Paratylenchus ilicis n. sp. (Nematoda: Paratylenchinae) Associated with Holly from the Netherlands and New Taxonomical and Phylogenetic Support for the Synonymization of Cacopaurus with Paratylenchus

During a survey of root-knot nematodes, several soil samples were collected from rhizospheres of holly (I. aquifolium L.) from Hilversum, the Netherlands, including root materials of the hosts. Nematodes were extracted from the soil and the roots following centrifugal flotation method (van Bezooijen, 2006) at the National Plant Protection Organization (NPPO), Wageningen, the Netherlands. The nematode extract was stored at 4°C during the course of the study.

Specimens of Paratylenchus collected from the extracts were picked out separately in a drop of water in glass cavity blocks and killed and fixed using hot 4% formaldehyde (about 60°C). The specimens were left in the fixative for 7 d at 4°C, then gradually transferred to anhydrous glycerin following the protocol described in Seinhorst (1959), and subsequently mounted on glass slides in glycerin medium. Morphological characterization of the nematodes was done using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Olympus C5060Wz camera and a drawing tube (Singh et al., 2018). Illustrations were improved using (Adobe Photoshop CS6, San Jose, CA).

For SEM, fixed specimens were washed in 0.1 M phosphate buffer (pH = 7.5) and dehydrated in a graded series of ethanol solutions, critical-point-dried with liquid CO₂, mounted on stubs with carbon tabs (double conductive tapes), coated with gold of 25 nm, and photographed with a JSM-840 EM (JEOL, Tokyo, Japan) at 12 kV (Singh et al., 2018).

Prior to DNA extraction, individual nematodes were temporarily mounted on glass slides in tap water and heat relaxed until they stopped movement. Morphological vouchers of the specimens were made using the above-mentioned camera equipped microscope, and then recovered in distilled water for further molecular characterization (Singh et al., 2021). The cuticle of each nematode was punctured using a metallic pin (used as worm-picking tool) and individually transferred to a PCR tube containing 20 ml of worm lysis buffer (50 mM KCl, 10 mM Tris at pH = 8.3, 2.5 mM MgCl₂, 0.45% NP 40 [Tergitol Sigma Belgium], and 0.45% Tween 20). The PCR tubes were then frozen at –20°C (10 min) followed by adding 1 ml proteinase K (1.2 mg/ml), incubation at 65°C (1 h) and 95°C (10 min), and finally centrifuging the lysate at 14,000 rcf for 1 min (Singh et al., 2018).

PCR amplifications of ITS and D2-D3 of 28S rRNA genes were done using the primer pairs, Vrain2F: 5´-CTT TGT ACA CAC CGC CCG TCG CT-3´/Vrain2R: 5´-TTT CACT CGC CGT TAC TAA GGG AAT C-3´ (Vrain et al., 1992) and D2A: 5´-ACA AGT ACC GTG AGG GAA AGT TG-3´/D3B: 5´-TCC TCG GAA GGA ACC AGC TAC TA-3´ (Nunn, 1992), respectively, following the thermal profiles described in Singh et al. (2019). For amplification of partial sequence of the mitochondrial cox1 gene, the primer pair JB3: 5´-TTT TTT GGG CAT CCT GAG GTT TAT-3´/JB4.5: 5´-TAA AGA AAG AAC ATA ATG AAA ATG-3´ was used according to Bowles et al. (1992). The PCR products were enzymatically cleaned with alkaline phosphatase (1 U/ml) and exonuclease I (20 U/ml) for 15 min at 37°C followed by 15 min at 80°C (Singh et al., 2020), and then sent for sequencing at Macrogen (https://dna.macrogen.com) The contigs were made from the newly produced forward and backward sequences using Geneious Prime 2020.0.5 (https://www.geneious.com) and were deposited in GenBank.

The obtained rRNA and cox1 gene sequences were analyzed with other relevant sequences available in GenBank using programs in Geneious Prime 2020.0.5. Multiple alignments of DNA sequences were made using MUSCLE in Geneious Prime 2020.0.5 with the default parameters and followed by manually trimming off the poorly aligned ends. Bayesian phylogenetic analysis (using MrBayes 3.2.6) was carried out using the GTR + I + G nucleotide substitution model; analyses were run under 1 × 10⁶ generations (four runs), Markov chains were sampled every 100 generations, and 20% of the converged runs were regarded as burnin (Huelsenbeck and Ronquist, 2001). Available sequences of nematode species from different genera, but within the same family (for D2–D3 and ITS) or superfamily (for cox1), were selected as outgroups.

Paratylenchus ilicis n. sp. Figures 1–3, Table 1

Figure 1

Figure 2

Figure 3

Paratylenchus ilicis n. sp. is characterized by slightly to fully obese females of light brown color with small body of 0.16 mm to 0.29 mm in length and 17 mm

to 35 mm in width. Bodies are ‘J’ or ‘C’ shaped or distorted or folded upon heat relaxation. The cuticle is up to 3 mm thick and refractive tubercles are present on cuticle throughout the body. The lateral field has three bands formed by four lines, and these bands widen into a characteristic ovoid to spindle-shaped field around vulva level. Deirids may be seen at the start of lateral field differentiation. The lip region is conical-truncate to occasionally rounded and is not offset, and the submedian lobes are not seen protruding. The stylet length is variable in length, between 70 mm and 100 mm long, its conus 80% to 90% of the total length. The reproductive tract is monodelphic with an outstretched to a reflexed ovary, a rounded sperm-filled spermatheca, and an oblique vagina which opens into a thick-walled, rounded uterine space. The relative position of vulva is at 89% to 95% of body length from the anterior end and both vulva lips are swollen and without vulva flaps. The tail is very short with a bluntly rounded terminus. The juvenile stylet length is variable (38 mm to 60 mm) depending on the life stage. The younger juvenile (J2) tail terminus has a characteristic finger-like tip. The males do not have a stylet. Spicules and gubernaculum are, respectively, about 15 mm and 4 mm long, and with a very thin bursa, which may be missed during observation. In both the juveniles and males, the SE pore is seen just above the hemizonid, and distinct and rather large deirids are present around the level of hemizonid.

Paratylenchus ilicis n. sp. is morphologically very similar to C. pestis. Both species have females that are slightly to fully obese, similar lateral field differentiation, cuticular ornamentation throughout the body, variable stylet length, a monodelphic reproductive track, sperm-filled spermatheca, spacious thick-walled rounded uterine chamber, swollen vulval lips, absence of vulva flaps, similar relative position of vulva, and similar tail shape and tip. However, the maximum body width of P. ilicis n. sp. females were found to be smaller compared to all the records for C. pestis, including type material (30 mm to 35 mm vs. 35 mm to 43 mm, compiled in Ghaderi et al., 2016). In the original description of C. pestis, a circular organ reminiscent of the scutellum of Hoplolaimus von Daday, 1905 was well illustrated in the oval-shaped lateral field differentiation. However, such structure is not observed in females of P. ilicis n. sp. The new species has both outstretched and reflexed ovaries, while C. pestis was always reported with flexures in the female ovaries. The juveniles of P. ilicis n. sp. have the characteristic finger-like tail tip while the juveniles of C. pestis have a bluntly rounded tail tip.

Females of the new species can also be easily separated from that of the molecularly closely related P. idalimus (Raski, 1962) Siddiqi and Goodey, 1964 and P. verus (Brzeski, 1995) Brzeski, 1998 (see molecular characterization part below) based on body (slender to obese vs. always slender), cuticle (ornamented with tubercles vs. non-ornamented), vulva flaps (absent vs. present but strongly reduced), rounded uterine chamber (present vs. absent), modification of lateral field at vulva level (into a spindle-shaped structure vs. no modification), V% (89% to 95% vs. 71% to 79%), and tail (very short vs. comparatively longer).

C. pestis was described from an orchard near Santa Clara, California, associated with Persian walnut (J. regia L.). It was subsequently reported in the southern parts of Europe and in Iran, found associated with the same and also newer hosts (Scotto La Massesee, 1971; Inserra, 1973; Bello and Belart, 1975; Sturhan, 1977; Inserra and Vovlas, 1981; Gharahkhani et al., 2007; Bahmani et al., 2013; Mokaram Hesar et al., 2019). However, this species has never been reported from the northern temperate regions. On the other hand, P. ilicis n. sp. was found in northern Europe (Hilversum, the Netherlands) with an annual temperature range of 0°C to 22°C and associated with the host plant holly (I. aquifolium L.). Most importantly, the rDNA and mtDNA fragments for the new species were found to be very different (see in molecular characterization part below) from that of the Iranian populations of C. pestis (Mokaram Hesar et al., 2019) but found close to those of P. idalimus and P. verus, other species with exceptionally long stylets, but without cuticular ornamentations (Ghaderi et al., 2016; Singh et al., 2021).