step toward developing effective management strategies to improve the productivity of this leguminous crop.

Assessment of Factors Associated with Molecular Quantification of Stubby Root Nematode Paratrichodorus allius from Field Soil DNA.

Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.

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.

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 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 in the 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 analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. 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.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

Specific Detection of the Root-Lesion Nematode Pratylenchus scribneri Using Conventional and Real-Time PCR.

Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological methods is an arduous task requiring extensive training on nematode taxonomy and years of experience. Thus, molecular methods for P. scribneri detection and identification were developed. Conventional and real-time polymerase chain reaction (PCR) assays with new species-specific primers were used in this study, which exclusively amplified DNA of P. scribneri but not DNA from other Pratylenchus spp. or non-Pratylenchus spp. tested. Compared with conventional PCR that was able to detect an equivalent to 1/4 of the DNA of a single nematode, real-time PCR was more sensitive and could amplify an equivalent to 1/128 of the DNA of one nematode. Both conventional and real-time PCR assays successfully identified P. scribneri and distinguished it from P. penetrans and P. neglectus isolated from field samples collected from various locations in North Dakota and Minnesota. The Blast-search based on the sequence information confirmed the reliability of the PCR assays for species identification. This is the first report of P. scribneri identification using a real-time PCR assay. The developed PCR assays are suitable for use in diagnostic laboratories and detection of field infestations with this nematode species.

Development of Real-Time and Conventional PCR Assays for Identifying Stubby Root Nematode Paratrichodorus allius.

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10-4 ng of DNA template could be detected by conventional PCR and 1.92 × 10-4 ng of DNA by real-time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.