Seinura (Fuchs, 1931) is a member of the family Aphelenchoididae (Skarbilovich, 1947) and is known for its predatory behavior on other nematode species. The word Seinura is derived from seios meaning move to and fro and oura meaning tail (Hunt, 1993). The genus contains over 50 species distributed across different climatic zones and environments (Kaisa, 2000; Bajaj, 2015; Adeldoost et al., 2016; Kanzaki et al., 2018). As Seinura species are not qualified as pest species, this group has not receive special attention in China. The outbreak of pine wilt disease accelerated the research on the aphelenchs in China, and several nationwide surveys were conducted on the pine trees that resulted in the documentation of Seinura species as well. Till now, S. aurangabadensis (Suryawanshi, 1971), S. elmiraensis (van der Linde, 1938; Goodey, 1960), S. filicaudata (Christie, 1939; Goodey, 1960), S. oahueensis (Christie, 1939; Goodey, 1960), S. oostenbrinki (Husain and Khan, 1967), S. steineri (Hechler, 1965 in Hechler and Taylor, 1965), S. tenuicaudata (de Man, 1895; Goodey, 1960), and S. tritica (Bajaj and Bhatti, 1982) have been documented from China (Jiang, 2000; Xie and Zhang, 2003; Huang and Ye, 2008; Feng, 2010; Ding et al., 2013; Zhang et al., 2013). The two previously described species S. lii (Huang and Ye, 2006) and S. wuae (Huang and Ye, 2006) from China are now transferred to Aphelenchoides (Fischer, 1894) and Bursaphelenchus (Fuchs, 1937), respectively (Gu et al., 2017; Kanzaki et al., 2018).
The present study describes a new Seinura species isolated from medium soil of imported Olea europaea L. from Italy. The species was compared with all related species and found to be a new member of the genus, being described herein as Seinura italiensis n. sp.
Materials and methods
Nematode isolation and morphological study
Medium soil collected from imported Olea europaea from Italy to Ningbo, China, was sent to the nematology laboratory for nematode detection. The nematodes were isolated by the modified Baermann funnel technique for 24 hr. Permanent slides were prepared by heat-killed and fixed nematodes with FA 4:1 and ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). Morphometrics, drawings, and light micrographs of nematodes were done with the aid of a Zeiss microscope equipped with a Zeiss AxioCam MRm CCD camera.
Molecular and phylogenetic analyses
DNA samples were prepared according to Li et al. (2008). Three sets of primers (synthesized by Majorbio, Shanghai, China) were used in the PCR analyses to amplify the near full-length SSU and D2-D3 expansion segments of LSU rDNA. The SSU region was amplified as two partially overlapping fragments; for the first fragment, the forward 988F (5′-CTC AAA GAT TAA GCC ATG C-3′) and reverse 1912R (5′-TTT ACG GTC AGA ACT AGG G-3′) primers were used and for the second part, the forward 1813F (5′-CTG CGT GAG AGG TGA AAT-3′) and reverse 2646R (5′-GCT ACC TTG TTA CGA CTT TT-3′) primers were used (Holterman et al., 2006). The LSU D2-D3 expansion segments were amplified with the forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and the reverse primer D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) (De Ley et al., 1999). PCR conditions were as described by Li et al. (2008) and Ye et al. (2007). PCR products were separated on 1.5% agarose gel and visualized by staining with ethidium bromide. PCR products of sufficiently high quality were sent for sequencing by Invitrogen, Shanghai, China.
The newly generated SSU and LSU sequences of Seinura italiensis n. sp. (accession numbers MN428135 and MN428136, respectively) were compared with those of other aphelenchoidid species available in GenBank using the BLAST homology search program. For reconstruction of SSU and LSU rDNA phylogenies, the homologous sequences of ektaphelenchid and seinurid species were retrieved from the database. The outgroup taxa were selected according to previous studies (Aliramaji et al., 2018, 2019). The selected sequences of both data sets were aligned using Clustal X2 (http://www.clustal.org/) with the default parameters. The editing of the resultant alignment was performed using MEGA (Tamura et al., 2011). The model of base substitution was selected using MrModeltest 2 (Nylander, 2004). The Akaike-supported model, a general time reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR+G+I) was used for both SSU and LSU analyses. Bayesian analyses were performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) with a random starting tree and running the chains for 5 × 106 generations for both data sets. After discarding burn-in samples, the remaining samples were retained for further analyses. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using the 50% majority rule. The convergence of model parameters and topology were assessed based on the average standard deviation of split frequencies and potential scale reduction factor values. The adequacy of the posterior sample size was evaluated using autocorrelation statistics as implemented in Tracer v.1.5 (Rambaut and Drummond, 2009). The output files of the phylogenetic trees were visualized using Dendroscope V.3.2.8 (Huson and Scornavacca, 2012) and re-drawn in CorelDRAW software version 2017. The Bayesian posterior probabilities (BPP) exceeding 0.50 are given on appropriate clades.
Results
Systematics
Seinura italiensis n. sp.
Figure 1:
Line drawings of Seinura italiensis n. sp. (A): pharynx; (B): female reproductive system; (C): anterior region; (D): male posterior body region (arrows showing the P1-P4 papillae); (E): lateral lines; (F): female tail; (G): spicule. (Scale bars = A – G = 10 μm).
Figure 2:
Light photomicrographs of Seinura italiensis n. sp. (A): entire female; (B): entire male; (C): lateral lines; (D, E): anterior region (arrows pointing on position of excretory pore); (F): vulval region; (G): female posterior region showing vulva and post-uterine sac; (H-J): female tail; (K, L): male tail (arrows showing position of caudal papillae); (M, N): spicules (Scale bars = A, B = 20 μm; C-N = 10 μm). exp = excretory pore; a = anus; P1 + P2 + P3 + P4 = caudal papillae.
Measurements
Measurements of the new species are given in Table 1.
Table 1.
Morphometrics of Seinura italiensis n. sp.
| Female | Male | ||
|---|---|---|---|
| Character | Holotype | Paratypes | Paratypes |
| n | – | 15 | 15 |
| L | 478 | 522 ± 36.3 (469 – 590) | 477 ± 41 (407 – 565) |
| a | 30.1 | 29.6 ± 1.6 (26.7 – 33.7) | 31.4 ± 2.1 (28.6 – 36.3) |
| b | 6.6 | 7.0 ± 0.4 (6.3 – 7.5) | 6.7 ± 0.5 (5.9 – 7.6) |
| b′ | 2.8 | 2.9 ± 0.2 (2.5 – 3.1) | 3.1 ± 0.2 (2.8 – 3.5) |
| c | 9.0 | 9.1 ± 1.3 (7.5 – 12.5) | 12.5 ± 1.2 (10.5 – 14.6) |
| c′ | 5.3 | 5.6 ± 0.7 (3.9 – 6.5) | 3.6 ± 0.3 (3.1 – 4.2) |
| V or T | 73.4 | 72.2 ± 1.5 (69.7 – 75.3) | 43.2 ± 11.2 (28.1 – 73.9) |
| Lip region height | 3.6 | 3.4 ± 0.4 (2.8 – 4.1) | 3.0 ± 0.3 (2.5 – 3.6) |
| Lip region width | 7.3 | 7.3 ± 0.4 (6.6 – 8.1) | 6.8 ± 0.6 (5.8 – 8.0) |
| Stylet length | 19.8 | 20.6 ± 1.6 (18.3 – 23.6) | 17.4 ± 1.5 (14.5 – 20.1) |
| Body diam. | 15.9 | 17.7 ± 1.4 (15.7 – 20.9) | 15.2 ± 1.2 (13.3 – 16.9) |
| Median bulb width | 9.6 | 10.2 ± 0.6 (9.4 – 11.8) | 8.7 ± 0.7 (7.5 – 10.2) |
| Median bulb length | 16.4 | 17.8 ± 1.5 (14.8 – 21.0) | 15.4 ± 1.0 (13.8 – 17.4) |
| Median bulb length/diam. ratio | 1.7 | 1.7 ± 0.1 (1.5 – 1.9) | 1.8 ± 0.1 (1.6 – 2.0) |
| Excretory pore from anterior end | 61.9 | 67.9 ± 4.9 (57.3 – 74.4) | 65.5 ± 3.8 (55.2 – 69.1) |
| Ovary or testis length | 175.6 | 180.2 ± 35.8 (113 – 232) | 206.0 ± 54.5 (143 – 340) |
| Post-uterine sac | 59.2 | 58.8 ± 5.6 (51.1 – 69.3) | – |
| Vulva to anus distance | 85.1 | 86.6 ± 7.4 (74.0 – 95.5) | – |
| Post-uterine sac length/vulva to anus (%) | 69.6 | 68.2 ± 7.7 (58.1 – 85.3) | – |
| Anal (cloacal) body diameter | 10.1 | 10.5 ± 0.7 (9.6 – 12.4) | 10.7 ± 0.6 (9.6 – 11.6) |
| Tail length | 53.1 | 58.3 ± 6.8 (43.6 – 72.0) | 38.5 ± 4.5 (29.3 – 45.2) |
| Spicules (curved median line) | – | – | 14.5 ± 1.0 (12.7 – 15.8) |
| Spicules (chord) | – | – | 14.1 ± 0.8 (12.6 – 15.0) |
| Hemizonid from anterior end | 90.7 | 84.4 ± 4.0 (77.6 – 91.8) | 81.9 ± 4.5 (73.1 – 89.7) |
| Pharyngo-intestinal junction from anterior end | 72.7 | 74.8 ± 3.6 (68.3 – 80.8) | 71.0 ± 3.6 (63.5 – 76.0) |
| Pharyngeal gland from anterior end | 170.7 | 182.1 ± 15.8 (156.8 – 222.2) | 154.1 ± 10.6 (132.5 – 175.2) |
