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Title: First report of the spiral nematode Helicotylenchus microlobus infecting soybean in North Dakota

item YAN, GUIPING - North Dakota State University
item PLAISANCE, ADDISON - North Dakota State University
item HUANG, DANQIONG - North Dakota State University
item Handoo, Zafar

Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/19/2017
Publication Date: 3/1/2017
Publication URL:
Citation: Yan, G.P., Plaisance, A., Huang, D., Handoo, Z.A. 2017. First report of the spiral nematode Helicotylenchus microlobus infecting soybean in North Dakota. Journal of Nematology. 49(1):1.

Interpretive Summary: Plant-parasitic nematodes are microscopic worms that attack plant roots and cause an estimated ten billion dollars of crop loss each year in the United States and 100 billion dollars globally. Spiral nematodes are an important problem damaging the roots of many kinds of plants worldwide. One problem with determining the extent of damage to crop plants is that the nematodes present in many areas are not known, such as in North Dakota. This brief report describes how a team of North Dakota State University and ARS scientists identified from a soybean field in Richland County, ND, a species of spiral nematode called Helicotylenchus microlobus by both morphological and molecular means. They also discovered how to distinguish this species from closely related species with molecular and anatomical features. This discovery is significant because it is the first report of this nematode in North Dakota and the molecular information obtained will allow this nematode to be more easily distinguished from closely related species. Therefore, this research will be used by scientists, growers, action agencies, and extension agencies involved in nematode research and control.

Technical Abstract: 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. 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 kg of soil. In June and August 2016, ten 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 kg of soil. One soil sample with 1,500 spiral nematodes per kg was used to inoculate two soybean cultivars Sheyenne and Barnes each in four replicates. After 15 weeks of growth at 22°C in a greenhouse, the population of spiral nematodes increased greatly. The final density was 9,300 ± 1,701 spiral nematodes per kg soil for Sheyenne and 9,451 ± 2,751 for Barnes. The reproduction 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 hand-picked 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), head end to posterior end of esophageal 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. DNA was extracted from single nematodes (n = 8) using the Proteinase K method. The ITS region of rDNA was amplified with the primers rDNA2/rDNA1.58S. The PCR products were then purified and sequenced. The consensus ITS rDNA sequence (Accession No. KY271078, 822 bp) that was deposited into GenBank shared 99% identity with two isolates of H. microlobus from California USA (KM506860.1, KM506859.1) and one isolate of H. microlobus from Spain (KM506862.1). It had only 91% sequence identity with seven isolates of H. pseudorobustus (KM506875.1, KM506880.1, KM506876.1, KM506874.1, KM506872.1, KM506879.1, KM506878.1) from California USA, Switzerland and New Zealand, a spiral nematode species very closely related to H. microlobus in morphology. The molecular tests confirmed the identity 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 soybean cultivars tested except Hawkeye were rated as hosts. To our knowledge, this is the first report of H. microlobus in North Dakota.