|YAN, GUIPING - North Dakota State University|
|PLAISANCE, ADDISON - North Dakota State University|
|HUANG, DANQIONG - North Dakota State University|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2016
Publication Date: 10/4/2016
Citation: Yan, G.P., Plaisance, A., Huang, D., Handoo, Z.A. 2016. First Report of the lance nematode Hoplolaimus stephanus from a soybean field in North Dakota. Plant Disease. 100(12):p.2536. https://doi.org/10.1094/PDIS-07-16-1012-PDN.
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. Lance 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 near Cogswell, Sargent County, ND, a species of lance nematode called Hoplolaimus stephanus by both morphological and molecular means. 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: Lance nematodes (Hoplolaimus spp.) are an important group of ecto-endo parasitic nematodes that feed on plant roots and cause damage to crop production. In August 2015, a soil sample was collected from a soybean field near Cogswell, Sargent County, ND to determine the occurrence of lance nematodes. Nematodes were extracted from soil using sugar centrifugal flotation method, revealing that this sample contained 210 lance nematodes per 100 cm3 of soil. In October 2015, four soil samples were collected from the same field and had lance nematodes ranging from 30 to 100 per 100 cm3 soil, along with seven other groups of plant-parasitic nematodes including lesion, pin, spiral, stunt, dagger, ring, and soybean cyst nematodes. Individual lance nematodes were hand-picked and characterized morphologically and molecularly for species identification. Morphological measurements of adult females (n = 8) included body length (range = 1048.0 to 1410.0 µm, mean = 1272.0 µm), stylet (42.5 to 48.0, 45.0), body width (45.0 to 60.0, 51.8), anterior end to basal bulb (140.0 to 190.0, 169.3), tail length (26.0 to 35.0, 31.0), a (22.2 to 27.3, 24.5), b (6.2 to 8.6, 7.4), c (32.2 to 52.1, 40.8), and V (51.5 to 57.5%, 53.6%). Morphological measurements of adult males (n = 4) were body length (935.0 to 1045.0, 1004.0), stylet (40.0 to 42.0, 40.7), body width (44.0 to 48.0, 46.0), anterior end to basal bulb (145.0 to 160.0, 152.5), tail length (30.0 to 32.0, 31.2), a (20.7 to 22.6, 21.7), b (6.2 to 7.2, 6.5), c (31.0 to 33.6, 32.0), spicules (36.0 to 40.0, 39.0), and capitulum (18.0 to 22.0, 20.5). The nematode species was identified as Hoplolaimus stephanus Sher, 1963 according to morphological and morphometric characteristics. DNA was extracted from single nematodes (n = 3) isolated from soil in 20 µl of extraction buffer (1×PCR buffer, 60 µg/ml Proteinase K). D1/D3 region of 28S rRNA and ITS1 of rDNA were amplified with primer pairs LSUD-1f/LSUD-2r and Hoc-1f/Hoc-2r, respectively. PCR products were cloned using pGEM-T easy vector and sequenced. The consensus sequence from each genomic region was deposited into the GenBank. The D1/D3 sequence (GenBank Accession No. KX347887, 1076 bp) was 99% identical to a population of H. stephanus (HQ678717) from Pennsylvania and was 96% or less identical to other Hoplolaimus spp. The ITS1 rDNA sequence (KX347888, 612 bp) was 98% identical with 13 H. stephanus isolates among which H. stephanus (KP303643) from South Carolina ranked the first in list. This sequence had no significant similarity with other Hoplolaimus spp. including H. galeatus. Sequence analysis here confirmed the species identity as H. stephanus. This species mainly has been reported in the southeastern USA and was found to be the most predominant lance nematode species on soybean in North Carolina. H. galeatus has been mentioned as common in the northern Great Plains, but H. stephanus has never been reported in the northern USA. This discovery is significant because it is the first report of H. stephanus in North Dakota, and the molecular information obtained will allow this nematode to be more easily distinguished from closely related species.