Danqiong Huang

First Report of the Stubby Root Nematode Paratrichodorus allius on Potato in North Dakota

6 Stubby root nematodes (Paratrichodorus and Trichodorus) are migratory ectoparasites that 7 feed on roots and vector tobraviruses (Riga et al. 2007). These nematodes are important to the 8 potato industry as they transmit Tobacco rattle virus (TRV) causing corky ringspot (CRS) disease 9 that has a direct economic impact on growers due to being abandoned or rejected by processing or 10 fresh markets (Charlton et al. 2010). TRV associated with CRS on potato in North Dakota was 11 reported but stubby root nematodes (SRN) were not investigated (David et al. 2010). In October 12 2014, three soil samples were collected from a potato field in Sargent County, ND to determine the 13 occurrence of SRN. Most of the field was harvested but a portion of the field was abandoned due 14 to 80 to 90% of the tubers (cv. Milva) exhibiting brown necrotic rings, arcs and spots, typical 15 symptoms of CRS. Diseased tubers from this field were tested and found to be infected with TRV. 16 Nematodes were extracted from soil using the sugar centrifugal flotation method and one of the 17 samples was found to contain SRN (44 per kg of soil). In April 2015, forty-nine soil samples were 18 collected from the same field and seven of the samples had SRN with population densities ranging 19 from 135 to 300 (mean = 175) per kg of soil. Nematodes were examined morphologically and 20 molecularly for species identification. Morphological measurements of adult females (n = 10) 21 included body length (range = 550.0 to 690.0 μm, mean = 606.8 μm), onchiostyle (40.0 to 47.5, 22 43.9), body width (35.0 to 58.0, 42.3), anterior end to basal bulb (90.0 to 150.0, 114.7), a (10.3 to 23 18.6, 14.7), b (4.0 to 6.7, 5.3), and V (50.0 to 60.0%, 53.8%). The anus and caudal pores were 24 subterminal. The nematode species was identified as Paratrichodorus allius (Jensen 1963) Siddiqi, 25 1974 according to morphological and morphometric characteristics (Decraemer 1980). DNA was 26 extracted from single nematodes (n = 7) isolated from three soil samples in 20 μl of extraction 27 buffer. The D2/D3 region of 28S rRNA, partial 18S rRNA, and ITS1 rDNA were amplified with 28 primer pairs D2A/D3B, SSUF07/SSUR26 and BL18/5818, respectively (Ye et al. 2015; Riga et al. 29 2007). PCR products were cloned using pGEM-T easy vector and sequenced. Since sequences 30 from all samples for each genomic region were identical, only one of the sequences from that 31 region was submitted to GenBank and thus represents a consensus sequence. The 18S rRNA 32 sequence (GenBank Accession No. KU094058, 919 bp) was 100% identical to one population of 33 P. allius (AJ439572) from Washington, 99% identical to P. teres, a closely related species of P. 34 allius, and less than 99% identical to other Paratrichodorus spp. The ITS1 rDNA sequence 35 (KU094059, 832 bp) was 99% homologous with two populations of P. allius from North Carolina 36 (KJ934124) and Washington (AM087124), but had no significant similarity with P. teres and other 37 Paratrichodorus spp. The 28S D2/D3 sequence (KU094057, 799 bp) was 91% or less homologous 38 with P. teres and other Paratrichodorus spp., but no P. allius sequence was available for 39 comparison. The molecular tests confirmed the identity as P. allius. P. allius is known to be the 40 most prevalent vector of TRV in Washington and Oregon (Riga et al. 2007). To our knowledge, 41 this is the first report of P. allius in North Dakota. 42

First Report of the Root-lesion Nematode Pratylenchus neglectus on Wheat (Triticum aestivum) in North Dakota

Root-lesion nematodes (Pratylenchus spp.) are important nematode pests that invade roots of plants and restrict productivity of wheat (Smiley et al. 2005). In August 2015, a soil sample was collected from a harvested wheat field in Walsh County, ND, and was found to contain 1,044 root-lesion nematodes per kg of soil using the sugar centrifugal flotation method. In October 2015, four soil samples were collected from the same field and had root-lesion nematodes ranging from 125 to 1,000/kg soil. One soil sample with 500 lesion nematodes/kg soil was used to inoculate hard red spring wheat cvs. Glenn and Faller. After 10 weeks of growth in a greenhouse room maintained at 22°C, wheat roots were harvested and washed, and light brown lesions were observed on lateral roots. The washed roots were cut into 1-cm segments for nematode extraction using the Whitehead tray method. After 48 h, lesion nematodes were recovered from the root tissues. Averages of 24 and 20 root-lesion nematodes per gram were found in the roots of Glenn and Faller, respectively. Nematodes from soil and wheat roots were examined morphologically and molecularly for species identification. Morphological measurements of adult females (n = 10) included body length (range = 402.0 to 495.0 μm, mean = 446.0 μm), stylet (15.0 to 17.5, 16.4), tail length (16.0 to 22.0, 18.8), body width (17.0 to 22.0, 19.6), anterior end to basal bulb (90.0 to 115.0, 101.6), a = (21.2 to 24.7, 23.1), b = (3.7 to 4.8, 4.4), c = (19.6 to 27.1, 23.8), and V (81.0 to 85.0%, 82.8%). The lip region had two annules, and was not set off, with anterior margins of apical lip annule convex, second annule was slightly wider than the first; lateral field had four lines with central zone of lateral field having oblique striae; tail terminus was smooth, rounded, or slightly oblique. The nematode species was identified as Pratylenchus neglectus (Rensch, 1924) Filipjev & Schuurmans Stekhoven, 1941 based on morphological and morphometric characteristics (Castillo and Vovlas 2007). DNA was extracted from single nematodes (n = 11) isolated from soil and wheat roots, and ITS region of rDNA was amplified (Tanha Maafi et al. 2003). PCR products from three nematodes were cloned using pGEM-T easy vector and sequenced, and the resulting ITS sequences were identical. The consensus sequence (GenBank Accession No. KU705392, 684 bp) was 99% homologous with one population of P. neglectus from China (JX046941) and 83% or less identical to other Pratylenchus spp. including P. thornei, a species closely related to P. neglectus. The specific primers from D3 28S rRNA and ITS rDNA were used to amplify DNA of eight nematodes and produced single bands specific for P. neglectus (Yan et al. 2008, 2013). The combination of the molecular tests confirmed the target species as P. neglectus. Two species of root-lesion nematodes, P. neglectus and P. thornei, were reported as damaging pathogens affecting wheat production in the Pacific Northwest (Smiley et al. 2005). To our knowledge, this is the first report of P. neglectus on wheat in North Dakota. The resistance levels of various wheat cultivars to this lesion nematode are being identified.

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.

First Report of a New Stunt Nematode Tylenchorhynchus sp. From a Soybean Field in North Dakota

Stunt nematodes in the genus Tylenchorhynchus are common plant-parasitic nematodes, and several species have been reported to damage crops (Handoo et al. 2014). In 2015 and 2016, eleven soil samples were collected from a soybean (Glycine max) field in Richland County, ND. Nematodes were extracted from soil using sugar centrifugal flotation method (Jenkins 1964). Nine samples contained the stunt nematodes ranging from 125 to 900 per kg of soil. One soil sample with 600 stunt nematodes per kg was planted with a soybean cultivar Sheyenne in three replicates. After three months of growth at 22°C under greenhouse conditions, the stunt nematode population increased to a final density of 1,953 ± 674 individuals per kg of soil. The reproductive factor of this nematode was 3.26. Soybean roots were washed with tap water and brown lesions were observed on the roots. Morphological measurements of adult females (n = 14) included body length (mean = 744.3 μm, range = 622.0-830.0), stylet (21.3 μm, 20.0-22.5), body widt...

First Report of the New Root-Lesion Nematode Pratylenchus sp. on Soybean in North Dakota

Root-lesion nematodes (RLN; Pratylenchus spp.) are important nematode pests on soybean (Glycine max (L.) Merr.). In May 2015 and 2016, sixtwo soil samples were collected from a soybean field in Walcott, Richland County, ND. Nematodes were extracted from soil using a sugar centrifugal flotation method., revealing these two All the samples contained RLN from 125 to 2, 000and 350 root-lesion nematodes per kg soil, respectively. In 2016, four soil samples were collected from the same field and all the samples had root-lesion nematodes ranging from 300 to 2,000. One soil sample with 350 RLN lesion nematodes per kg soil was planted withto soybean cultivar Barnes in four replicates each in a pot (6.4-cm × 25.4-cm) with 500 g soil(n = 4). After 15 weeks of growth at 22°C in a greenhouse room at 22°C, the root-lesion nematode RLN population was found to have increased greatly. The final population density in soil was 1,518 ± 541 lesion nematodesRLN per kg soil. Soybean roots were rinsed with water and brown lesion...

First Report of a New, Unnamed Lesion Nematode Pratylenchus sp. Infecting Soybean in North Dakota

First Report of a New, Unnamed Lesion Nematode Pratylenchus sp. Infecting Soybean in North Dakota | Plant Disease http://apsjournals.apsnet.org/doi/10.1094/PDIS-12-16-1749-PDN[7/13/2017 11:02:02 AM] 17.7 to 25.6), c’ (2.3, 2.2 to 2.4), spicule (17.5, 15.0 to 20.0), and gubernaculum (5.8, 4.5 to 7.5). DNA was extracted from single nematodes (n = 12) collected from soil and roots. D2-D3 of 28S rRNA (Subbotin et al. 2008) and ITS of rDNA (Yan and Smiley 2010) were amplified and sequenced. The ITS rDNA (KY200666, 981 bp) shared 95% sequence identity with P. alleni and low identity (≤87%) with other Pratylenchus spp. The D2-D3 (KY200665, 766 bp) had the highest identity of 96% with P. scribneri and 95% identity with P. hexincisus. No sequence of P. gibbicaudatus or P. flakkensis is available in GenBank. This species is very close to P. alleni but differs in having a slightly longer stylet, variations in tail shape, truncate to bluntly or broadly rounded tail with an annulated to occasionally smooth tail terminus, 4 to 6 lines in lateral field and 2 to 3 head annules. It is closely related to P. hexincisus, P. gibbicaudatus, and P. flakkensis but differs from them by one or more other characters: shape of head, number of head annules, tail shape, and V%. To our knowledge, this North Dakota Pratylenchus sp. population represents a new species of lesion nematode able to parasitize soybean.

First Report of the Lance Nematode Hoplolaimus stephanus From a Soybean Field in North Dakota

First Report of the Lance Nematode Hoplolaimus stephanus From a Soybean Field in North Dakota | Plant Disease http://apsjournals.apsnet.org/doi/10.1094/PDIS-07-16-1012-PDN[7/12/2017 10:41:33 AM] morphometric characteristics (Handoo and Golden 1992). DNA was extracted from single nematodes (n = 10) isolated from soil in 20 μl of extraction buffer. D1/D3 region of 28S rRNA and ITS1 of rDNA were amplified (Bae et al. 2008). PCR products from three nematodes were cloned using pGEM-T easy vector and sequenced. The consensus sequence from each genomic region was deposited into GenBank. The D1/D3 sequence (GenBank accession no. KX347887, 1,076 bp) was 99% identical to a population of H. stephanus (HQ678717) from Pennsylvania (Ma et al. 2011) 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 first in the list. This sequence had no significant similarity with other Hoplolaimus spp. including H. galeatus. Species-specific primers were used to amplify DNA of seven nematodes and produced single bands specific for H. stephanus (Ma et al. 2011). The molecular tests above confirmed the species identity as H. stephanus. This species mainly has been reported in the southeastern United States and was found to be the most predominant lance nematode species on soybean in North Carolina (Holguin et al. 2016). H. galeatus was mentioned as common in the northern Great Plains (Thorne and Malek 1968), but H. stephanus has never been reported in the northern United States. To our knowledge, this is the first report of H. stephanus in North Dakota.