University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Plant Pathology Plant Pathology Department 2016 Morphological and Molecular Characterization of Gracilacus wuae n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (Heracleum maximum) in Ontario, Canada Qing Yu Agriculture and Agri-Food Canada, [email protected] Weimin Ye North Carolina Department of Agriculture & Consumer Services Thomas O. Powers University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/plantpathpapers Part of the Botany Commons, Other Plant Sciences Commons, Plant Biology Commons, and the Plant Pathology Commons Yu, Qing; Ye, Weimin; and Powers, Thomas O., "Morphological and Molecular Characterization of Gracilacus wuae n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (Heracleum maximum) in Ontario, Canada" (2016). Papers in Plant Pathology. 376. http://digitalcommons.unl.edu/plantpathpapers/376 This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Journal of Nematology 48(3):203–213. 2016. Ó The Society of Nematologists 2016. Morphological and Molecular Characterization of Gracilacus wuae n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (Heracleum maximum) in Ontario, Canada 1 2 3 QING YU, WEIMIN YE, AND TOM POWERS Abstract: Gracilacus wuae n. sp. from soil associated with cow parsnip in Ontario, Canada is described and illustrated. Morpho- logically, females have a long stylet ranging from 80 to 93 mm long, the lip region not offset from the body contour, without lateral lips but with large and flat submedian lobes, the mouth opening slit-like elongated laterally and surrounded by lateral flaps, the excretory pore is anterior to the knobs of the stylet; males without stylet and the pharynx degenerated. The fourth-stage juveniles lack a stylet, the pharynx degenerated, and can be differentiated into preadult females and males based on the position of the genital primordia. The third-stage juveniles are similar to females but smaller. Phylogenetic studies using the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer (ITS) sequences collectively provide evidence of a grouping with other Gracilacus and some species of Paratylenchus with stylet length of females longer than 41 mm deposited in GenBank. Key words: Gracilacus, molecular, morphology, morphometric, new species, Paratylenchus, taxonomy. The genus Gracilacus was established by Raski (1962) 1959) Raski, 1962 in their analyses. Van Den Berg et al. to differentiate species of Paratylenchus Micoletzky, 1922, (2014) used 28S D2/D3 and ITS sequence to examine based on primarily stylet length and secondarily on body species groupings within Paratylenchus including the shape and the position of the excretory pore in females. above two Gracilacus species. Wang et al. (2015) de- Eight species with a female stylet longer than 48 mmwere scribed P. guangzhouensis, a species with the stylet aver- separated and placed in the genus Gracilacus primarily aging 47 mm long, and used ITS sequence to show that based on that single character. Shortly after the initial this new species is clustered with those four previously creation of Gracilacus, Siddiqi and Goodey (1964) syn- sequenced Gracilacus species. In another study, Wang onymized Gracilacus with Paratylenchus. Raski (1976) re- et al. (2016) also used ITS to demonstrate that their vised Gracilacus, lowered the minimum stylet length to newly described species P. nanjingensis with a 66 mm 41 um, and added 13 additional species to the genus. long stylet is grouped with G. bilineata and G. aculenta. Siddiqi (1986) established the subgenera Gracilacus and All these studies provided evidence of the possible Paratylenchus within the genus Paratylenchus based on existence of a genetic grouping of long-stylet-bearing stylet length. Thorne and Malek (1968) accepted Graci- paratylenchids. lacus when they described two new Paratylenchus species This study describes a new species of Gracilacus and evaluated the taxonomic position of G. audriellae from Canada. There are 43 species listed in the sub- (Brown, 1959) Raski, 1962. G. audriellae was later con- genus Gracilacus by Siddiqi (2000). Four species have sidered as a junior synonym of G. straeleni by Siddiqi been previously recorded from Canada (Brown, 1959; (2000). Geraert (1965) divided Paratylenchus sensu lato Wu, 1974). These include G. acicula (Brown, 1959) that includes Gracilacus into 10 groups based on the Raski, 1962, G. aculenta, G. audriellus,andG. robusta stylet length and the V-position and recently Ghaderi (Wu, 1974) Raski, 1976. The new species described et al. (2014) expanded it by dividing the genus into 11 herein, G. wuae n. sp., was found in soil associated groups based on stylet length, lateral fields, and advulval with wild parsnip (Heracleum maximum)inOntario, flaps. With the advent of molecular biology, phyloge- Canada. netic studies have been conducted to examine the re- lationships among paratylenchids including very few MATERIALS AND METHODS species of Gracilacus. Lopez et al. (2013) used the first ITS1 region to examine paratylenchid relationships in- Nematode samples: Soil samples associated with wild cluding G. bilineata Brzeski, 1995 and G. aculenta (Brown, parsnip were collected in Kanata, west of Ottawa, Ontario, Canada, with latitude 45818943.330N and lon- gitude 75855954.080W. The nematodes were extracted Received for publication May 5, 2016. 1Ottawa Research and Development Center, Agriculture and Agri-Food with the Baermann funnel method. Freshly extracted Canada, Ottawa, ON K1A 0C6, Canada. nematodes were processed for both morphological and 2Nematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services, Raleigh, NC 27607-6465. molecular studies. 3Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, Morphological study: The method used was as de- NE 68583-0722. This article was registered to the Official Register of Zoological Nomencla- scribed previously (Yu et al., 2014). The nematodes ture (ZooBank) as 16B8EBD7-4107-438E-A2D2-04AC63B3968B. were all fixed in TAF and mounted in dehydrated The authors would like to thank Dr. Z.A. Handoo, curator for the USDA nematode collection for lending the type materials of G. macrodorus, and Denise glycerin on slides for morphological studies. Specimens Chabot at the Ottawa Research and Development Center for helping the SEM were examined using a Leica DM5500 B compound works. Tom Powers would like to acknowledge funding from NSF DEB-1145440. E-mail: [email protected]. microscope (Wetzlar, Germany) using differential in- This paper was edited by Sergei A. Subbotin. terference contrast and pictures were taken with a Leica 203 204 Journal of Nematology, Volume 48, No. 3, September 2016 DFC 420 digital camera. Measurements were made using PCR and sequencing: Internal transcribed spacer was a Leica micro application system on the images. Scanning amplified with the primers ITS-F (59-TTGATTACGTC electron microscope (SEM) pictures were taken with a CCTGCCCTTT-39) and ITS-R (59-TTTCACTCGCCGT FEI Quanta 600 SEM. TACTAAGG-39) (Vrain et al., 1992) using the following thermocycling conditions: an initial denaturation at Molecular study 958C for 3 min, followed by 40 cycles of denaturation (30 DNA extraction: DNA extraction solution consisted sec at 958C), annealing (30 sec at 568C), and extension of 13 PCR buffer containing Proteinase K at a concen- (1 min at 688C), and a final extension step (3 min at tration of 0.25 mg/ml. Five nematodes were crushed in 688C). 18S rRNA gene (18S) was amplified with the 50 ml of the DNA extraction solution and incubated primers 18S-F (59-CGATCAGATACCGCCCTAG-39)and at 658C for 2.5 hr, followed by incubation at 958C for 18S-R (59-TACAAAGGGCAGGGACGTAAT-39)(Powers 15 min and on ice for 5 min. The DNA extracts were et al., 2010) using the following thermocycling condi- stored for later use at 2208C. tions: an initial denaturation at 958C for 3 min, followed FIG. 1. Line drawing of Gracilacus wuae n. sp. associated with cow parsnip in Ontario, Canada, A. Head and pharynx of the third-stage juvenile. B. Head and pharynx of female. C. Female body. D. Male genital system. E. Posterior half of preadult female. F. Posterior half of preadult female. Gracilacus wuae n. sp.: Yu et al. 205 by 30 cycles of denaturation (30 sec at 958C), annealing Biotechnology Information’s GenBank database using (30 sec at 628C), and extension (1.5 min at 688C), and a Basic Local Alignment Search Tool (BLAST). final extension step (3 min at 688C). D2/D3 region of the Phylogenetic study: Phylogenetic analysis was per- 28S rRNA gene (28S D2/D3) was amplified with the prim- formed as described previously (Zeng et al., 2013). The ers D2A (59-GACCCGTCTTGAAACACGGA-39)(Nunn, sequences were deposited into the GenBank database. 1992) and D3B (59-TCGGAAGGAACCAGCTACTA-39) DNA sequences were aligned by Clustal W using default (De Ley et al., 1999), using the following thermocycling settings. The DNA sequences of G. wuae n. sp. were conditions: an initial denaturation at 958C for 3 min, fol- compared with those of the other nematode species lowed by 40 cycles of denaturation (30 sec at 958C), an- available at the sequence database using the BLAST nealing (30 sec at 508C), and extension (1 min at 688C), homology search program. The model of base sub- and a final extension step (3 min at 688C). stitution was evaluated using MODELTEST, and the All PCR reactions were prepared using the Titanium Akaike information criterion (AIC) supported model, Taq PCR kit (Clontech Laboratories Inc., Mountain the base frequencies, the proportion of invariable sites, View, CA) and contained 5 or 10 ul of DNA, 2.5 ml and the gamma distribution shape parameters and of 103 PCR buffer, 1.0 ml of 2.0 mM dNTPs, 0.4 ml substitution rates were used in phylogenetic analyses.