Description and Molecular Characterization of a New Dorylaimid Nematode, Mesodorylaimus pini n. sp. (Nematoda: Dorylaimidae) from Korea

The studied nematode population was extracted from the bark and cambium layer of a wilted and dead pinewood nematode-infected black pine (Pinus thunbergii) tree stand on Gonri Island in Tongyeong, Gyeongsangnam-do Province, Korea. Nematodes were extracted from the cuttings using the Baermann funnel method (Baermann, 1917). The collected nematode suspension was examined under a Nikon SMZ 1000 stereomicroscope (Nikon) and specimens belonging to Mesodorylaimus were picked out and subsequently characterized based on inferences from morphometric and DNA barcode data.

The 31 female and 32 male specimens of the recovered population were heat-killed, fixed, and mounted to pure glycerin (Seinhorst, 1959). Morphometric data and photomicrographs were taken using a Zeiss Imager Z2 microscope (Carl Zeiss) fitted with AxioVision, a material science software for research and engineering (Carl Zeiss). Line drawing of the specimens was made under a drawing tube before being redrawn using CorelDRAW® software version 24. Species delineation and diagnosis were done following the keys presented by Andrássy (1986, 1988) and the various original species descriptions by Loof (1969); Thorne (1974); Botha and Heyns (1992); Ahmad (1993); Nedelchev and Peneva (2000); Peña Santiago and Abolafia (2000); and Tauheed and Ahmad (2010).

Ribosomal DNA was extracted from single female, male, and juvenile specimens using the DNA extraction kit WizPure™, as described by Iwahori et al. (2000). The nearly full-length 18S-rRNA gene was amplified as two partially overlapping fragments using two primer sets: 988F (5′-CTCAAAGATTAAGCCATGC-3′) and 1912R (5′-TTTACGGTCAGAACTAGGG-3′), 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and 2646R (5′-GCTACCTTGTTACGACTTTT-3′), (Holterman et al., 2006). The D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primer set (Nunn, 1992) was used in the amplification of the D2-D3 expansion segment of 28S-rRNA. Polymerase chain reaction (PCR) was performed with a PCR cycler (T100™, Bio-Rad), and the PCR thermal profiles for the D2A/D3B, 988F/1912R, and 1813F/2646R primer sets were as described by Mwamula et al. (2023a). The PCR products were purified using a PCR purification kit (Qiagen) and quantified using a quickdrop spectrophotometer (Molecular Devices). The purified PCR products were subsequently used for direct sequencing in both directions using the same primers as specified above. DNA sequencing was done at Macrogen. The edited DNA sequences were submitted to the GenBank database under the accession numbers PP525782-PP525784 (for 18S-rRNA) and PP525785-PP525788 (for 28S-rRNA).

The edited sequences (18S-rRNA and 28S-rRNA gene) were aligned using ClustalX (Thompson et al., 1997) along with the sequence data sets of Mesodorylaimus, Calcaridorylaimus, and other species from related genera that are published in GenBank (Holterman et al., 2006; Nedelchev et al., 2014; Vinciguerra et al., 2016; Kagoshima et al., 2019; Swart et al., 2020; Zhang et al., 2023). The partial sequences of Paravulvus hartingii (de Man, 1880; Heyns, 1968) (AY552976) and Nygolaimus cf. parvus (Thorne, 1974) (AY552974) were selected as the outgroup taxa for the 18S-rRNA gene. Mononchus tunbridgensis (Bastian, 1865) (AY593063) and Anatonchus tridentatus (de Man, 1876; De Coninck, 1939) (AY593065) were the outgroup for 28S-rRNA gene. The generated alignments were analyzed with Bayesian Inference (BI) using MrBayes 3.2.7 (Ronquist et al., 2012) under the GTR + I + G model. Bayesian inference analysis for each gene was initiated with a random starting tree and run with four chains for 1 × 10⁶ generations. The Markov chains were sampled at intervals of 100 generations. After discarding burn-in samples, a consensus tree was generated with a 50% majority rule. The generated trees were visualized and edited using FigTree v1.4.4 software. Posterior probabilities (PP) exceeding 50% are given on appropriate clades. Intraspecific and interspecific sequence variations were examined using PAUP* v4.0a169 (Swofford, 2003).

See Table 1.

Female (n = 31):

General habitus is slightly curved ventrally. Cuticle is 2–3 μm thick at the base of pharynx, 3–4 μm at midbody, and 4.0–5.5 μm at postanal region. Lateral chords are ca 1/3–1/4 of body diameter. Lips are angular, with lip region offset by a depression, 11.0–13.5 μm wide, 4.5–6.0 μm high. The amphidial aperture is wide, 5.5–6.5 μm wide or about half as wide as the lip region diameter; the amphidial fovea is typically stirrup-shaped. Odontostyle is typical of the genus, ca 1.4–1.7 times the lip region diameter, and its aperture occupies 28%–37% of its length. Odontophore are simple, rod-like, ca 1.1–1.3 times the odontostyle length. The guiding ring is thin and single, located at 8.5–9.5 μm from the anterior end. The nerve ring encircles the corpus part of the pharynx at ca 31–38% of total pharynx length from anterior end. The pharynx is 333–398 μm long, ca 0.5–0.6 times as long as the distance between pharynx and vulva, posteriorly expanding to a muscular expanded part. Pharyngeal expansion begins at ca 52–59% of the total neck length, appearing almost uniform in width along its length and slightly wider at the posterior end. Body diameter at the posterior end of pharyngeal expansion is ca 3 (2.7–3.5) times the lip region diameter. DN large, 4.0–4.5 μm, globular, is located near the anterior end of the expanded part, 10–25 μm from the anterior-most part of the expanded part. The two S₁N are located in the posterior half of the distance DN-S₂N, the anterior one (S₁N₁) appearing smaller (1.5–2 μm in diameter) than S₁N₂ (2–4 μm in diameter). S₂N visible, globular shaped, 2–3 μm in diameter located in the posterior portion of the expanded part. The pharyngeal gland nuclei with their orifices are located as follows: (n = 8): DO = 57–59; DN = 60–62; DO-DN = 2.1–2.8; S₁N₁ = 79–82; S₁N₂ = 81–84; S2N = 88–91; S2O = 90–92 (for terminology and formulae, see Loof and Coomans (1970)).

Cardia is typically conoid, variable in length, 14.5–27.5 μm long. Reproductive system is didelphic-amphidelphic. Ovaries are reflexed, generally variable in length, not reaching the vulva level, and identical as illustrated in Figure 1A. The anterior branch occupies 14–20% of the total body length, and the posterior branch 15–21%. Each branch is basically arranged from ovary to gonoduct (oviduct outstretched, joining ovary sub-terminally; sphincter weak, present at oviduct-uterus junction, joining an undifferentiated uterus) and vagina. The uterus is filled with well-developed, elongated, and sausage-shaped sperms. The vulva is a transverse slit. Pars distalis vaginae 1.5–2.5 μm long. Pars refringens vaginae has two elongate drop-shaped to spindle-shaped sclerotizations, each measuring 5.0–8.0 μm deep and 2.5–3.5 μm wide, with a combined width of 6.0–9.5 μm. Pars proximalis are 6.0–9.0 μm wide and 12.5–16.0 μm long. Intestine-prerectum junction has a long anteriorly directed conical or tongue-like projection measuring 32–50 μm long, or about 1.1–1.7 times as long as the corresponding body diameter. The rectum and prerectum are ca 1.1–1.5 and 3.0–4.3-times anal body diameter long, respectively. Anterior tail part (ca 25–38 μm from anal opening) is convex-conoid, with two pairs of caudal pores, one subventral and the other subdorsal; the tail continues gradually and tapers to a smoothly rounded tail terminus. The posterior part of the tail appears straight. Total tail length ca 4.9–7.0 times anal body diameter.

Male (n = 32):

They are similar to females in general morphology except for sexual characteristics and short conoid tail. Their body is curved ventrally to open in a J-shape when heat-killed and fixed. The genital system is diorchic, and testes are opposed and well-developed with well-developed elongated and sausage-shaped sperms. Spicules are ventrally curved, 48–57 μm long, or ca 1.7–2.1 times the cloacal body diameter. Lateral guiding pieces are 10.0–13.5 μm long, tapering at the end. Supplements consist of an adcloacal pair and 7–8 regularly spaced ventromedians. The posterior-most ventromedian supplement located at 33–50 μm from the adcloacal pair. Intestine-prerectum junction begins mostly at the level of the last supplement or slightly extending beyond the range of supplements by 0–25 μm. The rectum and prerectum are ca 1.2–1.8 and 3.5–5.4-times cloacal body diameter long, respectively. The tail appears dorsally conoid and broadly rounded. Tail length is less than cloacal body diameter (ca 0.6–0.8 times cloacal diameter).

Mesodorylaimus pini n. sp. is characterized by having a medium-sized body (L = 1.50–1.89 mm), lip region angular and offset by a depression; a relatively long odontostyle (17.0–19.0 μm), 1.4–1.7 times as long as the lip region diameter; expanded part of pharynx 142–186 μm long, vulval opening a transverse slit, positioned slightly posteriorly (V = 50–55); pars refringens vaginae with two elongated drop-shaped to spindle-shaped sclerotizations; intestine-prerectum junction with a long anteriorly directed conical or tongue-like projection, 1.1–1.7 times as long as the corresponding body diameter; a relatively long female tail (115–187 μm, c = 10.0–13.6; c’ = 4.9–7.0), spicules 48.0–57.0 μm long, 2–3 times the tail length; and the regularly spaced 7–8 ventromedian supplements.

Mesodorylaimus pini n. sp. is very close to M. subtilis (Thorne & Swanger, 1936; Andrássy, 1959), M. subtiliformis (Andrássy, 1959; Andrássy 1959), M. pseudosubtilis (Basson & Heyns, 1974), M. shamimi (Ahmad & Araki, 2003), and M. noreasus (Tauheed & Ahmad, 2010). It differs from all by the presence of a long anteriorly directed conical or tongue-like projection in the intestine-prerectum junction. In addition, Mesodorylaimus pini n. sp. differs from M. subtilis by the relatively longer body length (1.37–1.78 vs 1.3 mm in males and 1.50–1.89 vs 1.4–1.5 mm in females), longer odontostyle (17.0–19.0 μm vs 12 μm), shorter tail (c = 10.0–13.6, c’ = 4.9–7.0 vs c = 8.3–8.5, c’ = 8), a relatively more posteriorly positioned vulva (V = 50–55 vs 44–50), tail tapering to a smoothly rounded tail terminus vs subfiliform, and longer spicules (ca 2–3.0 times tail length vs hardily longer than the tail); from M. subtiliformis by the longer body length (1.50–1.89 vs 1.3 mm), lip region set off by a depression vs not set off or only barely set off, longer odontostyle (17.0–19.0 μm vs 14–15 μm), a straight tail that tapers to a smoothly rounded tail terminus vs filiform and pointed at the terminal end, longer spicules (48–57 μm vs 44.5 μm), and relatively fewer number of ventromedian supplements (7–8 vs 10); from M. pseudosubtilis by longer odontostyle (17.0–19.0 μm vs 10–14 μm), a relatively more posteriorly positioned vulva (V = 50–55 vs 46–50), tail tapering to a smoothly rounded tail terminus vs filiform with acute terminus, longer spicules (48–57 μm vs 41 μm), a relatively fewer number of ventromedian supplements (7–8 vs 10), and the prerectum-intestine junction either slightly anterior to range or almost at level of ventromedian supplements vs prerectum terminating well within the range of supplements; from M. shamimi by the relatively longer body length (1.37–1.78 vs 1.12–1.29 mm in males and 1.50–1.89 vs 1.2–1.52 mm in females), longer odontostyle (17.0–19.0 μm vs 13.5–16 μm), pars refringens vaginae with two elongated drop-shaped to spindle-shaped sclerotizations, 5.0–8.0 μm deep vs short (4.0–4.5 μm) triangular sclerotization pieces, female tail tapering to a smoothly rounded tail terminus vs acute terminus, longer prerectum in males (111–152 μm vs 105 μm), and relatively longer spicules (48–57 μm vs 45–49 μm); and from M. noreasus by longer odontostyle (17.0–19.0 μm vs 14–16 μm), pars refringens vaginae with two elongated drop-shaped to spindle-shaped sclerotizations, vs two well separated rectangular sclerotizations, longer prerectum in females (3.0–4.3 times the anal body diameter vs 2.4–3.0 times the anal body diameter), longer spicules (48–57 μm vs 42–43 μm), and the prerectum-intestine junction either slightly anterior to range or almost at level of ventromedian supplements vs prerectum extending far beyond the range of supplements.

Among species with a long anteriorly directed conical or tongue-like projection in the intestine-prerectum junction, Mesodorylaimus pini n. sp. is close to M. trapaefructus Andrássy, 1986; M. baeticus Peña-Santiago & Abolafia, 2000; and M. nevadensis Peña-Santiago & Abolafia, 2000. Mesodorylaimus pini n. sp. differs from M. trapaefructus by the lip region shape (lip region angular and offset by a depression, wider than the adjacent neck region vs narrowing head, not offset, narrower than adjacent neck region), pars refringens vaginae with two elongated drop-shaped to spindle-shaped sclerotizations vs heavily cuticularized and peculiarly characteristic in shape, resembling the crop of the water-chestnut, longer tail, posterior part straight with a smoothly rounded terminus vs shorter tail, spicate posterior part always curved dorsally (c = 10.0–13.6 vs c = 14–18), longer prerectum (3.0–4.3 times anal body diameter vs 2.6–2.8 times as long as anal body diameter), thick cuticle 3–4 μm at mid-body, and 4.0–5.5 μm at postanal region vs cuticle smooth and thin, 1.5 μm on mid-body and 2.5 μm on anterior end of tail, and males abundant vs no males; from M. baeticus by lip region set off by a depression vs lip region continuous or offset by a weak depression, longer odontostyle (17.0–19.0 μm vs 13–15 μm), relatively longer tail, convex-conoid, continuing gradually, and tapering to a smoothly rounded tail terminus vs tail elongated, tapering abruptly at first, then more gradually to a cylindrical thickened terminal portion (c = 10.0–13.6 vs c = 12–19), and males abundant vs no males; and from M. nevadensis by the long odontostyle (17.0–19.0 μm vs 12.5–14 μm), cardia typically conoid variable in length vs cardia enveloped by intestinal tissue forming a tongue-like extension, long spicules (48–57 μm vs 33.5–37.5 μm in one population and 41–47 μm in paratypes), and the pars refringens vaginae with two elongated drop-shaped to spindle-shaped sclerotizations, 5.0–8.0 μm deep vs pars refringens vaginae with two divergent and usually well separated triangular to drop-shaped sclerotizations measuring 3 x 5 μm.

The nematode population was recovered from the bark and cambium layer of a wilted and dead pinewood nematode-infected black pine (Pinus thunbergii) tree stand on Gonri Island in Tongyeong, Gyeongsangnam-do Province, Korea. GPS coordinates: 34°46′38″N, 128°21′45″E).

Holotype female, 16 female, and 18 male paratypes were deposited in the Nematode Collection of Kyungpook National University (KNU), South Korea; 10 female and 10 male paratypes were deposited in the Nematode Collection of Makerere University, Kampala, Uganda; and 5 females and 4 males were deposited in the Nematode Collection in the Ghent University Zoology Museum, Gent, Belgium.

Mesodorylaimus pini n. sp. was isolated from the bark and cambium layer of a dead black pine tree. Thus, the species epithet pini is derived from the tree name.

The amplification of the nearly full-length 18S-rRNA and the D2-D3 expansion segment of the 28S-rRNA gene yielded single fragments of approximately 1,700 and 780 bp, respectively. The three newly obtained 18S-rRNA gene partial sequences (PP525782-PP525784) exhibited no intraspecific variation (0.0%), and the top 5 hits in GenBank BLAST homology search for these three sequences comprised closely homologous sequences of unidentified Mesodorylaimus isolate (MG921251), Mesodorylaimus cf. nigritulus (AJ966490), Calcaridorylaimus isolates (MG921242 and MG921244), Ecumenicus sp. (MK292127), and Aporcelaimellus obtusicaudatus (DQ141212), all with percent identities of over 98%. In the 18S-rRNA gene phylogeny, Mesodorylaimus pini n. sp. sequences were grouped in a well-supported subclade (PP = 100) with M. pseudobastiani (AY919195), Mesodorylaimus cf. simplex (AY146504), Calcaridorylaimus signatus (LC457654), Mesodorylaimus sp. (MG921251), Calcaridorylaimus sp. (MG921242), Calcaridorylaimus sp. (MG921244), Mesodorylaimus cf. nigritulus (AJ966490), Mesodorylaimus japonicus (AJ966489), M. bastiani (AJ966488 and MF325117), Mesodorylaimus subtilis (MG921247), and Calcaridorylaimus castaneae (KF717497) differing by 8 bp (1.2%), 9 bp (1.4%), 13 bp (1.5%), 27 bp (1.6%), 27 bp (1.6%), 28 bp (1.6%), 29 bp (1.7%), 33 bp (1.9%), 21–39 bp (2.4–2.8%), 47–48 bp (2.8%), and 47 bp (2.9%), respectively.

The four D2-D3 sequences from the different life stages and sexes of Mesodorylaimus pini n. sp. (PP525785-PP525788) were identical, with no intraspecific sequence variation (0.0%). Similar to 18S-rRNA gene phylogeny, the D2-D3 sequences of Mesodorylaimus pini n. sp. were grouped in a well-supported subclade with the sequences from Mesodorylaimus bastiani isolates (MF325232 and MF325241), an unidentified Mesodorylaimus sp. (MG921253 and MG921254), Mesodorylaimus subtilis (MG921249 and MG921250), Dorylaimidae sp. (MH590274), Calcaridorylaimus heynsi (MH062988), Calcaridorylaimus sp. (MG921245 and MG921246), and Calcaridorylaimus castaneae (KF717498 and OR625214), differing by 17 bp (5.3%–5.5%; with 39.8% coverage), 43 bp (5.6%), 44 bp (5.7%), 44 bp (6.0%), 48 bp (6.6%), 50 bp (6.5%), and 71 bp (9.2–10.0%), respectively. Sixty-seven 18S-rRNA and 67 28S-rRNA gene sequences from various member species of Mesodorylaimus, Calcaridorylaimus and other related genera, inclusive of the newly obtained sequences and outgroup taxa, constituted the sequence dataset for phylogenetic analyses. Phylogenetic relationships, as inferred from Bayesian analysis of the dataset with GTR + I + G substitution model, are shown in Figures 3 and 4.

Figure 3.

Bayesian tree inferred under the GTR + I + G model from 18S-rRNA gene sequences of Dorylaimid species. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Paravulvus hartingii and Nygolaimus cf. parvus.

Figure 4.

Bayesian tree inferred under the GTR + I + G model from LSU D2–D3 partial sequences of Dorylaimid species. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Mononchus tunbridgensis and Anatonchus tridentatus.