JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2018-027 Issue 3 | Vol. 50 (2018) Description of Xiphinema parachambersi n. sp. (Nematoda: Longidoridae) from Imported Ornamental Plants in Japan with a Key to Xiphinema Species in Group 1 Munawar Maria,1 Weimin Ye, 2 3 4 Qing Yu, and Jianfeng Gu * Abstract 1Institute of Biotechnology, College of Agriculture & Biotechnology, Xiphinema parachambersi n. sp. recovered in Ningbo, China, from Zhejiang University, Hangzhou the rhizosphere of ornamental plants (Gardenia jasminoides and 310058, Zhejiang, China. Euonymus hamiltonianus) imported from Japan is described. The new species is characterized by a long female body 1,830 to 2,109 μm 2Nematode Assay Section, North long, odontostyle 105 to 116 μm long, reproductive system mono- Carolina Department of Agriculture, opisthodelphic, vulva located anteriorly at 25.2 to 27.7% of total Raleigh, NC. body, long ovary 119 to 292 μm with simple uterus and Z-organ 3Ottawa Research and Develop- absent, female tail elongated conoid with a rounded terminus. Only ment Centre, Agriculture and two juvenile stages were available to study and no male was found. Agri-Food Canada, Ottawa, ON The polytomous identification codes for this new species are A1, K1A 0C6, Canada. B4, C2, D23, E1, F2, G2, H2, I2, J2, K?, L1 and it belongs to the morphospecies group 1. Phylogenetic analysis based on the 18S, 4Technical Centre, Ningbo Entry- ITS1 and 28S D2/D3 sequences of the new species showed close Exit Inspection and Quarantine relationships with X. chambersi. Morphologically, the new species is Bureau, Ningbo 315012, Zhejiang, similar to X. chambersi, X. hangzhouense, and X. winotoi but can be P.R. China. differentiated by morphological characters and DNA sequences. To *E-mail: [email protected]. help identify the species, a diagnostic key to the group 1 species is This article was edited by Zafar presented. Ahmad Handoo. Received for publication March 18, Key words Molecular, morphology, morphometrics, nematode, new species. 2018. Dagger nematodes (Xiphinema Cobb, 1913) contain X. tarjanense (Lamberti and Bleve-Zacheo, 1979) are more than 260 species (Palomares-Rius et al., 2017). known to transmit nepoviruses, which cause additional They are polyphagous and ectoparasites on a variety indirect damages to plants (Hunt, 1993; Decraemer of cultivated and wild plants. Their feeding behavior and Robbins, 2007). Because of their economic causes considerable mechanical damage to plants importance, species in Xiphinema have received con- due to its excessive long stylet. The root symptoms siderable attention. Virus-transmitting Xiphinema spe- include darkening of tissues, cortical hyperplasia, lat- cies are listed as quarantine pests in many countries eral root proliferation, tip galling, and necrosis (Hunt, including China. 1993). In addition, nine Xiphinema species, three spe- During a routine quarantine inspection, a Xiphin- cies from Xiphinema non-americanum group including ema population was detected from the soil sam- X. index (Thorne and Allen, 1950), X. diversicaudatum ples from imported ornamental plants, Gardenia (Micoletzky, 1927) (Thorne, 1939), and X. italiae (Meyl, jasminoides J. Ellis and Euonymus hamiltonianus 1953) and six putative species in the X. americanum Wall. from Japan. The preliminary morphological group including X. americanum s. str., X. californicum investigation revealed that the species has a medium (Lamberti and Bleve-Zacheo, 1979), X. bricolense size body, opisthodelphic reproductive system, ante- (Ebsary et al., 1989, X. intermedium (Lamberti and riorly located vulva and elongated tail, very similar to Bleve-Zacheo, 1979), X. revesi (Dalmaso, 1969), and North American species X. chambersi (Thorne, 1939). 369 © The Society of Nematologists 2018. Description of Xiphinema parachambersi n. sp. (Nematoda: Longidoridae) from Imported Ornamental Plants in Japan In order to make the final species identification, a et al., 2006). Primers for amplification of ITS1 were detailed morphological and DNA sequencing analysis forward primer V1 (Ferris et al., 1993) and reverse was conducted which resulted in a new species and primer 5.8S (Cherry et al., 1997). Primers for ampli- was herein described as X. parachambersi n. sp. The fication of 28S D2/D3 were forward primer D2A and objectives of the present study were to: (i) provide a reverse primer D3B (De Ley et al., 1999). The 25-µl PCR morphological description of the new species and was performed using Master Mix DNA polymerase compare it with other similar species; (ii) characterize (Invitrogen, Shanghai, China) according to the the species molecularly, using three DNA markers, manufacturer’s protocol in a thermocycler. The 18S, ITS1, and 28S D2/D3 ribosomal (iii) examine the thermal cycler program for 28S was as follows: phylogenetic relationships of the new species with denaturation at 95 °C for 5 min, followed by 35 cy- other species in Xiphinema. cles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 2 min. Materials and methods A final extension was performed at 72 °C for 10 min. The thermal cycler program for 18S and ITS was as Nematode samplings, extraction, follows: denaturation at 95 °C for 5 min, followed by and morphological study 35 cycles of denaturation at 95 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 2 min. A Xiphinema specimens were collected from the rhiz- final extension was performed at 72 °C for 5 min as osphere of Gardenia jasminoides (sample number: described by Ye et al. (2007) and Li et al. (2008). PCR 2186-1) and Euonymus hamiltonianus (sample products were separated and visualized on 1% aga- number: 2186-2) from the same container using rose gels and stained with ethidium bromide. PCR modified Baermann funnel method for 24 to 48 hr. products of sufficiently high quality were sequenced Measurements were made on specimens fixed in TAF by Invitrogen (Shanghai, China). and processed to glycerin following the method of Seinhorst’s (1959). The nematodes were measured Phylogenetic analysis using AxioVs40 (v4.6.3.0) of Zeiss company. All the abbreviations used are as defined in Decraemer and The sequences were deposited into the GenBank Hunt (2006). Light micrographs were made using a database. DNA sequences were aligned by MEGA7 Zeiss Imager Z1 microscope equipped with a Zeiss (Kumar et al., 2016.) using default settings. The DNA AxioCam MRm CCD camera. Drawings were made sequences were compared with those of the other with a drawing tube. Juvenile stages were determined nematode species available at the GenBank by a plot with scattergraph method of the lengths of sequence database using the BLAST homology odontostyles and replacements. search program. The model of base substitution was evaluated using MODELTEST (Posada and Criandall, Molecular analyses 1998; Huelsenbeck and Ronquist, 2001). The Akaike-supported model, the base frequencies, the For DNA extraction, a single nematode was trans- proportion of invariable sites and the gamma distri- ferred to worm lysis buffer (WLB: 20 mM Tris-HCl bution shape parameters and substitution rates were pH 8.0, 100 mM KCl, 3.0 mM Mg2Cl, 2.0 mM DTT, used in phylogenetic analyses. Bayesian analysis was 0.9% Tween) and crushed with a sterilized pi- performed to confirm the tree topology for each gene pette tip. The crushed nematode was pipetted into separately using MrBayes 3.1.0 (Huelsenbeck and Ronquist, 2001) running the chain for 1 × 106 gener- 8 µl ddH2O with 2 µl proteinase K (60 µg/ml) in an Eppendorf tube, which was then briefly spun and ations and setting the “burnin” at 2,500. We used the stored at −70°C for at least 10 min. Subsequently, Markov Chain Monte Carlo (MCMC) method within a the Eppendorf tube was incubated at 65°C for 1 Bayesian framework to estimate the posterior prob- to 2 h and the proteinase K was denatured at 95°C abilities of the phylogenetic trees (Larget and Simon, for 10 min. Finally, the DNA suspension was cooled 1999) using 50% majority rule. to 4°C and used for conducting PCR (Li et al., 2008). Three sets of primers (synthesized by In- Results vitrogen, Shanghai, China) were used in the PCR analyses to amplify the partial 18S, ITS1, and 28S SYSTEMATICS rDNA D2/D3. Primers for amplification of 18S were Xiphinema parachambersi n. sp. forward primer K4f and reverse primer K1r (Penas (Table 1; Figs. 1–4) 370 JOURNAL OF NEMATOLOGY Table 1. Morphometrics data for Xiphinema parachambersi n. sp. All measurements are in µm and in the form of mean ± s.d. (range). Holotype Paratype Character/ratios Female Female J2 or J3 J3 or J4 n 1 15 5 11 L 1,918 2,008 ± 78.7 1,127.0 ± 52.6 1,419.6 ± 38.5 (1,830.0–2,109.0) (1,046.0–1,210.0) (1,349.0–1,587.0) a 45.6 47.2 ± 2.3 (44.2–53.1) 42.7 ± 3.6 (36.7–46.3) 48.3 ± 3.5 (41.60–55.5) b 5.4 5.5 ± 0.2 (5.1–6.0) 3.9 ± 0.5 (3.0–4.4) 4.7 ± 0.3 (4.2–5.0) c 18.1 17.9 ± 1.0 (16.1–19.6) 11.8 ± 1.0 (10.5–12.8) 13.2 ± 0.6 (12.0–15.1) c’ 4.4 4.9 ± 0.4 (4.2–6.0) 6.4 ± 0.3 (5.9–6.8) 5.9 ± 0.4 (5.2–6.9) V 27 26.2 ± 0.6 (25.2–27.7) – – Lip diam. 10 10.3 ± 0.6 (8.8–11.2) 8.6 ± 0.3 (8.2–9.0) 8.9 ± 0.5 (8.2–10.1) Lip height 5 4.1 ± 0.7 (2.9–5.2) 3.6 ± 0.4 (3.0–4.2) 3.8 ± 0.4 (3.1–4.7) Odontostyle 115 110.4 ± 2.6 (105.0–115.6) 71.0 ± 3.8 (64.4–75.6) 85.5 ± 2.0 (79.5–90.0) Odontophore 67 65.5 ± 1.6 (61.0–68.1) 46.1 ± 1.4 (44.5–47.8) 53.8 ± 1.0 (52.2–54.8) Re placement – – 88.0 ± 2.5 (85.0–92.0) 107.0 ± 7.9 odontostyle (90.5–116.3) Total stylet 173 176 ± 2.9 (169.0–181.2) 116.6 ± 4.7 (109.0–120.5) 140.0 ± 2.2 (137.2–142.5) Flanges width 10 10.4 ± 0.7 (8.8–11.5) 8.1 ± 0.4 (7.7–8.7) 9.0 ± 0.6 (7.6–10.8) Esophagus 358 366.6 ± 10.6 (348.4–387.2) 289.6 ± 28.2 (272.2–345.6) 311.4 ± 14.7 (275.0–344.2) Es ophageal bulb 78 81.5 ± 2.1 (77.4–85.1) 63.7 ± 2.2 (60.1–66.1) 70.4 ± 3.0 length (64.8–76.5) Esophageal bulb diam.