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.

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

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.

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.

First Report of the Spiral Nematode Helicotylenchus microlobus Infecting Soybean in North Dakota.

Spiral nematodes (Helicotylenchus spp.) are common plant-parasitic nematodes in fields of many crops. In June 2015, two soil samples were collected from a soybean field in Richland County, ND. Nematodes were extracted from soil using the sugar centrifugal flotation method (Jenkins, 1964). Plant-parasitic nematodes were identified to genus based on morphological features and counted. Both samples contained spiral nematodes from 1,500 to 3,300 per kilogram of soil. In June and August 2016, 10 soil samples were collected from the same field. Nematodes were extracted, and nine of the samples had spiral nematodes ranging from 125 to 3,065 per kilogram of soil. One soil sample with 1,500 spiral nematodes per kilogram was used to inoculate two soybean cultivars Sheyenne and Barnes each in four replicates. After 15 wk of growth at 22°C in a greenhouse room, the population of spiral nematodes was found to have increased greatly. The final density was 9,300 ± 1,701 spiral nematodes per kilogram of soil for Sheyenne and 9,451 ± 2,751 for Barnes. The reproductive factor in Sheyenne and Barnes was 6.2 and 6.3, respectively, indicating that this spiral nematode infects and reproduces well on these two soybean cultivars. Infected soybean roots had small brown lesions on the surface. Individual spiral nematodes were handpicked and examined morphologically and molecularly for species identification. Morphological measurements of adult females (n = 15) included body length (mean = 708.5 µm, range = 600.0-812.0 µm), stylet (27.6, 26.0-29.0), body width (28.3, 25.0-33.0), lip region end to posterior end of pharyngeal glands (142.5, 130.0-152.0), anal body width (15.8, 14.0-17.5), tail length (20.3, 15.0-25.0), tail annules (11.6, 10.0-14.0), a (25.0, 21.4-27.1), b (5.0, 4.4-5.7), c (35.4, 30.2-41.7), c' (1.3, 1.0-1.6), and V (61.8%, 60.0-63.7). The spiral nematode was identified as Helicotylenchus microlobus according to morphological and morphometric characteristics (Subbotin et al., 2015). DNA was extracted from single nematodes (n = 8) using the Proteinase K method (Kumari and Subbotin, 2012). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers rDNA2/rDNA1.58S (Cherry et al., 1997). The PCR products were then purified and sequenced. The consensus ITS rDNA sequence (accession no. KY271078, 822 bp) that was deposited into the GenBank shared 99% identity with two isolates of H. microlobus from California (KM506860.1 and KM506859.1) and one isolate of H. microlobus from Spain (KM506862.1) (Subbotin et al., 2015). It had only 91% sequence identity with seven isolates of H. pseudorobustus (KM506875.1, KM506880.1, KM506876.1, KM506874.1, KM506872.1, KM506879.1, and KM506878.1) from California, Switzerland, and New Zealand, a spiral nematode species very closely related to H. microlobus in morphology. The molecular tests confirmed the identity of this spiral nematode as H. microlobus. The H. microlobus nematode was reported as one of the most commonly observed spiral nematodes in soil samples in the state of Minnesota, and all 13 soybean cultivars tested except Hawkeye were rated as hosts (Taylor, 1960). To our knowledge, this is the first report of H. microlobus in North Dakota.