**Preface to "The Systematics, Morphological, and Molecular Characterization of Economically Important Plant–Parasitic Nematodes: A Themed Issue in Honor of Dr. Gary Bauchan"**

This is a compilation of articles published in the Special Issue "The Systematics, Morphological, and Molecular Characterization of Economically Important Plant–Parasitic Nematodes: A Themed Issue in Honor of Dr. Gary Bauchan" in *Plants*. It includes a series of original research (seven) and review articles (four) focused on plant-parasitic nematodes, including two new species descriptions, *Pratylenchus dakotaensis* n.sp. and *Xiphinema malaka* n. sp. Several original articles present integrative taxonomy and molecular phylogeny methods to identify plant-parasitic nematodes of the genera *Paratylenchus*, *Xiphinema*, *Pratylenchus*, and *Rotylenchulus*. A few articles present reports of plant-parasitic nematodes found in major economically important crops such as soybean and potato, while some other articles describe new sensitive and rapid detection methods for *Meloidogyne hapla*, *Globodera pallida* and *Globodera rostochiensis*. We would like to express our gratitude to all the authors who submitted their work to be included in this Special Issue.

> **Zafar Handoo and Mihail Kantor** *Editors*

### *Article* **Morphometric and Molecular Diversity among Seven European Isolates of** *Pratylenchus penetrans*

**Mesfin Bogale 1,†, Betre Tadesse 2,†, Rasha Haj Nuaima <sup>3</sup> , Bernd Honermeier <sup>2</sup> , Johannes Hallmann <sup>2</sup> and Peter DiGennaro 1,\***


**Abstract:** *Pratylenchus penetrans* is an economically important root-lesion nematode species that affects agronomic and ornamental plants. Understanding its diversity is of paramount importance to develop effective control and management strategies. This study aimed to characterize the morphological and genetic diversity among seven European isolates. An isolate from the USA was included in the molecular analyses for comparative purposes. Morphometrics of the European *P. penetrans* isolates generally were within the range of the original descriptions for this species. However, multiple morphometric characteristics, including body length, maximum body width, tail length and length of the post-vulval uterine sac showed discrepancies when compared to other populations. Nucleotide sequence-based analyses revealed a high level of intraspecific diversity among the isolates. We observed no correlation between D2-D3 rDNA- and COXI-based phylogenetic similarities and geographic origin. Our phylogenetic analyses including selected GenBank sequences also suggest that the controversy surrounding the distinction between *P. penetrans* and *P. fallax* remains.

**Keywords:** *Pratylenchus penetrans*; *Pratylenchus fallax*; root-lesion nematode; genetic diversity; morphometrics; COXI; D2-D3 rDNA; PP5; *β*-1,4-endoglucanase

#### **1. Introduction**

With a global distribution and significant economic impact [1], sometimes requiring quarantine measures [2], species within the plant parasitic nematode genus *Pratylenchus* are some of the most agriculturally important pests. Species identification within the genus is traditionally based on morphological and morphometric characterization [1,2]. The main diagnostic characteristics are presence/absence of males, body length, head shape, stylet length, and other cuticular characters including the number of lip annules, the number of lateral field lines, the presence/absence of areolated bands on the lateral fields within the vulval region, the length and structure of the post-vulval uterine sac and shape of the spermatheca, the shape of the female tail and tail tip, and de Man's indices [3–6].

Identification and delineation of *Pratylenchus* species using these anatomical and morphometric features alone can pose many issues due to interspecific similarity and intraspecific variability of some of these characters [1,7,8]. For example, the high intraspecific morphological variations that exist within *P. penetrans* and *P. fallax* have contributed to the taxonomic confusion of these species. *P. fallax* was separated from *P. penetrans* by Seinhorst [7], only to be considered conspecific later by Tarte and Mai [8], who attributed the variations to environmental factors. The separation of the two species was confirmed using breeding experiments [9], isozyme [10] and PCR Restriction Fragment Length Polymorphism (PCR-RFLP; [11]) analyses. The presence/absence of males also does not appear

**Citation:** Bogale, M.; Tadesse, B.; Nuaima, R.H.; Honermeier, B.; Hallmann, J.; DiGennaro, P. Morphometric and Molecular Diversity among Seven European Isolates of *Pratylenchus penetrans*. *Plants* **2021**, *10*, 674. https://doi.org/ 10.3390/plants10040674

Academic Editors: Zafar Handoo and Mihail Kantor

Received: 31 January 2021 Accepted: 25 March 2021 Published: 31 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to be a robust taxonomic characteristic as some asexual species such as *P. thornei, P. neglectus* and *P. hippeastri* have been reported to occasionally have males though these males may not play a role in reproduction [12]. The large number of species (110 species) described within the genus [13] is also a contributing factor owing to the limited number of distinguishing morphological features that are available. Consequently, different molecular methods have been developed for species identification and assessment of genetic variation within and between species of *Pratylenchus.* Commonly used molecular methods include quantitative PCR (qPCR; [14]), Amplified Fragment Length Polymorphism (AFLP; [15,16]), RFLP [11,17], Random Amplified Polymorphic DNA (RAPD; [18–20]), Sequence Characterized Amplified Region (SCAR; [16,21]), Single Nucleotide Polymorphism (SNP; [22]) and Simple Sequence Repeats or Variable Number Tandem Repeats (SSR or VNTR, respectively; [23,24]).

One of the most economically important species within this genus is *P. penetrans*, which affects a wide range of agronomic and ornamental plants, and has the potential to parasitize over 400 plant species [1,25]. *P. penetrans* is cosmopolitan though more significant in temperate regions, harbours high morphological variation, and it is considered to represent a species complex [26]. The objective of this work was to determine the diversity among seven populations of *P. penetrans* that were collected from different geographical regions in Europe based on morphometric and molecular analyses. An isolate (VA) obtained from Virginia, USA, was also included in nucleotide sequence analyses for comparative purposes.

#### **2. Results**

#### *2.1. Morphometrical Observations*

Significant similarities and differences in morphometric characters were observed amongst the seven *P. penetrans* isolates (Table 1). The ratio (b') of body length (L) to length of pharynx (from anterior end to posterior end of pharyngeal gland) was the largest for NL, FR and UK, and the smallest for MN, WZ and BL. The ratio (c) of body length to tail length (tail) ranged from 14.10 in BN to 23.30 in FR. These isolates were significantly different from each other in terms of this ratio. The excretory pore (EP) was most anterior in MN, WZ and some UK isolates, and most posterior in BL, FR, NL and some BN isolates. Ovary length (Ovary) was significantly different between MN and BL isolates. MN and WZ isolates had shorter tails than BN, BL, FR and NL. Some morphological characters varied among the seven populations, but no distinct groupings were observed in terms of these characters. Such characters included stylet length (Stylet), pharynx length (Ph-L; anterior end to end of pharyngeal gland) and length of pharyngeal overlap (Ph-O). The distance of vulva from anterior end divided by body length (V) did not vary significantly among the seven populations.

Coefficient of variation (CV) for the various morphometric characters ranged from 2.40% to 14.92% (Table 1). CV was the lowest for Stylet length (2.40%) and a value (2.85%); and the highest for ovary length (14.92%) and length of post-vulval uterine sac (PUS; 14.59%).

#### *2.2. Nucleotide Sequence Analysis*

For each of the eight isolates, we sequenced the partial β-1,4-endoglucanase gene, the D2-D3 expansion of rDNA and the partial mitochondrial COXI gene region. The rDNA amplicon for each isolate was cloned (see below) and two transformed bacterial colonies were sequenced to check for the presence/absence of gene variants and/or intrapopulation variants. Both colonies that were sequenced for each isolate's rDNA fragment had identical D2-D3 sequences. We included in our sequence alignments selected GenBank sequences spanning the D2-D3 rDNA expansion and the mitochondrial COXI sequences for which our sequences found the highest hits during nucleotide Basic Local Alignment Search Tool (BLASTn) analysis (Table 2). We also included *P. neglectus* sequences for outgroup purposes (Table 2). The aligned D2-D3 and COXI sequences (each consisting of 23 taxa, including our eight isolates; Supplementary Data S1) were analyzed as a combined dataset. The β-1,4-endoglucanase sequences were not included in the phylogenetic analyses for lack of related sequences in the public databases for use as references.

**Table 1.** Morphometry of the seven European *Pratylenchus penetrans* female isolates and their geographical origins.


<sup>1</sup> Average and standard error (*n* = 10), <sup>2</sup> Different letters between columns in the same row indicate significant differences according to generalized linear models and estimated marginal means with Sidak corrections for multiple comparison of means at *<sup>p</sup>* <sup>≤</sup> 0.05, <sup>3</sup> Range, <sup>4</sup> Coefficient of variation.



Aligned sequences were trimmed at the 50 - and 30 -ends such that nucleotide sequences including the primer sequences, or their complimentary nucleotides were excluded. This was to match the regions that we sequenced for our isolates. In the case of COXI sequences, this was also to exclude the two nucleotide differences that we observed in the middle of the JB3 binding sites (see below; indicated by boldface letters) in some GenBank sequences. In some (accession numbers MK877993–MK877996, MK877985–MK877987) the JB3 binding site had the sequence 50 -TTT TTT GG**T** CAT CC**G** GAG GTT TAT-30 , while in others (accession numbers MN453207–MN453217) this sequence was 50 -TTT TTT GG**G** CAT CC**T** GAG GTT TAT-30 . A third group of sequences (accession numbers MK877989–MK877992) had a JB3 site 50 -TTT TTT GG**T** CAT CC**A** GAG GTT TAT-30 . The D2-D3 and COXI datasets incorporated 692-and 321-characters including alignment gaps, respectively.

Maximum Likelihood and Maximum Parsimony analysis of the concatenated D2-D3 rDNA and COXI dataset resulted in the trees presented in Figures 1 and 2. The MP and ML trees had the same general topology though the level of bootstrap support for the two lineages and branches in these lineages differed. Both ML and MP analyses resolved the ingroup into two well-supported lineages, one of which (Lineage 2) exclusively consisted of three of our eight *P. penetrans* isolates (UK, MN and WZ) and *P. fallax* sequences from GenBank. The remaining five of our isolates fell in Lineage 1 either within well-supported groups or scattered throughout this branch. Both analyses used the General Time Reversible model [28] and all nucleotide positions were included.

**Figure 1.** ML tree based on the combined D2-D3 rDNA and COXI dataset. Bootstrap values > 50 are shown. Scale bar indicates number of substitutions per site.

**Figure 2.** Maximum Parsimony tree generated using the combined D2-D3 and COXI dataset. Bootstrap values > 50 are indicated above nodes. Scale bar indicates number of changes.

#### **3. Discussion**

*3.1. Morphometrical Observations*

Morphometric measurements of the seven *P. penetrans* populations studied here were within the range of the original descriptions [29,30]. Most of these measurements also largely corresponded with those described for populations from China [31,32]; Colombia, Ethiopia, France, Japan, Rwanda, The Netherlands, and USA [15]; and Morocco [33]. However, remarkable differences were also observed for some characters.

Average ratios of body length to maximum body width (a) observed in the isolates examined here (25.10–27.70) were comparable to those described by Janssen et al. [15] (24.00–27.00),

but lower than those reported by Chen et al. [31] (29.90–32.00) and Mokrini et al. [33] (29.20–33.00). The range of ratios of body length to pharynx length from anterior end to posterior end of pharyngeal gland (b') in our isolates (4.33–4.98) was comparable to those described by Mokrini et al. [33] (4.40–5.00). Average body length to tail length ratios (c) ranged from 17.00 to 19.90 among our isolates. Most of these values were lower than those measured for population(s) of Wu et al. [32] (21.40), Chen et al. [31] (20.20–22.10) and Janssen et al. [13] (20.00–25.00). The *P. penetrans* isolates we studied were shorter (437–545 µm) than those described by Wu et al. [32] (666 µm), Chen et al. [31] (540–610 µm) and Janssen et al. [13] (593–684 µm). Position of the vulva relative to body length (V) in our isolates was comparable to those described by Chen et al. [31], Wu et al. [32], Mokrini et al. [33] and Janssen et al. [13]. Similarly, positions of the excretory pore (EP), maximum body width (MBW; Table 1) and tail length in the isolates we studied were comparable to those reported for other populations by Mokrini et al. [33]. Except for MBW, which was considerably higher in our isolates, EP and tail length among our isolates were also comparable to those studied by Chen et al. [31] (69.00–80.00 µm, 9.40–10.40 µm and 25.00–28.00 µm, respectively). However, measurements for these three morphometrical features were shorter in populations described by Wu et al. [32] (91.90 µm, 25.40 µm and 31.40 µm, respectively) and Janssen et al. [13] (97–120 µm, 21–28 µm and 29–32 µm, respectively). The isolates we studied had a shorter post-vulval uterine sac (PUS; 19.60–23.60 µm) than those of Mokrini et al. [33] (26.20–30.90 µm) and Wu et al. [32] (24.90 µm).

Stylet length was the least variable character among our isolates. Previous studies on *P. penetrans* [5,32] and other *Pratylenchus* species [34,35] also reported the same. This suggests that stylet length is a stable characteristic that may allow for clear demarcations among different populations of *P. penetrans* and species of *Pratylenchus*. On the contrary, ovary length and length of the post-vulval uterine sac (PUS) showed high CV among our isolates, confirming previous studies by Román and Hirschmann [5], Tarjan and Frederick [34] and Wu et al. [32]. Ph-L and Ph-O were also among the morphometric characters with high variability that we observed (Table 1). These characteristics with high CVs would be of less value in the morphological taxonomy of *P. penetrans* owing to this high variability.

#### *3.2. Sequence Analysis*

Mekete et al. [36] designed primer set PP5F/PP5R based on aligned *β*-1,4-endoglucanase sequences from GenBank for the purpose of identifying *P. penetrans* isolates via amplification of a species-specific 520-bp-fragment. The authors tested the primer set using isolates representing *P. penetrans, P. crenatus, P. scribneri, Helicotylenchus pseudorobustus, Hoplolaimus galeatus, Xiphinema americanum* and *X. rivesi*, where it resulted in amplification of the expected 520-bp-product only in *P. penetrans* isolates, indicating specificity of the primer set. Similarly, the authors developed a second set of primers (PSC3) that was specific to *P. scribneri* and amplified a 280-bp-fragment only in isolates of this species. In our study, PP5 amplified a PCR product in all the eight isolates. However, the size of the PP5 product among our isolates was only ~346 bp, as opposed to the expected 520 bp. BLASTn analysis of PP5-sequenes of our isolates returned *P. penetrans* β-1,4-endoglucanase as the only one or two significant match(es) from among the eight *Pratylenchus* β-1,4-endoglucanase sequences currently available in GenBank; unfortunately, Mekete et al. [36] did not sequence their PP5 PCR products. To rule out the possibility that Mekete et al. [36] confused amplicon sizes of PP5 and PSC3 in their report, we tested primer set PSC3 in our isolates. PSC3 did not produce amplification products at any of the annealing temperatures reported for this primer set [36]. While we cannot discount the usefulness of PP5 for the identification of *P. penetrans* isolates based on amplification of a PCR product, we can, however, confirm that the size of the amplicon may not always be 520 bp.

Three of our eight isolates which are grouped in Lineage 2 (UK, WZ and MN) shared several morphological characteristics apart from the remaining five isolates. The three isolates had the most anterior excretory pores, 71.60 ± 1.27 µm, 67.70 ± 1.23 µm, and

70.60 ± 1.26 µm, respectively. This was in sharp contrast to that described for *P. fallax* by Janssen et al. [13]. This measurement for *P. fallax* isolates by Janssen et al. [13] were 87 ± 8.3 µm (Ysbrechitum F2455), 91 ± 11 µm (Uddel F0689) and 108 ± 14 µm (Doornenburg– Type locality). Body and tail length in UK, WZ and MN isolates were in the short end of the spectrum for our seven isolates and matched that reported by Janssen et al. [13] for two of their *P. fallax* populations. The third *P. fallax* population (Ysbrechtum F2455), however, had much longer bodies (527 ± 32 µm). The range of pharynx length (Ph-L) reported for *P. fallax* [13] was much wider than what we found among our seven isolates. Stylet length, which showed the least variation among isolates of *P. penetrans* [this study; 6,31] and other *Pratylenchus* species [34,35], did not correlate with phylogenetic groupings. Janssen et al. [13] have attempted to resolve the controversy surrounding the separation of *P. fallax* from *P. penetrans* using morphology and sequence information. However, our findings suggest that *P. fallax* may remain to be a cryptic species along several others in the *P. penetrans* species complex [26].

Phylogenetic resolution of the seven European isolates we studied did not correspond with the geographical origins of these isolates. For example, the three German isolates that were collected not more than 40 km away from each other, grouped in two different lineages. Isolate BN grouped in Lineage 1, while isolates WZ and MN grouped in Lineage 2. On the other hand, isolates UK and WZ, which had the largest distance between their geographical origins (861 km), grouped together in Lineage 2. The isolate from the USA also grouped in Lineage 1, together with some of the European isolates, confirming that geographical origin did not correspond with phylogenetic grouping. The *P. penetrans* group [13] is known to include several more cryptic species than that represented by the two lineages here.

The separation of *P. fallax* from *P. penetrans* was based on breeding experiments that produced infertile interspecific offspring [9], and distinctive isozyme [10] and ITS-RFLP [37] patterns. We have not done any of these analyses using our isolates and cannot confirm or refute the validity of these techniques for the separation of the two species. However, the morphological variations that we observed among our Lineage 2 isolates, and the variation that Janssen et al. [13] reported among their *P. fallax* populations, taken together with the fact that MN, WZ and UK isolates grouped with *P. fallax* isolates in a stronglysupported-Lineage 2, indicates that neither morphology nor D2-D3 rDNA- and COXI-based phylogenetic analyses are sufficient to separate the two species.

#### **4. Materials and Methods**

#### *4.1. Nematode Isolates and Microscopy*

Seven of the isolates were collected from soils in different regions in Europe, multiplied from single females on carrot disc cultures for two–three generations (Table 3; [38]) and used in morphometric and molecular analyses. The eighth isolate (VA) obtained from Virginia, USA, was used in the nucleotide sequence analyses for comparative purposes.


**Table 3.** Isolate designation, geographical origin, and distance (km) between geographical origins of the seven European isolates.

Killing, fixing, and mounting of nematode specimens was done following Hooper et al. [39]. For each isolate, nematode suspensions were transferred into 10 mL glass vials in ~2 mL of water. A double-strength TAF fixative stock solution consisting of 10 mL formalin (35% formaldehyde), 1 mL triethanolamine and 56 mL aqua dest was prepared and heated to 70 ◦C in a water bath. Two mL of the hot fixative was then dispensed into each of the vials containing nematode suspensions, which were then left at room temperature for 24 h. The TAF fixative was removed from the vials leaving ~1 mL nematode suspension, which were then transferred onto 5 cm sterile plastic Petri dishes. The Petri dishes were filled with a solution consisting of 30% ethanol, 67% aqua dest and 3% glycerine, and placed in a wooden cabinet at room temperature for 5–7 weeks, covered only partially to allow evaporation. Specimens were permanently mounted in anhydrous glycerol.

The selection of morphometric characters studied was in accordance with Decraemer and Hunt [40] and Castillo and Vovlas [1]. Ten females were evaluated for each nematode sample. Measurements were performed using a Nikon ECLIPSE Ni-U microscope at 100X magnification with the aid of a Nikon DS Fi-2 camera and exclusive NIS-Elements image analysis software (Nikon, Tokyo, Japan). Morphometric data were analysed using generalized linear models using Gaussian (for homogeneous) or quasipoisson (inhomogeneous variances) families. Estimated marginal means (R version 4.0.2; [41]) were used to generate means and standard errors as well as for separation of treatments at *p* ≤ 0.05.

#### *4.2. DNA Extraction*

For each isolate, DNA was extracted following Holterman et al. [42] from ten nematodes (4-stage juveniles and adults). Nematodes were transferred individually into 0.2 mL PCR tubes using micropipette in a total of 25 µL. An equal volume of lysis buffer (25 µL) consisting of 0.2 M NaCl, 0.2 M Tris-HCl (pH 8.0), 1% *v/v* β-Mercaptoethanol, 0.8 mg/mL Proteinase K was then added to each sample. The tubes were briefly centrifuged at 16,000 rpm and incubated at 65 ◦C and 750 rpm for 2 h followed by 10 min at 100 ◦C in a Thermomixer (Eppendorf, Hamburg, Deutschland). Nematode lysates were used immediately or stored at −20 ◦C till used.

#### *4.3. Nucelotide Sequence Analysis*

Amplicons of ~2000 base pair (bp), ~350 bp and ~286 bp of the genes encoding for the 28S rDNA, the mitochondrial COXI gene and "PP5 region" were amplified using primer pairs 18S CL-F2 [43] and D3B [44], JB3 and JB4.5 [45], and PP5F and PP5R [36], respectively. The reaction and cycling conditions for the COXI and PP5 gene regions were as described by Bowles et al. [45] and Mekete et al. [36], respectively. These fragments were sequenced using the same primers as for the respective PCRs. The PCR cycles for the 28S rDNA consisted of an initial denaturation at 95 ◦C for 4 min followed by 35 cycles of denaturation at 95 ◦C for 45 s, annealing at 64 ◦C for 30 s and extension at 72 ◦C for 2 min; and a final extension at 72 ◦C for 10 min. The resulting fragments were cloned using a NEB PCR Cloning Kit (New England Biolabs Inc., Ipswich, MA, USA) following the manufacturer's recommendations. For each isolate, two colonies were PCRamplified using the primers supplied with the kit and sequenced using the D3B primer [44]. All amplification reactions were performed on a GeneAmp PCR System 2700 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). PCR products were purified using QIAquick PCR purification kit (QIAGEN, Germantown, MD, USA), and sequenced at Eurofins USA (https://www.eurofinsgenomics.com (accessed on 1 February 2021).

For COXI and D2-D3 rDNA gene regions sequenced in this study, selected sequences were obtained from GenBank and included here for reference and outgroup purposes (Table 2). DNA sequences generated in this study have been deposited in GenBank (Table 2). Nucleotide sequences were assembled using Geneious (Version 11.1.5, Biomatters Ltd., Auckland, New Zealand), and aligned using Clustal Omega [46], after which the alignments were manually corrected where needed using Phylogenetic Analysis Using Par-

simony (PAUP, Version 4.0b 10; [47]). Maximum Parsimony (MP) and Maximum Likelihood (ML) analyses were done on the concatenated D2-D3 and COXI dataset using MEGA-X [48]. Heuristic searches based on 1000 random addition sequences and tree bisection-reconnection were used for this purpose, with the branch swapping option set on 'best trees' only. Bootstrap analysis [49] was based on 1000 replications.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/plants10040674/s1, Data S1: D3 rDNA and COXI sequences of 37 taxa (including our eight isolates) still groups MN, WZ and UK isolates in a strongly supported branch together with *P. fallax* isolates.

**Author Contributions:** Conceptualization, M.B., B.T., P.D. and J.H.; methodology, M.B., B.T., R.H.N. and B.H.; formal analysis, M.B., B.T. and R.H.N.; investigation, M.B., B.T. and R.H.N.; resources, B.H., J.H. and P.D.; data curation, M.B., B.T. and R.H.N.; writing—original draft preparation, M.B. and B.T.; writing—review and editing, M.B., B.T., R.H.N., B.H., J.H. and P.D.; supervision, B.H., J.H. and P.D.; project administration, B.H., J.H. and P.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or Supplementary Materials. The data presented in this study are available in Supplementary Data S1 and sequences produced can be found in GenBank, accessions listed in Table 2.

**Acknowledgments:** We would like to acknowledge the contributions of Peter-Jan Jongenelen from Joordans Zaden in acquiring nematode samples throughout Europe that significantly increased the breadth of this work, as well as Jan Henrik Schmidt from Julius Kühn-Institut for his support in statistical analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Integrative Taxonomy and Molecular Phylogeny of the Plant-Parasitic Nematode Genus** *Paratylenchus* **(Nematoda: Paratylenchinae): Linking Species with Molecular Barcodes**

**Phougeishangbam Rolish Singh 1,\*, Gerrit Karssen 1,2, Marjolein Couvreur <sup>1</sup> , Sergei A. Subbotin 3,4 and Wim Bert <sup>1</sup>**


**Abstract:** Pin nematodes of the genus *Paratylenchus* are obligate ectoparasites of a wide variety of plants that are distributed worldwide. In this study, individual morphologically vouchered nematode specimens of fourteen *Paratylenchus* species, including *P. aculentus, P. elachistus, P. goodeyi, P. holdemani, P. idalimus, P. microdorus, P. nanus, P. neoamblycephalus, P. straeleni* and *P. veruculatus*, are unequivocally linked to the D2-D3 of 28S, ITS, 18S rRNA and *COI* gene sequences. Combined with scanning electron microscopy and a molecular analysis of an additional nine known and thirteen unknown species originating from diverse geographic regions, a total of 92 D2-D3 of 28S, 41 ITS, 57 18S rRNA and 111 *COI* new gene sequences are presented. *Paratylenchus elachistus*, *P. holdemani* and *P. neoamblycephalus* are recorded for the first time in Belgium and *P. idalimus* for the first time in Europe. *Paratylenchus* is an excellent example of an incredibly diverse yet morphologically minimalistic plantparasitic genus, and this study provides an integrated analysis of all available data, including coalescence-based molecular species delimitation, resulting in an updated *Paratylenchus* phylogeny and the corrective reassignment of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 *COI* gene sequences that were previously unidentified or incorrectly classified.

**Keywords:** D2-D3 of 28S; ITS; 18S; *COI*; morphology; morphometrics; *Paratylenchus*; plant-parasitic nematodes; phylogeny; taxonomy

#### **1. Introduction**

The plant-parasitic nematode (PPN) genus *Paratylenchus* Micoletzky, 1922, commonly known as pin nematodes, are obligate ectoparasites of a wide variety of plants, including herbs, shrubs and trees, that are distributed worldwide and cause various symptoms in their host plants [1–5]. This genus was reviewed by Tarjan [6], who provided the first key to the species. In subsequent years, several attempts were made to split the genus and group its representatives into new genera. The genus *Gracilacus* Raski, 1962, was proposed for members of the *Paratylenchus* species with stylet lengths longer than 48 µm [7]. The validity of *Gracilacus* was supported by Thorne and Malek [8], Raski and Luc [9], Maggenti et al. [10], Raski [11], Esser [12], Andrássy [13] and Yu et al. [14], while Siddiqi [15] treated it as a subgenus of *Paratylenchus*. *Gracilacus* was synonymised with *Paratylenchus* by Brzeski [16], and it was recognized in further works of Siddiqi and Goodey [17], Geraert [18], Brzeski [19], Nguyen et al. [20], Decraemer and Hunt [21], Van den Berg et al. [22], Ghaderi et al. [23],

**Citation:** Singh, P.R.; Karssen, G.; Couvreur, M.; Subbotin, S.A.; Bert, W. Integrative Taxonomy and Molecular Phylogeny of the Plant-Parasitic Nematode Genus *Paratylenchus* (Nematoda: Paratylenchinae): Linking Species with Molecular Barcodes. *Plants* **2021**, *10*, 408. https://doi.org/10.3390/ plants10020408

Academic Editor: Francesc Xavier Sorribas Royo

Received: 15 January 2021 Accepted: 15 February 2021 Published: 22 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Hesar et al. [24] and Maria et al. [25]. The genus *Paratylenchoides* Raski, 1973 was assigned to *Paratylenchus* species with stronger cephalic sclerotisations, dorso-ventrally narrower heads and small narrow rounded protrusions on the anterior surface of conoid lip region [26]. Siddiqi [15] subsequently lowered *Paratylenchoides* to a sub-generic level for *Paratylenchus*, while Raski and Luc [9] synonymised the two genera owing to the apparent lack of morphological differences between them and Siddiqi [2] accepted this. It was proposed that another genus, *Gracilpaurus* Ganguly and Khan, 1990, included four species displaying long stylets and tubercles on the surface of the cuticle [27]. However, Brzeski [19] did not consider cuticular ornamentation as a generic characteristic, a decision that led to the synonymising of *Gracilpaurus*. The monotypic genus *Cacopaurus* Thorne, 1943 was also proposed and distinguished from *Paratylenchus* by the obese female body, tubercles on annuli of the female cuticle and sessile parasitism [28]. Although Goodey [29] synonymised *Cacopaurus* with *Paratylenchus* due to the lack of consistent differential traits—apart from the female of the former sometimes being sessile and slightly swollen—*Cacopaurus* has been, nevertheless, accepted by Raski [7], Raski and Luc [9], Ebsary [30], Raski [11], Brzeski [19], Siddiqi [2], Andrassy [13] and Ghaderi et al. [23,31].

Nematodes of the genus *Paratylenchus* in a broad sense or *sensu lato* are characterised by: small size (<0.7 mm); females being vermiform to obese; C, J or 6 shapes when heat relaxed; two to four lateral lines; cuticle with or without ornamentations; often continuous cephalic regions of rounded to conoid, truncate or trapezoid shapes; protruding or non-protruding submedian lobes; stylet lengths ranging between 10 and 120 µm; welldeveloped valves of median bulb, slender isthmuses and rounded to pyriform end bulbs in female pharynges; secretory-excretory pores are often at the level between median bulb and end bulb; spermathecae with or without sperm cells; commonly swollen prevulval region; vulvae with or without lateral flaps; presence or absence of a short post-vulval uterine sac; tails ranging from conoid to hemispherical with variable tail termini. The diagnostic traits of juveniles and males are less frequently used for identification, except for looking for the presence of a stylet and looking at the length of the spicules of males.

Recently, Ghaderi et al. [23] recognized 117 species of *Paratylenchus sensu lato* (excluding *Cacopaurus*), six species of *inquirendae* and four of *nomina nuda*. The nominal species were pragmatically divided into eleven groups based on stylet length, number of lateral lines and absence vs. presence of vulval flaps in females. Since then, seven more species of *Paratylenchus* have been described and linked to DNA sequences [14,25,32–36]. Molecular work on this nematode group is gaining momentum and provides an attractive solution to difficulties encountered in species identification, as well as phylogenetic relationships among species. Subbotin et al. [37], Chen et al. [38,39] and van Megen et al. [40] started to molecularly characterise some *Paratylenchus* spp. using the D2-D3 of 28S rRNA, ITS rRNA and 18S rRNA gene sequences, respectively. Lopez et al. [41] used ITS rRNA gene to examine phylogenetic relationships among four nematode genera; two of the included genera were *Paratylenchus* and *Gracilacus*. Van den Berg et al. [22] conducted the first comprehensive phylogenetic study including several *Paratylenchus* spp. by using 58 28S rRNA and 40 ITS rRNA gene sequences. Several other studies provided additional molecular characterisations, phylogenetic analyses and descriptions of new *Paratylenchus* species [14,25,32–36,42–50]. In a study by Hesar et al. [24], 28S rRNA and ITS rRNA gene sequences of several *Paratylenchus* spp. as well as *Cacopaurus pestis* Thorne, 1943, were updated. In addition to providing the first molecular characterisation of *C*. *pestis*, their phylogenetic analyses based on the two partial gene sequences did not support the monophyly of the genera *Cacopaurus*, *Gracilacus* and *Gracilpaurus* that were all found embedded within the clade of *Paratylenchus.*

Despite these recent efforts to integrate and include molecular information in species descriptions and species delineations of *Paratylenchus*, several taxonomic challenges still remain. This is often the case in the field of nematology in general, but the genus *Paratylenchus* is a perfect case in point. Most of the traditionally described species are not yet linked to molecular data, numerous sequences that are currently available are not linked to

established species and/or morphological information, sequences are often misplaced and the existence of cryptic species within the genus is common.

Species boundaries in *Paratylenchus* are sometimes difficult to delimit based solely on morphology because of the limited diagnostic features and morphological plasticity. As of December 2020, only 40 *Paratylenchus sensu lato* species have been linked to molecular data in the GenBank and this database also includes several putative, new, unidentified and incorrectly classified sequences. These misidentified sequences may result in a cascade of erroneous interpretations, including incorrect morphological identification [51] and flawed interpretations of species identity based on relationships in phylogenetic trees. Cryptic species are also likely to represent a component of *Paratylenchus* diversity [22]. It is important to note that correct differentiation of species belonging to agricultural nematode pests from its sibling species has gained importance for a number of reasons, including food security, quarantine regulations and nonchemical pest management strategies [52].

The aims of this study are to: (1) provide and update molecular barcodes of several known and unknown *Paratylenchus* species using four partial sequences—D2-D3 of 28S, ITS and 18S rRNA gene and *COI* gene of mtDNA; (2) link these molecular data to comprehensive morphological information, including light microscopy (LM) and scanning electron microscopy (SEM) images and morphometrics; (3) reconstruct an updated *Paratylenchus* phylogeny; (4) provide a molecular species delimitation for all four markers; (5) reassign unidentified and/or incorrectly classified GenBank sequences to the appropriate species.

#### **2. Results**

#### *2.1. Species Identification, Characterisation and Delimitation*

Ten identified and four unidentified *Paratylenchus* species, recovered from soil samples collected in Belgium, were morphologically and molecularly characterised. The identified species were *Paratylenchus aculentus* Brown, 1959, *Paratylenchus elachistus* Steiner, 1949, *Paratylenchus goodeyi* Oostenbrink, 1953, *Paratylenchus holdemani* Raski, 1975, *Paratylenchus idalimus* (Raski, 1962) Siddiqi and Goodey, 1964, *Paratylenchus microdorus* Andrássy, 1959, *Paratylenchus nanus* Cobb, 1923, *Paratylenchus neoamblycephalus* Geraert, 1965, *Paratylenchus straeleni* (De Coninck, 1931) Oostenbrink, 1960 and *Paratylenchus veruculatus* Wu, 1962. The unidentified *Paratylenchus* spp. were *Paratylenchus* sp.2, *Paratylenchus* sp.BE11, *Paratylenchus* sp.D, and *Paratylenchus* sp.F. *Paratylenchus elachistus, P. holdemani, P. idalimus* and *P. neoamblycephalus* were reported for the first time in Belgium and *P. idalimus* was recorded for the first time in Europe. Additional sequences of *Paratylenchus aquaticus* Merny, 1966, *Paratylenchus dianthus* Jenkins and Tylor, 1956, *Paratylenchus hamatus* Thorne and Allen, 1950, *Paratylenchus leptos* Raski 1975, *P. nanus, Paratylenchus projectus* Jenkins 1956, *Paratylenchus shenzhenensis* Wang, Xie, Li, Xu, Yu and Wang, 2013, *P. straeleni* and *Paratylenchus tenuicaudatus* Wu, 1961 and thirteen unidentified *Paratylenchus* species that originated from diverse geographic regions are also provided (Table 1). In total, 68 D2-D3 of 28S, 38 ITS, 57 18S rRNA and 84 *COI* gene sequences were linked to morphological data of the abovementioned ten known and four unknown species collected from Belgium, and 24 D2-D3 of 28S, 3 ITS rRNA and 27 *COI* gene sequences were added to the other nine known and thirteen unidentified species.

**Table 1.** List of *Paratylenchus* populations included in this study. Accession numbers of three ribosomal RNA genes (D2-D3 of 28S, ITS and 18S) and a mitochondrial gene (*COI*) fragments are provided for 18 identified and 14 unidentified *Paratylenchus* species. Accession numbers in italics are ones generated in this study.





**Table 1.** *Cont.*




#### 2.1.1. *Paratylenchus aculentus*

*Females* (Sample BE9; Figure 1, Table 2): Heat relaxed specimens open C- to J-shape. Lateral field with three lateral lines. Deirids not observed (not necessarily an indication that they are absent). Cephalic region rounded, low, sometimes appearing slightly truncated, submedian lobes not protruded. Stylet 52–61 µm long, cone 80–91% of stylet length, knobs 2–4 µm across. Pharynx well developed, about one-third of body length. Secretoryexcretory pore between median bulb and isthmus level. Spermatheca rounded to slightly oval and filled with sperm cells. Prevulval swelling not prominent. Vulval flaps very small and can be visible under LM. Vulval located at 71–76% of body length from anterior end. Vagina straight to slightly oblique, reaching to almost half of body width. Anus obscure. Tail 18–25 µm long, tapers gradually to a finely or bluntly rounded terminus.

*Molecular characterisation:* Three D2-D3 of 28S, two ITS, two 18S rRNA and four *COI* gene sequences were generated without intraspecific sequence variations. The D2-D3 of 28S and the 18S sequences, respectively, were found to be similar to KP966492 (99% similarity; 4 out of 544 bp difference) and KP966494 (100% similarity; 800 bp) of *P. colinus* from Iran after Hesar et al. [24].

*Remarks:* Males were not found. Female morphology and morphometrics matched very well with *P. aculentus*. This species has been reported earlier in Belgium [53]. Although the D2-D3 and 18S sequences pointed towards *P. colinus*, the current population had no cuticular ornamentations present in the anterior part of the body and female bodies were not swollen and submedian lobe protrusions were not seen, which are important characteristics for *P. colinus*. According to Ghaderi et al. [23], *P. aculentus* is part of Group 9 of the *Paratylenchus* species with stylet lengths longer than 40 µm, three lateral lines and absence of vulval flaps. Here, we confirm the presence of small vulval flaps in *P. aculentus*, clearly supported by SEM. This was also an observation originally made by Brzeski [19]. *Paratylenchus aculentus* should, therefore, be placed in Group 8 with *P. colinus* and *P. idalimus*; furthermore, the close affinity of our *P. aculentus* population with *P. colinus* is also molecularly supported by the very conserved 18S rRNA gene fragment.

**Figure 1.** Light and scanning electron microscopy images of *Paratylenchus aculentus* females: (**A**) face view; (**B**,**D,E**) anterior region; (**C,J**) total body; (**G**) vulva region; (**F**,**H**,**I**) tail region.


**Table 2.** Female morphometrics of *Paratylenchus aculentus*, *Paratylenchus goodeyi*, *Paratylenchus idalimus* and *Paratylenchus straeleni* from fixed specimens mounted in glycerine. All measurements except for ratios and percentages are given in µm and in the form mean ± stdev (range).

#### 2.1.2. *Paratylenchus elachistus*

*Females* (Sample BE15; Figure 2, Table 3): Heat relaxed specimens open C- to J-shape. Lateral field with four lateral lines. Deirids visible under SEM. Cephalic region conicalrounded to sometimes truncated. *En face* square-shaped, showing poorly developed submedian lobes, two pronounced lateral ridges and small indistinct dorso-ventral ridges around oral opening, two slit-like amphidial openings laterally. Stylet 20–22 µm long, cone 61–68% of stylet length and knobs 3–4 µm across. Pharynx well developed, about one-fourth of body length. Hemizonid commonly above secretory-excretory pore about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded to oval and filled with sperm cells. Vulval flaps rounded, prominent. Vulva located at 80–83% of body length from anterior end. Vagina oblique, reaching to half of body width. Tail 21–29 µm long, conical, thin and terminus from spicate to pointed or minutely rounded.

*Molecular characterisation:* Two D2-D3 of 28S, four ITS, four 18S rRNA and four *COI* gene sequences were generated for the first time from this species without intraspecific sequence variations.

*Remarks:* Males were not found. This species is reported for the first time in Belgium and has only been recorded in Poland and Slovakia before in Europe [19,54,55]. Female morphology and morphometrics agree well with the original description [56] and also with descriptions of other populations [19,31]. *Paratylenchus elachistus* can be separated from its closest species, *Paratylenchus minutus*, Lindford in Lindford, Oliveira & Ishii, 1949, by a longer body length (0.23–0.34 mm vs. 0.19–0.31 mm), a more robust and longer stylet (19–25 µm vs. 15–21 µm) and a more slender tail, commonly with spicate to finely rounded tail termini.

**Figure 2.** Light and scanning electron microscopy images of *Paratylenchus elachistus* females: (**A**,**B**) face view; (**C**,**D**,**G**–**I**) anterior region; (**E**,**F**) vulva region; (**J**) total body; (**K**–**Q**) tail region; arrows pointed to deirid in (**J**) and spermatheca in (**M**).


**Table 3.** Female morphometrics of *Paratylenchus elachistus*, *Paratylenchus holdemani*, *Paratylenchus microdorus* and *Paratylenchus veruculatus* from fixed specimens mounted in glycerine. All measurements except for ratios and percentages are given in µm and in the form mean ± stdev (range).

#### 2.1.3. *Paratylenchus goodeyi*

*Females* (Sample BE22; Figure 3, Table 2): Heat relaxed specimens C- to J-shape. Lateral field with four lateral lines. Deirids observed under LM. Cephalic region conical-rounded, submedian lobes not protruding except in two freshly killed specimens where small protrusions were seen under LM. Stylet 47–59 µm long, cone 78–90% of stylet length, stylet guide faintly seen, knobs 3–5 µm across. Pharynx well developed, about one-third of body length. Secretory-excretory pore around median bulb level. Spermatheca oval to elongate, filled with sperm cells. Vulval flaps present. Vulva located at 78–82% of body length from anterior end, in one female a short post-vulva sac observed. Vagina oblique and reaching to two-third of body width. Tail 26–32 µm long, conoid with variable terminus from finely rounded to bluntly rounded and rarely pointed.

*Molecular characterisation:* Three D2-D3 of 28S, one ITS, two 18S rRNA and three *COI* gene sequences were generated for the first time for this species without intraspecific sequence variations.

*Remarks:* Males were not found. Females morphology and morphometrics agree well with former *P. goodeyi* descriptions [18,19,57]. This species was originally described from the Netherlands and has been reported from many European countries, including Belgium. *Paratylenchus goodeyi* is one of the 22 species of the Group 10 of *Paratylenchus* after Ghaderi et al. [23] with stylet length more than 40 µm, four lateral lines and presence of vulval flaps. This species is comparable to other members of the group namely *Paratylenchus ivorensis* Luc & de Guiran, 1962, *Paratylenchus pandatus* (Raski, 1976) Siddiqi, 1989 and *P. straeleni* with females having more or less conical-rounded heads, stylet lengths in the range 40–61 µm (except for *P. pandatus* for which, a stylet length up to 68 µm was rarely reported). However, the vulvae of *P. goodeyi* and *P. straeleni* are located more posteriorly (77–88%) than that of the other two species (70–78%); *P. ivorensis* and *P. goodeyi* have been reported with variable tail termini, while *P. pandatus* and *P. straeleni* have been found usually with finely rounded to sub-acute female tail termini.

**Figure 3.** Light microscopy images of *Paratylenchus goodeyi* females: (**A**,**B**) total body; (**C**–**G**) anterior region; (**H**,**I**) lateral field; (**J**–**L**) tail region; arrows pointed to spermatheca in A, protruding submedian lobe in G and post-vulva sac in K.

#### 2.1.4. *Paratylenchus holdemani*

*Females* (Sample AR3; Figure 4, Table 3): Heat relaxed specimens C- to J-shape. Lateral field with four lateral lines. Deirids not observed. Cephalic region slightly truncated, submedian lobes not protruded. *En face* showing four small submedian lobes, four irregular ridges around oral opening, slit-like lateral amphidial openings. Stylet 19–26 µm long, cone 61–77% of stylet length, knobs 3–4 µm across. Pharynx well developed, about one-fourth of body length. Secretory-excretory pore commonly between mid-isthmus and end bulb level. Spermatheca rounded, filled with sperm cells. Vulval flaps prominent. Vulva located at 81–90% of body length from anterior end. Vagina oblique and reaching to two-third of body width. Tail 20–30 µm long, conoid with regularly finely rounded to sometimes bluntly rounded or digitate terminus.

**Figure 4.** Light and scanning electron microscopy images of *Paratylenchus holdemani* females: (**A**,**F**) face view; (**B**–**D**,**G**,**H**) anterior region; (**E**,**I**) total body; (**J**–**O**) tail region; arrow pointed to deirid in (**E**).

*Males*: Two males were obtained from Sample AR3 and one from Sample BE20. Their conspecificity with the females was confirmed by identical D2-D3 of 28S rRNA and *COI* gene sequences identified from the AR3 and BE20 males, respectively. The males had an average stylet length of approximately 12 µm and spicule length of 21 µm.

*Molecular characterisation:* Three D2-D3 of 28S, one 18S rRNA and three *COI* gene sequences were generated from the AR3 females, whereas two D2-D3 of 28S, one ITS, one 18S rRNA and four *COI* gene sequences were generated from the BE20 females. No sequence from either population showed any intraspecific variations. The D2-D3 sequences were found to be identical to *P. bukowinensis* sequences that originated from Italy [37] and Belgium [47]; however, morphological data for these populations are not available for comparison and both are considered here as representatives of *P. holdemani*.

*Remarks:* This species has been reported for the first time in Belgium and has only been reported in the Czech Republic in Europe [19]. The morphology and morphometrics of the AR3 population agree well with the original description [58] and with the population from the Czech Republic [19]. Although our D2-D3 sequences were identical to a *P. bukowinensis* sequence (AY780943), the female morphology of this Belgian population is different from *P. bukowinensis* descriptions.

Most importantly, the average stylet length (22.5 µm) of our population is shorter than for many previously reported *P. bukowinensis* populations. In addition, the cephalic region of *P. bukowinensis* is more rounded than that of *P. holdemani. Paratylenchus holdemani* is comparable to *P. hamatus* and *Paratylenchus baldaccii* Raski, 1975, but is distinguishable from both species by a shorter stylet length of 22.5 ± 2.0 (19–26) µm vs. always above 26 µm. In this study, greater variation in the tail termini was observed in our *P. holdemani* population compared to the other two species.

#### 2.1.5. *Paratylenchus idalimus*

*Females* (Samples BE19 and BE20, two nearby localities; Figures 5 and 6, Table 2): Heat relaxed specimens J- or open C-shape. Lateral field with three lateral lines. Deirids not observed. Cephalic region conical-truncate, submedian lobes well developed and protruding. Stylet 84–93 µm long of which 83–89% is cone, prominent stylet guide, knobs about 4 µm across. Pharynx well developed, occasionally reaching up to half of body length. Secretory-excretory pore around level of stylet knobs which is above median bulb level. Spermatheca small, rounded, usually filled with sperm cells. Vulval lips slightly protruding. Vulval flaps reduced and small, sometimes easily overlooked. Vulva located at 77–79% of body length from anterior end. Vagina oblique, often reaching to two-third of body width. Anus obscure. Tail 22–28 µm long, conoid with subacute to finely rounded terminus.

*Molecular characterisation:* Two identical D2-D3 of 28S and two identical 18S rRNA gene sequences were generated from the BE20 population, whereas one D2-D3 sequence, identical to that of the BE20 sequence, was generated from the BE19 population. These sequences were generated for the first time for this species.

*Remarks:* Males and swollen females were not found. Only one juvenile was recorded from the BE20 population with a stylet length of 42 µm. This is the first time the species has been reported in Europe. Female morphology and morphometrics based on seven females from both populations (three from BE19 and four from BE20) agree well with the description of the slender female by Raski [7] in the USA. This species and *P. colinus* are the only two members of Group 8 of *Paratylenchus* [23]. It differs from *P. colinus* in having a longer stylet (84–93 µm vs. 56–72 µm), more pronounced protrusion of submedian lobes, slightly posterior position of vulva (77–79% vs. 69–78%) and absence vs. presence of cuticular ornamentation in anterior body.

**Figure 5.** Light microscopy images of *Paratylenchus idalimus* females from sample BE19: (**A**) total body; (**B**–**D**) anterior region; (**E**–**G**) lateral field and tail region; arrow pointed to secretory–excretory pore in D.

**Figure 6.** Light microscopy images of *Paratylenchus idalimus* females from sample BE20: (**A**,**F**) total body; (**B**–**E**) anterior region; (**G**–**J**) tail region.

#### 2.1.6. *Paratylenchus microdorus*

*Females* (Sample BE9; Figure 7, Table 3): Body small, heat relaxed specimens open C- to 6-shape. Lateral field with four lateral lines. Deirids not observed. Cephalic region conical-truncate, submedian lobes sometimes slightly protruding. *En face* showing four submedian lobes and slit-like lateral amphidial openings. Stylet 11–15 µm long, cone 45–60% of stylet length. Pharynx about one-fifth of body length. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded, empty or filled with sperm cells. Vulval flaps prominent. Vulva located at 79–83% of body length from anterior end. Vagina oblique, reaching to half of the body width. Post-vulval uterine sac not seen. Tail 28–36 µm long, conoid and terminus pointed to subacute to sometimes finely rounded.

**Figure 7.** Light and scanning electron microscopy images of *Paratylenchus microdorus* females: (**A**) face view; (**B**) total body; (**C**–**F**) anterior region; (**G**–**J**) tail region.

*Molecular characterisation:* Three D2-D3 of 28S, three ITS, four 18S rRNA and three *COI* gene sequences were generated without intraspecific variability; the ITS and the *COI* sequences are new for this species. Only 300 bp of the D2-D3 sequences were found to be homologous with four *P. microdorus* sequences from Germany (MF325254–MF325257; 98% similarity; 5 bp difference). The 18S rRNA sequences are 98–99% similar with *P. microdorus* from the Netherlands (AY284632 and AY284633; 8–15 out of 880 bp difference).

*Remarks:* Males were not found. Female morphologies and morphometrics agree well with the original description [59] and other populations [16,31], except for a slightly shorter stylet length (11–15 µm vs. 13–18 µm). Wide variations in the tail termini have been reported for this species [31]. However, for the BE9 population, finely rounded to subacute female tail termini were commonly observed. This species is comparable to *Paratylenchus recisus* Siddiqi, 1996, *Paratylenchus variabilis* Raski, 1975 and *P. veruculatus*, with

a female stylet length within 11–17 µm, four lateral lines, presence of vulval flaps, secretory– excretory pore at the posterior part of pharynx and vulva located at 78–87% of body length. However, they differ from each another in having conical-truncate heads with sometimes slightly protruded submedian lobes in *P. microdorus*, broadly rounded to truncated head with central swallow depression in *P. recisus*, rounded to almost hemispherical head in *P. variabilis* and low and broadly rounded head in *P. veruculatus.* Only *P. microdorus* and *P. veruculatus* males have been reported to have weak stylets, while stylets in the males of the other two species are degenerated.

#### 2.1.7. *Paratylenchus nanus*

*Females* (Sample BE11; Figure 8, Table 4): Heat relaxed specimens open C- to J-shape. Lateral field with four lateral lines. Deirids not observed. Cephalic region conical-rounded, in some specimens with sloping sides to rounded end, submedian lobes not protruding under LM. *En face* square shaped, revealing four submedian lobes, four distinct ridges around oral opening, lateral ridges slightly larger than dorso-ventral ridges, and two slit-like lateral amphidial openings. Stylet 27–31 µm long, cone 67–78% of stylet length and knobs 3–5 µm across. Pharynx well developed, about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretoryexcretory pore between isthmus and end bulb level. Spermatheca rounded and filled with sperm cells. Vulval flaps present. Vulva located at 82–86% of body length from anterior end. Vagina oblique, reaching up to half of body width. Tail 19–26 µm long, conoid, often more pronounced curvature on dorsal side ending with sub-acute to finely rounded terminus.

*Molecular characterisation:* Seven D2-D3 of 28S, four ITS, four 18S rRNA and seven *COI* gene sequences were generated without any intraspecific sequence variations among four *P. nanus* populations—AR3, BE1, BE11 and BE18. The D2-D3 and the ITS sequences were, respectively, identical to KF242194, KF242197 and KF242267, KF242268 of *P. nanus* from Van den Berg et al. [22]

*Remarks:* Only in the BE11 population was a sufficient number of females recovered to allow morphological and morphometrical data comparisons, which agreed well with the original description [60] and subsequent descriptions of *P. nanus* [19,22,58]. Van den Berg et al. [22] reported two sibling species of *P. nanus* with different genotypes type A and type B (the latter of which was recently transferred to *P. projectus*) [61]. This correction suggests that the available 28S (MN720102–MN720103) and *COI* (MN734387 and MN734388) sequences of *P. nanus* from South Korea [48] were misidentified as they were found to be identical to the *P. projectus* sequences. *Paratylenchus nanus* is very similar to *P. projectus* and *P. neoamblycephalus*. It differs from *P. projectus* in having a conical-rounded vs. more trapezoid head shape and sperm-filled vs. empty spermathecae. It is differentiated from *P. neoamblycephalus* by more rounded vs. oval spermathecae and a conoid tailwith pronounced curvature on the dorsal side ending with a subacute or finely rounded terminus vs. a conoid tail with subacute terminus or almost acute tip. Furthermore, in our study we also observed that the ridges around the oral opening of the freshly killed specimens protruded more in *P. neoamblycephalus* compared to *P. nanus* when observed under LM.

**Figure 8.** Light and scanning electron microscopy images of *Paratylenchus nanus* females: (**A**,**E**,**F**,**L**) face view; (**B**–**D**,**G**,**H**,**J**,**K**,**M**) anterior region; (**I**) total body; (**N**,**O**,**P**–**U**) tail region.


**Table 4.** Female morphometrics of *Paratylenchus nanus*, *Paratylenchus neoamblycephalus*, *Paratylenchus* sp.2, *Paratylenchus* sp.D and *Paratylenchus* sp.F from fixed specimens mounted in glycerine. All measurements except for ratios and percentages are given in µm and in the form mean ± stdev (range).

#### 2.1.8. *Paratylenchus neoamblycephalus*

*Females* (Sample BE10; Figure 9, Table 4): Heat relaxed specimens open C-shape. Lateral field with four lateral lines. Deirids clearly visible on SEM images. Cephalic region truncated-rounded, submedian lobes sometimes very slightly protruding under LM. *En face* showing four rounded to oval submedian lobes, four ridges around oral opening, lateral ridges thicker than dorso-ventral ridges and seen as a protruding bi-lobed structure under LM. Stylet 32–34 µm long, cone 63–73% of stylet length, knobs 4–5 µm across. Pharynx about one-fourth of body length. Secretory-excretory pore between mid-isthmus and end bulb level, with swellings sometimes seen at the duct near the opening in freshly killed specimens. Spermatheca oval and filled with sperm cells. Vulval flaps present. Vulva located at 81–84% of body length from anterior end. Vagina oblique and reaching up to half of body width. Tail 20–28 µm long, conoid and terminating to sub-acute to almost acute tip.

**Figure 9.** Light and scanning electron microscopy images of *Paratylenchus neoamblycephalus* females: (**A**,**C**) face view; (**B**) total body; (**D**–**H**) anterior region; (**I**–**O**) tail region; arrows pointed to secretory–excretory pore in (**H**) and deirid in (**G**).

*Males:* Two males were obtained with very thin stylets in freshly killed specimens, which were not visible after fixation, and spicules of 24 µm long. Their conspecificity with the females was confirmed by identical *COI* and D2-D3 sequences.

*Molecular characterisation:* Four D2-D3 of 28S, five ITS, six 18S rRNA and eight *COI* gene sequences were generated without intraspecific sequence variations. The 18S and the *COI* sequences are new for this species. The D2-D3 sequences were found to be identical to

KF242189 and KF242190 of an unidentified *Paratylenchus* sp.6 from the USA [22], which is considered here as *P. neoamblycephalus*. However, the D2-D3 sequences were only 89% similar (79 out of 710 bp difference) with MG925221 and 92% similar (43 out of 546 bp difference) with MK506807 of *P. neoamblycephalus* from the USA and Iran, and named here as type A and type B, respectively. Interestingly, we observed 17 ambiguous nucleotide sites in the American *P. neoamblycephalus* type B sequence, which was found to be similar to *P. projectus* (previously *P. nanus* type B; KF242198–KF242201; 98% similarity; 16–20 out of 690 bp difference) after Van den Berg et al. [22,61]. On the other hand, the Iranian *P. neoamblycephalus* sequence [24] was similar to *P. nanus* (KF242194 and KF242197; 95% similarity; 27 out of 575 bp difference) [22]. Furthermore, our ITS sequences were only 74% similar (222 out of 865 bp difference) to MK506794 of *P. neoamblycephalus* type A generated from the same Iranian population.

*Remarks:* This species is reported for the first time in Belgium. Female morphology and morphometrics agree well with the original description from Germany [18] and to subsequent descriptions from Poland [19]. *Paratylenchus neoamblycephalus* is very similar to *P. nanus* and a comparison is provided above.

#### 2.1.9. *Paratylenchus straeleni*

*Females* (Sample BE15; Figure 10, Table 2): Heat relaxed specimens J- to C-shape. Lateral field with four lateral lines. Deirids clearly visible under SEM. Cephalic region conical-rounded to sometimes slightly truncated, submedian lobes not protruded. Stylet straight to slightly curved, 54–59 µm long, cone 76–83% of stylet length, knobs 3–5 µm across. Pharynx roughly one-fourth of body length. Secretory-excretory pore between isthmus and end bulb level. Spermatheca rounded to sometimes slightly ovoid and filled with sperm cells. Vulval flaps distinct. Vulva located at 80–84% of body length from anterior end. Vagina oblique, occasionally reaching to two-third of body width. Tail conical, 31–41 µm long, and terminus sharply pointed to minutely rounded.

*Males*: Two males were recovered without stylets and with spicule lengths of 20 and 22 µm, respectively. Their conspecificity with the females was confirmed by identical *COI* sequences.

*Molecular characterisation:* One D2-D3 of 28S, two ITS (99% similarity; 4 out of 830 bp difference), three identical 18S rRNA and five identical *COI* gene sequences were generated from the BE15 population. From another population (BE11), single D2-D3, ITS and 18S sequences and three identical *COI* sequences were also generated. All the sequences from both populations showed no intraspecific variation, except for the ITS sequences. The 18S sequences were 99% similar (3–5 out of 930 bp difference) with *P. straeleni* from the Netherlands (AY284630 and AY284631). The D2-D3 sequences were 97–99% similar (11–18 out of 700 bp difference) with four *P. straeleni* sequences—i.e., MK506804 from Iran [24], KM875547 from Turkey [42], and KF242235 and KF242236 from the USA [22]. The *COI* sequences were generated for the first time for this species. Remarkably, the Belgian ITS sequences were only 62% similar (295 bp difference) to the Iranian *P. straeleni* sequence (MK506791) of Hesar et al. [24].

*Remarks:* Female morphology and morphometrics agree well with the original description, also from Belgium [62], and subsequent descriptions of globally distributed *P. straeleni* populations [31]. This species is comparable to *P. goodeyi* as described above.

**Figure 10.** Light and scanning electron microscopy images of *Paratylenchus straeleni* females: (**A**) whole body; (**B**,**D**) face view; (**C**,**G**–**J**) anterior region; (**E**) vulva region; (**F**,**K**,**L**) tail region; (**M**) total body; arrows pointed to deirids in (**A**,**C**).

#### 2.1.10. *Paratylenchus veruculatus*

*Females* (Sample BE20; Figure 11, Table 3): Heat relaxed specimens open C-shape to slightly ventrally curved. Lateral field with four lateral lines. Deirids not observed. Cephalic region broadly rounded, submedian lobes not protruding. *En face* rectangular with indistinct submedian lobes, four irregular ridges around oral opening and lateral amphidial openings. Stylet 13–15 µm long, cone 60–65% of stylet length, knobs about 3 µm across. Pharynx roughly one-fourth of total body length. Hemizonid two body annuli long, usually visible just above secretory-excretory pore. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded and filled with sperm cells, young females with empty spermatheca also seen. Vulval flaps prominent. Vulva located at 84–90% of body length from anterior end. Vagina oblique and long, reaching up to three-fourth of body width. Tail 14–19 µm long, conoid with often broadly rounded to sometimes finely rounded terminus.

**Figure 11.** Light and scanning electron microscopy images of *Paratylenchus veruculatus* females: (**A**–**C**) face view; (**D**–**G**) anterior region; (**H**,**O**) total body; (**I**–**N**) lateral field and tail region.

*Molecular characterisation:* Five D2-D3 of 28S (99% similarity; 1–2 out of 720 bp difference) and two identical 18S rRNA and seven *COI* gene (97–100% similarity; 12–13 out of 410 bp difference) sequences were generated for the first time for this species.

*Remarks:* Female morphology and morphometrics agree well with the original description [63] and with other populations [19,31]. This species is comparable to other species with a short stylet such as *P. microdorus*, *P. recisus* and *P. variabilis* (see also above).

#### 2.1.11. *Paratylenchus* sp.2

*Females* (Sample BE15; Figure 12, Table 4): Heat relaxed specimens open C-shape. Lateral field with four lateral lines. Deirids observed under SEM. Cephalic region conicalrounded, sometimes slightly trapezoid, submedian lobes not protruding under LM. *En face* square-shaped, showing four rounded, poorly separated submedian lobes, four ridges around oral opening, lateral ridges more prominent and larger than dorso-ventral ridges. Stylet 27–31 µm long, cone 64–71% of stylet length, knobs about 4 µm across. Pharynx well developed, about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded to occasionally slightly ovoid, filled with sperm cells. Vulval flaps prominent. Vulva located at 81–84% of body length from anterior end. Vagina oblique, reaching up to two-third of body width. Tail 23–29 µm long, conoid, slender and terminating with finely rounded tip.

*Molecular characterisation:* Two identical sequences each of D2-D3 of 28S, ITS, 18S rRNA as well as the *COI* gene were generated. The D2-D3 and ITS sequences were found to be, respectively, identical to KF242220 and KF242221 and 99% similar (five out of 750 bp difference) to KF242243 of *Paratylenchus* sp.2, which was identified as a member of the *P. hamatus* species complex [22]. The 18S and *COI* sequences were generated for the first time.

*Remarks:* Males were not found. The female morphology and morphometrics are in agreement with the description of *P. hamatus* [64]. Based on morphology and D2-D3 and ITS sequences, Van den Berg et al. [22] considered *P. hamatus* as a species complex containing several species, including *P. hamatus sensu stricto* collected from the type locality, and *Paratylenchus* sp.1 and *Paratylenchus* sp.2, collected from other places in California. *Paratylenchus* sp.1 is identified as representative of *P. tenuicaudatus. Paratylenchus* sp.2 is morphologically similar with *P. hamatus sensu stricto* but differs based on D2-D3 and ITS sequences [22], and this species appears to be not only present in the USA (California) but also in Belgium and Kyrgyzstan.

**Figure 12.** Light and scanning electron microscopy images of *Paratylenchus* sp.2 females: (**A**,**B**) face view; (**C**) total body; (**D**–**I**) anterior region; (**J**–**O**) tail region; arrows pointed at deirids in (**C**,**I**).

#### 2.1.12. *Paratylenchus* sp.BE11

*Females* (Sample BE11; *n* = 3; Figure 13): Body about 0.3 mm long with maximum body width of about 15 µm, heat relaxed specimens open C- to 6-shape. Lateral field with four lateral lines. Deirids not observed. Head broadly rounded, submedian lobes not protruded, cephalic sclerotization strong. Stylet about 15 µm long, cone 60% of stylet length, knobs 3 µm across. Pharynx about one-fourth of body length. Secretory-excretory pore at the

level of pharyngeal end bulb or about 70 µm from anterior end. Spermatheca rounded and filled with sperm cells. Vulval flaps small and rounded. Vulva located at 80–82% of body length from anterior end. Tail 25–32 µm, conoid with bluntly rounded tip.

**Figure 13.** Light microscopy images of *Paratylenchus* sp.BE11 females: (**A**) total body; (**B**–**E**) anterior region; (**F**–**H**) tail region; (**I**) total body; (**J**) lateral field.

*Molecular characterisation:* Three identical D2-D3 of 28S, one ITS rRNA and two identical *COI* gene sequences were generated.

*Remarks:* No males were found. Female description is based on only three freshly killed specimens, while sufficient specimens are needed for a comprehensive species characterisation. The female morphology is close to *P. variabilis*, *P. veruculatus* and *Paratylenchus vexans* Thorne and Malek, 1986. These four species have more or less broadly rounded heads with non-protruding submedian lobes, stylet lengths in the range of 12–18 µm, four lateral lines, sperm-filled spermathecae, vulval flaps and conoid tails with more or less rounded termini. However, our population appears to have a stronger cephalic sclerotisation and slightly more anteriorly located vulvae (80–82% vs. 80–87%) compared to the other three species. This species is a sister to *P. microdorus* in the D2-D3 tree (96% similarity; 27 out of 740 bp difference), ITS tree (93% similarity; 37 out of 530 bp difference) as well as the COI tree (91% similarity; 36 out of 420 bp difference). It can, however, be readily morphologically distinguished from *P. microdorus* (see above).

#### 2.1.13. *Paratylenchus* sp.D

*Females* (Sample BE20; Figure 14, Table 4): Heat relaxed specimens open C-shape. Lateral field with four lateral lines. Cephalic region conical-rounded to sometimes slightly trapezoid, submedian lobes not protruding under LM. Deirids visible under SEM. *En face* showing four well-separated rounded submedian lobes and four ridges around oral opening. Stylet 26–29 µm long, cone 61–67% of stylet length, knobs about 4 µm across. Pharynx about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca empty. Vulval flaps prominent, commonly rounded. Vulva located at 82–85% of body length from anterior end. Vagina oblique reaching up to half of body width. Tail 18–26 µm long, conoid with finely rounded to bluntly rounded terminus, sometimes dorsally sinuate.

*Molecular characterisation:* Seven D2-D3 of 28S (99% similarity; one out of 730 bp difference), four ITS, five 18S rRNA and eleven *COI* gene sequences were generated without intraspecific sequence variation.

*Remarks:* Males were not found. The female morphology and morphometrics is close to *P. projectus* and *Paratylenchus neoprojectus* Wu and Hawn, 1975. The cephalic region of females were seen with both rounded to trapezoid shape, secretory-excretory pore located between mid-isthmus to end bulb level, empty spermatheca and tail termini which fit both the above two species. However, the molecular data appears to be different from any available sequences including that of *P. projectus*. A comparative study of this species with type specimens of *P. neoprojectus* and its molecular information should further confirm whether or not this species is *P. neoprojectus*.

**Figure 14.** Light and scanning electron microscopy images of *Paratylenchus* sp.D females; (**A**,**G**,**H**) face view; (**B**–**F**,**M**,**N**) anterior region; (**I**,**L**) total body; (**J**,**K**,**O**–**Q**) tail region; arrow pointed to deirid in L.

#### 2.1.14. *Paratylenchus* sp.F

*Females* (Sample BE22; Figure 15, Table 4): Heat relaxed specimen open C- to 6-shape. Lateral field with four lateral lines. Deirids present. Cephalic region conical-truncate, slightly rounded in few specimens, submedian lobes not protruding under LM. *En face* square-shaped, showing four rounded submedian lobes, four ridges around oral opening, dorso-ventral ridges much larger than the lateral ridges, slit like amphidial apertures laterally. Stylet 25–30 µm long, cone 65–70% of stylet length, knobs about 4 µm across. Pharynx about one-fourth of body length. Hemizonid just above secretory-excretory pore,

about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca oval to elongated and filled with sperm cells. Vulval flaps rounded to oval and very prominent. Vulva located at 81–84% of body length from anterior end. Vagina oblique, reaching up to two-third of body width. Tail 20–26 µm long, conoid with regularly bluntly rounded terminus.

**Figure 15.** Light and scanning electron microscopy images of *Paratylenchus* sp.F females: (**A**–**C**) face view; (**D**,**E**,**L**,**M**,**P**,**Q**) anterior region; (**F**,**G**) vulva region; (**H**–**K**,**O**,**R**–**T**) tail region; (**N**) total body.

*Males*: Heat relaxed specimen curved slightly ventrally, about the same body length as females but slightly slender. Cephalic region conoid and rounded. Stylet and pharynx degenerated. Secretory-excretory pore at about one-fifth of body length from anterior end. Spicule arcuate ventrally, about 21.5 µm in length. Gubernaculum 3–5 µm long. Tail conical with finely rounded tip. Conspecificity of males with females was confirmed by identical D2-D3, 18S and ITS sequences.

*Molecular characterisation:* Five D2-D3 of 28S, three ITS, four 18S rRNA and three *COI* gene sequences were generated without intraspecific sequence variations. The D2-D3, 18S and *COI* sequences were found to be identical, respectively, to MN783707, MN783708, MN783668–MN783670 and MN782407–MN782413 of *Paratylenchus* sp.F [47], while the ITS sequences were generated for the first time.

*Remarks:* Specimens belong to the same population as *Paratylenchus* sp.F in Etongwe et al.'s work [47]. Detailed morphological reanalysis revealed very close similarity to *P. nanus*. Nevertheless, the submedian lobes of this species appear to be somewhat more rounded than that of *P. nanus* based on SEM images and the vulval flaps also appear to be more pronounced and rounded compared to that of the latter. However, these characteristics need careful additional observations based on more specimens from both species. All four gene sequences of *Paratylenchus* sp.F were closest to the sequences of *P. elachistus* and phylogenetic analysis revealed their highly supported (PP > 90%) sister relationship. However, this species is morphologically different from *P. elachistus en face*, with rounded vs. poorly differentiated submedian lobes, stylet lengths of 25–30 µm vs. 20–22 µm and bluntly rounded vs. spicate to pointed tail termini.

#### *2.2. Phylogenetic and Species Delimitation Analysis*

The D2-D3 domains of the 28S rRNA gene alignment (744 bp long) included 128 sequences of 31 *Paratylenchus* species and three outgroup species. Forty-nine new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the analysis of the D2-D3 alignment contained three highly supported major clades and a weakly supported one (Figure 16, PP < 70%). The molecular species delimitation based on the generalized mixed-yule coalescent (GMYC) and Poisson tree process (bPTP) methods revealed 66 and 63 putative species, respectively, a result that is largely congruent with former species delineations. However, *P. projectus*, *P. straeleni*, *P. minor* and *P. shenzhenensis* were further divided into 6, 5 (four according to bPTP), 2 and 2 separate lineages, respectively.

The ITS rRNA gene alignment (995 bp long) included 99 sequences of 37 *Paratylenchus* species and three outgroup species. Thirty-six new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the analysis of the ITS alignment contained four highly supported major clades (Figure 17). Results of molecular species delimitation showed a high discrepancy between the models used—i.e., 48 putative species based on GMYC vs. 56 species based on bPTP. Additionally, molecular species delimitation based on the GMYC and bPTP methods did not correspond to species demarcation based on morphology and clade support; for example, virtually all individual sequences of *P. chongqingensis* and *P. shenzhenensis* were delineated as separate species.

**Figure 16.** Phylogenetic relationships within populations and species of *Paratylenchus*, as inferred from Bayesian analysis using the D2-D3 of 28S rRNA gene sequence dataset with the GTR + I + G model. Posterior probability of more than 70% is given for the appropriate clades. Newly obtained sequences are indicated in bold. <sup>1</sup> = originally identified as *P. nanus*, <sup>2</sup> = originally identified as *P. bukowinensis*, <sup>3</sup> = originally identified as *Paratylenchus* sp., <sup>4</sup> = originally identified as *Paratylenchus* sp.8, <sup>5</sup> = originally identified as *Paratylenchus* sp.E, <sup>6</sup> = originally identified as *Gracilacus* sp. <sup>7</sup> = originally identified as *Paratylenchus* sp.5 and <sup>8</sup> = originally identified as *Paratylenchus* sp.6. Black and grey bars represent species boundaries estimated by generalized mixed-yule coalescent (GMYC) and Poisson tree process (bPTP) methods, respectively (only differences with GMYC provided).

**Figure 17.** Phylogenetic relationships within populations and species of *Paratylenchus* as inferred from Bayesian analysis using the ITS rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. <sup>1</sup> = originally identified as *P. nanus* and <sup>2</sup> = originally identified as *Paratylenchus* sp. Black and grey bars represent species boundaries estimated by GMYC and bPTP methods, respectively.

The 18S rRNA gene alignment (899 bp long) included 88 sequences of 31 *Paratylenchus* species and two outgroup species. Fifty-four new sequences were obtained for this study. The Bayesian 50% majority rule consensus tree inferred from the analysis of the partial 18S sequence alignment contained four highly supported major clades (Figure 18). Molecular species delimitation failed to delimit well established species—for example *P. goodeyi, P. veruculatus, P. nanus* and *P. neoamblycephalus* were identified as belonging to the same species. Furthermore, both models provided highly varied results (14 putative species according to GMYC vs. 26 according to bPTP), reducing the confidence in said results.

**Figure 18.** Phylogenetic relationships within populations and species of *Paratylenchus*, as inferred from Bayesian analysis using the 18S rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. <sup>1</sup> = originally identified as *P. dianthus* and <sup>2</sup> = originally identified as *P. nanus*. Black and grey bars represent species boundaries estimated by GMYC and bPTP methods, respectively.

The *COI* gene alignment (745 bp long) included 130 sequences of 31 *Paratylenchus* species and three outgroup species. Seventy-one new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the analysis of the *COI* sequence alignment contained four moderate (Figure 19, PP = 70–90%) or highly supported major clades. Both employed species delineation methods, GMYC and bPTP, provided exactly the same 54 putative species delineations. These results were largely consistent with those obtained using other methods. However, *P. enigmaticus*, *P. microdorus* and *P. veruculatus* were subdivided into different species despite these sequences originating from the same population and their corresponding D2-D3 sequences being similar. *Paratylenchus straeleni* was appointed as a species complex with nine putative species. Statistical parsimony networks showing the phylogenetic relationships between different isolates of *P. straeleni* and *P*. *enigmaticus* based on *COI* sequences are given in Figure 19B,C. The maximum variation of sequences for *P. straeleni* was found to be 9.1%.

Taking both morphological and molecular evidence together, we have been able to reassign a total of 49 *Paratylenchus* sequences, including 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 *COI* gene sequences, to their appropriate species (Table 5). However, we cannot exclude that in future, the identification of *Paratylenchus* species made in this study may be improved in light of new datasets.

**Table 5.** List of some existing unidentified or incorrectly classified *Paratylenchus* sequences on the GenBank reassigned to corrected species. In total, 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 *COI* gene sequences have been reassigned.


**Figure 19.** (**A**). Phylogenetic relationships within populations and species of *Paratylenchus*, as inferred from Bayesian analysis using the *COI* gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. <sup>1</sup> = originally identified as *Paratylenchus* sp., <sup>2</sup> = originally identified as *P. nanus*, <sup>3</sup> = identified as *Paratylenchus* sp.E, <sup>4</sup> = originally identified as *Gracilacus* sp., <sup>5</sup> = originally identified as *Paratylenchus* sp.8, <sup>6</sup> = originally identified as *Paratylenchus* sp.B; (**B**). Statistical parsimony network showing the phylogenetic relationships between *COI* haplotypes for *P. straeleni*; (**C**). Statistical parsimony network showing the phylogenetic relationships between *COI* haplotypes for *P*. *enigmaticus*. Pies (circles) represent the sequences with the same haplotype and their size is proportional to the number of these sequences in the samples. Numbers of nucleotide differences between the sequences are indicated on lines connecting the pies. Small black circles represent missing haplotypes. Bars represent species boundaries estimated by both GMYC and bPTP methods (identical results).

#### **3. Discussion**

The genus *Paratylenchus sensu lato*, with 124 valid species, is an important plantparasitic group consisting of several commonly occurring and economically important species such as *P. bukowinensis, P. dianthus, P. hamatus, P. nanus, P. neoamblycephalus* and *P. projectus*, which are difficult to separate solely based on morphology [14,23,25,31–36]. Female morphological traits are the most commonly used features for the identification of *Paratylenchus* populations, with the relative lengths of stylet cones and the positions of the secretory-excretory pores and vulvae as the most informative traits [31,67], while several ratios such as a, c and c' show high intraspecific variation. Given the limited species-specific female traits, some characteristics of males and juveniles—such as the presence or absence of stylet and male spicule length—may also be used to supplement the available data. However, care must be taken—for example, the occasional observance of a thin stylet in freshly killed juveniles or males that was invisible once the specimens were fixed highlights the importance of reporting this characteristic from both freshly killed and fixed specimens. Further complicating *Paratylenchus* taxonomy is the presence of mixture of species within one locality and sample [31]—an observation which calls for precaution concerning the conspecificity of several life stages. Indeed, the presence of multiple species in a soil sample was amply illustrated in our study. Seventy five percent of our investigated soil samples contained multiple species, with up to five different *Paratylenchus* species present in the same sample. This is, to the best of our knowledge, one of the highest numbers of species of one plant-parasitic nematode genus present in a single soil sample. More suitable morphological characters such as ridges around the oral opening or distinct to fused submedian lobes in face view also appear to be usefully informative but were only clearly revealed in our study with supporting evidence from SEM; additionally, the small vulval flaps in *P. aculentus* confirmed in this study have often been overlooked in previous studies under LM. Scanning electron microscopy is known to be important in nematode taxonomy [68–70], and this is especially true for the genus *Paratylenchus* as demonstrated in this study.

Nevertheless, even if all existing morphological tools are carefully employed, it remains impossible for all *Paratylenchus* species to be morphologically delineated, owing to the existence of cryptic species such as *P. aquaticus* [22]. The extensive use of new molecular data in the current study has demonstrated a remarkable molecular diversity in *Paratylenchus*, with several additional cryptic species being potentially present. The most obvious example is *P. straeleni*, which comprises 9, 5 and 4 putative species according to *COI-* (both GMYC and bPTP), D2-D3 (GMYC) and D2-D3 (bPTP)-based molecular species delimitation methods, respectively. It is noteworthy that the *P. straeleni COI* sequences have clearly clustered according to geographical location, as revealed by the COI haplotype network. The problems of morphologically delineating the *Paratylenchus* species have been further demonstrated in our study by the difficulties experienced in distinguishing between *Paratylenchus* sp.2, *Paratylenchus* sp.D and *Paratylenchus* sp.F, which were found to be very similar to *P. hamatus, P. projectus*/*P. neoprojectus* and *P. nanus*, respectively. A formal description with an appropriate diagnosis can only be developed for these putative new species following detailed observations of additional specimens and a thorough comparison with type materials of the known species.

Taken together, it is abundantly clear that molecular data are essential in advancing *Paratylenchus* taxonomy. Unfortunately, the several sequences published for *Paratylenchus* have serious limitations. One such issue is that the majority of the available D2-D3, 18S and ITS rRNA sequences have either not been linked to morphological data or have been associated with poor morphological data, thereby rendering them unreliable for use in identification purposes. For example, sequences of *P. aculentus*, *P. leptos, P. microdorus, P. neoamblycephalus*, etc., are not currently linked to reliable and clear morphological data and any subsequent identification based on these sequences may, therefore, lead to the deposition of further sequences under incorrect names [51]. An additional problem identified with the currently available 18S sequences, which are often relatively short (700–800 bp), is

that several *Paratylenchus* species were detected with almost identical sequences. It is clear that to render these conserved sequences useful, complete or nearly complete lengths of the 18S rRNA gene (1600–1800 bp) will be required to allow species delimitation [71–74].

In the present study, we have also applied DNA-based species delimitation approaches to infer putative species boundaries on a given phylogenetic input, based on two different models [75,76] and four gene fragments (D2-D3 of 28S, ITS, 18S rRNA and *COI*). These coalescence-based species delimitation methods are rapidly gaining popularity in studies on closely related species that are difficult to distinguish based on phenotypic features, and have been applied to various eukaryotic groups [77,78]. However, despite plant-parasitic nematodes being a morphologically minimalistic group par excellence, such methods have been rarely applied to this group; nevertheless, they appear to be largely congruent with traditional methods [79–81]. Conversely, we have observed a remarkable discrepancy among the genes used, showing a poor link between DNA species delimitation and other methods, including a discrepancy between the employed models. The ITS and 18S rRNA genes gave, respectively, a likely overestimation and underestimation of the number of putative species, while for *COI* and D2-D3 of 28S rRNA genes, we observed, to a certain extent, an agreement with traditional methods, albeit with a likely overestimation of the number of species in several cases. This was not unexpected, as it has been exemplified by several studies that methods of species delimitation based on the coalescent model tend to overestimate phylogenetic lineages [52,77,82]. Both approaches (bPTP and GMYC) are similar in the fact that they identify significant changes in the pace of branching events on the tree. However, GMYC uses time to identify branching rate transition points, whereas, in contrast, bPTP directly uses the number of substitutions. Based on real and simulated data, both methods yield, in general, similar results [76,83]. This is the case for our COI-based output (identical results) and the D2-D3-based output (two differences, bPTP being more conserved). If differences have been observed, bPTP usually yields a more conservative delimitation than GMYC [76,80,83]. This is contrary to our unexpected ITS and 18S results and reduces the trust in the latter. Counterintuitively, the mutation rate of a chosen marker does not have a direct influence on its effectiveness to detect species. Mitochondrial markers reveal clearer discontinuities between interspecific divergence and intraspecific variation because of their faster coalescence within species lineages compared with nuclear loci, not necessarily because of their higher mutation rates [84,85]. The discrepancy between ITS and other delimitation methods in this study agrees with previous observations pointing to an unclear transition between species-level and population-level genetic distance for ITS [78]. Furthermore, it has been indicated that species delimitation based on single gene trees has serious limitations due to gene tree-species tree incongruence—confusions caused by processes including incomplete lineage sorting, trans-species polymorphism, hybridisation and introgression [78]. Multilocus approaches provide a posteriori double-check for contamination, sequencing errors or mitochondria-specific pitfalls [86]—for example, the high *COI* gene sequence variations within *P*. *enigmaticus*, *P. microdorus* and *P. veruculatus* observed in this study, despite these sequences originating from the same population. Although both nuclear and mitochondrial sequences were provided consistently from the same morphologically vouchered individuals, this study was restricted to the use of only single-locus data since only a limited number of other *Paratylenchus* individuals (and plant-parasitic nematodes in general) are linked to the same two genes. A further rigorous acquisition of both D2-D3 of 28S and *COI* gene sequences, which appear most promising for species delimitation in plant-parasitic nematodes (see [74]), will allow for more substantiated coalescence-based, multilocus species delimitation in plant-parasitic nematodes. Nevertheless, based on all obtained evidence, our findings support the proposition of Puillandre et al. [87], Padial et al. [88] and Qing et al. [80], that DNA-based species delimitation methods are important tools for the exploration of species delineation in diverse groups, but that identification of any new putative species will require further corroboration by an integrative taxonomic approach.

#### **4. Materials and Methods**

#### *4.1. Nematode Populations*

Nematode samples used in this study were collected from various localities (Table 1). Bulk soil samples of about 500 mL from 15–20 cm depths were collected from twelve locations in Belgium using a shovel. They were subsequently stored at 4 ◦C until nematode extraction. Nematodes were extracted from soil using a modified Baermann's method [89] or a rapid centrifugal flotation method [90]. Nematode extracts were observed under a stereo microscope. *Paratylenchus* populations were picked out in an embryo glass dish and stored in tap water at 4 ◦C for further analysis.

#### *4.2. Morphological Study*

Morphological study of nematodes was carried out using both heat relaxed and fixed specimens mounted on temporary and permanent slides, respectively. For preparation of a temporary mount of a nematode, a Cryo-Pro label (VWR International) was cut into two halves and stuck at the centre of a glass slide creating a small parallel gap between them. A single nematode was then transferred in a drop of distilled water to the glass slide in the centre of the gap. The nematode was then heat relaxed by passing over a flame a few times and covered with a glass coverslip. The specimen was then examined, photographed and measured using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Olympus C5060Wz camera [91]. After recording morphological data, the specimen was recovered from the slide by adding a few drops of water from one end of the gap and collecting the nematode that was flushed out on the other end of the gap. The recovered specimens were subsequently used to extract genomic DNA as described in the next section.

A small nematode suspension of the remaining nematodes was heated in an embryo glass dish with a few drops of Trump's fixative ((2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Sorenson buffer (sodium phosphate buffer at pH = 7.5)) in a microwave (700 Watts) for 3–4 sec and leaving it at room temperature for 1 h and at 4 ◦C for 24 h and followed by gradually transferring to anhydrous glycerine, as described in Singh et al. [92]. The fixed specimens were then mounted in glycerine on glass slides and were studied as above using the camera-equipped microscope. Species identification was carried out both at Nematology Research Unit of Ghent University and National Plant Protection Organization, Wageningen, the Netherlands.

For scanning electron microscopy, specimens fixed in Trump's fixative 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) at 12 kV [92].

#### *4.3. Extraction of DNA, PCR and Sequencing*

Genomic DNA was extracted from individual heat relaxed nematode specimen, which had been morphologically vouchered. The cuticle of the specimen was punctured using a fine entomological pin mounted on a thin bamboo stick, which was also used as nematode picking tool and the nematode was subsequently transferred to a PCR tube with 20 µL of worm lysis buffer (50 mM KCl, 10 mM Tris at pH = 8.3, 2.5 mM MgCl2, 0.45% NP 40 (Tergitol Sigma), 0.45% Tween 20) and incubated at −20 ◦C (at least 10 min). This was followed by adding 1 µL proteinase K (1.2 mg/mL), incubation at 65 ◦C (1 h) and 95 ◦C (10 min) and ending by centrifuging the mixture at 14,000 rpm for 1 min [92]. Genomic DNA from a single nematode was used to amplify four DNA fragments—D2-D3 of 28S, partial ITS and partial 18S rRNA gene and partial *COI* gene of mtDNA. PCR and sequencing were completed in two laboratories: Nematology Research Unit, Gent University, Belgium and Nematology lab, Plant Pest Diagnostic Center, CDFA, Sacramento, California, USA. For PCR amplifications of the D2-D3 of 28S, ITS and 18S rRNA gene sequences, the primer pairs D2A: 50 -ACA AGT ACC GTG AGG GAA AGT TG-30/D3B: 50 -TCC TCG GAA GGA

ACC AGC TAC TA-30 [93], Vrain2F: 50 -CTT TGT ACA CAC CGC CCG TCG CT-30/Vrain2R: 5 0 -TTT CAC TCG CCG TTA CTA AGG GAA TC-30 [94] or TW81: 50 -GTT TCC GTA GGT GAA CCT GC-30/AB28: 50 -ATA TGC TTA AGT TCA GCG GGT-30 [95], and SSU18A: 5 0 -AAA GAT TAA GCC ATG CAT G-30/SSU26R: 50 -CAT TCT TGG CAA ATG CTT TCG-3 0 [96] were used, respectively, with thermal profiles described by Singh et al. [97] and Tahna Maafi et al. [98]. Partial *COI* gene was amplified using the primer pairs JB3: 50 -TTT TTT GGG CAT CCT GAG GTT TAT-30/JB4.5: 50 -TTT TTT GGG CAT CCT GAG GTT TAT-30 according to Bowles et al. [99] or COI-F5: 50 -AAT WTW GGT GTT GGA ACT TCT TGA AC-30/COI-R9: 50 -CTT AAA ACA TAA TGR AAA TGW GCW ACW ACA TAA TAA GTA TC-30 according to Powers et al. [100]. The PCR products were purified [101] and sent to Macrogen [102] and Genewiz [103] for sequencing. New sequences were assembled using Geneious Prime 2020.0.5 and deposited to the GenBank under the accession numbers given in Table 1.

#### *4.4. Phylogenetic and Species Delimitation Analysis*

The new sequences for each gene (D2-D3 of 28S, ITS, 18S rRNA and *COI*) were aligned using Clustal X 1.83 [104] with their corresponding published gene sequences [22,24,32–39,42–50]. Outgroup taxa for each dataset were chosen based on previously published data [105]. Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.1.2 [106] under the GTR + I + G model. BI analysis was initiated with a random starting tree and was run with four chains for 1.0 <sup>×</sup> <sup>10</sup><sup>6</sup> generations for 18S and ITS rRNA gene alignments, 5.0 <sup>×</sup> <sup>10</sup><sup>6</sup> generations for D2-D3 of 28S rRNA gene alignment and 9.0 <sup>×</sup> <sup>10</sup><sup>6</sup> generations for *COI* gene alignment. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The log-likelihood values of the sample points stabilised after approximately 1,000 generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PPs) are given on appropriate clades. Sequence analyses of alignments were performed with PAUP∗ 4b10 [107]. Pairwise divergences between taxa were computed as absolute distance values and as percentage mean distance values based on whole alignment with adjustments for missing data.

The *COI* gene alignments for *P. straeleni* and *P. enigmaticus* were used to construct phylogenetic network estimation using statistical parsimony, as implemented in POPART software [108].

Species delimitation of *Paratylenchus* in this study was undertaken using an integrated approach that considered morphological and morphometric evaluations combined with molecular-based phylogenetic inference (tree-based methods) and coalescent-based molecular species-delimitation methods. Putative species boundaries on a given phylogenetic input tree were inferred using a Bayesian implementation of the Poisson tree processes (bPTP) method [76] and using the generalized mixed-yule coalescent (GMYC) method [75]; see Qing et al. [80] for more details. Ultrametric trees were constructed using BEAST v1.10.4 [109] based on D2-D3, ITS, 18S and *COI* sequences, respectively. Default prior distributions were used and analyses were run for 1 <sup>×</sup> <sup>10</sup><sup>7</sup> generations, saving trees every <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>3</sup> generations. The final trees were produced after removing 2,000 samples (20%) as burn-ins, and the maximum clade credibility tree was calculated using TreeAnnotator 1.10.4 [109]. Finally, for the bPTP method, an unrooted Bayesian 50% majority-rule consensus tree, containing only ingroups and unique haplotypes, was uploaded on the online server [110] and 1 <sup>×</sup> <sup>10</sup><sup>5</sup> Markov chain Monte Carlo (MCMC) generations were performed. The same tree was also uploaded on the GMYC web server [111] using the single threshold method.

#### **5. Conclusions**

An integrative approach by linking DNA sequences and morphological characters represents the best way to move nematode taxonomy forward. Creating this link involves

the rigorous generation of multiple DNA sequences from individual morphologically vouchered nematode specimens, which, in the current study, resulted in the first molecular characterisations for five species, the first *COI* sequences for eight species and, most importantly, the reassignments of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 *COI* gene sequences, which had been unidentified or misidentified.

This study showed that *Paratylenchus* is a case in point, representing an incredibly diverse yet morphologically minimalistic plant-parasitic genus. Our recommendations for future protocol in *Paratylenchus* taxonomy, which are also valid for integrative nematode taxonomy, are: (1) to include SEM in new descriptions or re-descriptions; (2) to use juvenile and male traits after their conspecificity is irrefutably proven using molecular data; (3) to unequivocally link elaborate morphological data with both nuclear D2-D3 of 28S rRNA and mitochondrial *COI* gene sequences; (4) to employ caution when performing molecular identification using partial 18S rRNA gene fragments only; (5) to make use of the promising molecular species delineation methods to establish species boundaries, but base this on multilocus data and merely use it as one of the elements of integrative taxonomy.

**Author Contributions:** Conceptualisation, P.R.S., W.B. and S.A.S.; methodology, P.R.S., M.C., W.B. and S.A.S.; software, P.R.S., W.B. and S.A.S.; validation, P.R.S., G.K., W.B. and S.A.S.; formal analysis, P.R.S., G.K., W.B. and S.A.S.; investigation, P.R.S., G.K., W.B. and S.A.S.; resources, P.R.S., G.K., M.C., W.B. and S.A.S.; data curation, P.R.S., G.K. and S.A.S.; writing—original draft preparation, P.R.S.; writing—review and editing, W.B., S.A.S. and G.K.; visualisation, P.R.S. and M.C.; supervision, W.B. and G.K.; project administration, W.B.; funding acquisition, W.B. and S.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ghent University, Belgium, UGent BOF01D05918 and the Plant Pest Diagnostic Center, CDFA, USA.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated during and/or analyzed during the currentstudy are available from the corresponding author on reasonable request.

**Acknowledgments:** The authors thank E. Van den Berg, A. Westphal, J. Zogfar, C.M. Etongwe and A.W. Aseffa for providing nematode specimens and soil samples.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode,** *Meloidogyne hapla***, Using Recombinase Polymerase Amplification Assays**

**Sergei A. Subbotin \* and Julie Burbridge**

Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA 95832, USA; burbridge.julie@gmail.com

**\*** Correspondence: sergei.subbotin@cdfa.ca.gov

**Abstract:** Rapid and reliable diagnostics of root-knot nematodes are critical for selections of effective control against these agricultural pests. In this study, recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the northern root-knot nematode, *Meloidogyne hapla.* The RPA assays using TwistAmp® Basic, TwistAmp® exo and TwistAmp® nfo kits (TwistDx, Cambridge, UK) allowed for the detection of *M. hapla* from crude extracts of females, eggs and juveniles without a DNA extraction step. The results of the RPA assays using real-time fluorescence detection (real-time RPA) in series of crude nematode extracts showed reliable detection after 13 min with a sensitivity of 1/100 of a second-stage juvenile and up to 1/1000 of a female in reaction tubes. The results of the RPA assays using lateral flow dipsticks (LF-RPA) showed reliable detection within 30 min with a sensitivity of 1/10 of a second-stage juvenile and 1/1000 of a female in reaction tubes. The RPA assay developed here is a successful tool for quick, accurate and sensitive diagnostics of *M. hapla*. The application of the LF-RPA assay has great potential for diagnosing infestation of this species in the lab, field or in areas with a minimal laboratory infrastructure.

**Keywords:** diagnostics; root-knot nematode; recombinase polymerase amplification

#### **1. Introduction**

The northern root-knot nematode, *Meloidogyne hapla* is one of the four most common root-knot nematode species worldwide. This nematode is extremely polyphagous, attacking a wide variety of crops and weeds. *Meloidogyne hapla* causes important economic losses for several horticultural, vegetable and pasture crops, including carrots, lettuce, lucerne, onion, potato, rose, sugarbeet, strawberry, white clover and others [1,2].

Accurate and rapid identification of nematodes is essential for their control. It has been shown that sequences of nuclear ribosomal genes: 18S rRNA, ITS rRNA, the D2–D3 of 28S rRNA, IGS rRNA and mitochondrial genes: *COII*-16S rRNA fragment, *COI* and *COII* clearly differentiate *M. hapla* from all other root-knot nematodes [3]. Several specific primers have been designed for the diagnostics of this species using conventional PCR [4–7]. Several authors also developed a TaqMan real-time PCR assay with species-specific primers for the detection of *M. hapla* from root galls and soil samples [8–12]. Recently, Peng et al. [13] developed loop-mediated isothermal amplification methods (LAMP) combined with a Flinders Technology Associates card for the identification of *M. hapla*.

Recombinase polymerase amplification (RPA), an isothermal in vitro nucleic acid amplification technique, has recently appeared as a novel molecular technology for simple, robust, rapid, reliable, and low-resource diagnostics. RPA represents a hugely versatile alternative to PCR [14–16]. RPA uses a highly efficient displacement polymerase that amplifies a few copies of target nucleic acid in 20 min at a constant temperature (37–42 ◦C). It does so by utilizing three core enzymes: recombinase, single-stranded binding protein (SSB), and strand-displacing polymerase. The recombinase enzyme forms a complex with

**Citation:** Subbotin, S.A.; Burbridge, J. Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode, *Meloidogyne hapla*, Using Recombinase Polymerase Amplification Assays. *Plants* **2021**, *10*, 336. https://doi.org/10.3390/ plants10020336

Academic Editor: Zafar Handoo Received: 15 January 2021 Accepted: 8 February 2021 Published: 10 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

a primer to facilitate their binding to the targeted DNA template. Then, the SSB binds to the displaced strands of DNA and prevents the displacement of the recombinase–primer complex by branch migration. The strand-displacing polymerase then recognises the bound recombinase–primer complex and initiates DNA synthesis. Like PCR, RPA produces an amplicon constrained in size to the binding sites of the primers. The advantages of RPA include highly efficient and rapid amplification and a low constant operating temperature. RPA products can be detected by agarose gel electrophoresis or carried out by using fluorescent probes in real time (real-time RPA) or lateral flow strips (LF-RPA). RPA assays show high sensitivity and specificity for detecting various plant viruses, bacteria, fungi, vertebrate parasitic trematodes, nematodes and other organisms [17–22]. Real-time RPA detection assay of plant parasitic nematodes was first designed and published by Subbotin et al. [23] for *Meloidogyne enterolobii.* RPA assays were also developed for *Meloidogyne javanica*, *M. arenaria* and *M. incognita* [24,25] and *Bursaphelenchus xylophilus* [26,27]. Recently, Song et al. [28] described diagnostics of *Meloidogyne hapla* using RPA combined with a lateral flow dipstick assay, where species-specific primers and a probe were designed based on the effector gene *16D10* sequence. This LF-RPA assay allows detecting *M. hapla* from infested plant roots and soil samples and the entire detection process can be completed within 1.5 h.

In our study, we developed real-time RPA and LF-RPA assays for the detection of *Meloidogyne hapla* using crude nematode and infected plant root extracts, with results within 13–30 min. Species-specific primers and probes were designed based on the IGS ribosomal RNA gene sequence.

#### **2. Results**

#### *2.1. RPA Primers and Probe Design*

All available sequences of the IGS rRNA for *M. hapla* and other *Meloidogyne* were downloaded from the Genbank and aligned with ClustalX. Several regions with high sequence dissimilarity between *M. hapla* and other *Meloidogyne* were assessed and several species-specific *M. hapla* candidate primers sets and probes were manually designed. The Blastn search of these species-specific candidate primer sequences and probe sequences showed high similarity (100%) only with the IGS rRNA fragments of *M. hapla* deposited in the GenBank.

#### *2.2. RPA Detection*

Nine primer combination candidate sets were screened for the best performance under the same RPA conditions. The species-specific forward F3-IGS-Hapl and the species-specific reverse R3-IGS-Hapl primers were found to be optimal with clearly visible bands and had no cross-reactions with other root-knot nematodes (Table 1). The final sequences of primers and probes used for the assays are listed in Table 2 and are indicated in the IGS rRNA gene alignment in Figure 1. This primer set reliably and specifically amplified the target gene fragment, approximately 164 bp in length from the IGS region (Figure 2) and was also confirmed by a direct sequencing of the product. Additional non-specific weak additional bands having other sizes were observed sometimes in experiments with *M. hapla* as well as samples with other root-knot nematode species (data not shown).


**Table 1.** Samples of *Meloidogyne hapla* and other root-knot nematodes tested in the present study.

**Table 2.** RPA primers and probe for amplification of *Meloidogyne hapla* DNA.


\* FAM—fluorophore, THF—tetrahydrofuran, BHQ—quencher, C3—spacer block.

#### *2.3. Real-Time RPA Detection Assays*

Using the results of nine experimental runs, which included positive and negative controls with water and non-target DNA, the threshold level for reliable *M. hapla* detection was established as equal to 8 cycles (~3 min) with a baseline of 250,000 (∆Rn) fluorescence using the TwistAmp® exo kit on the Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System (Figures 3 and 4A). Samples that produced an exponential amplification curve above the threshold were considered as positive for *M. hapla* and below the threshold were considered as negative. Detection of *M. hapla* was confirmed with all samples.

*Plants* **2021**, *10*, x FOR PEER REVIEW 3 of 12

curve above the threshold were considered as positive for *M. hapla* and below the threshold were considered as negative. Detection of *M. hapla* was confirmed with all samples.

curve above the threshold were considered as positive for *M. hapla* and below the threshold were considered as negative. Detection of *M. hapla* was confirmed with all samples.

**Figure 1.** The fragment of alignment of the IGS rRNA gene sequences for several root-knot nematodes, *Meloidogyne*, with the positions of recombinase polymerase amplification (RPA) primers and probes used in the present study. **Figure 1.** The fragment of alignment of the IGS rRNA gene sequences for several root-knot nematodes, *Meloidogyne*, with the positions of recombinase polymerase amplification (RPA) primers and probes used in the present study. **Figure 1.** The fragment of alignment of the IGS rRNA gene sequences for several root-knot nematodes, *Meloidogyne*, with the positions of recombinase polymerase amplification (RPA) primers and probes used in the present study.

**Figure 2.** RPA amplicon of the partial IGS rRNA gene on agarose gel. Lanes: M: 100 bp DNA marker (Promega, Madison, WI, USA); 1: RPA amplicon obtained after 24 min at 39 °C with F3- IGS-Hapl and R3-IGS-Hapl primers using TwistAmp® Basic kit; 2: negative control. **Figure 2.** RPA amplicon of the partial IGS rRNA gene on agarose gel. Lanes: M: 100 bp DNA marker (Promega, Madison, WI, USA); 1: RPA amplicon obtained after 24 min at 39 °C with F3- IGS-Hapl and R3-IGS-Hapl primers using TwistAmp® Basic kit; 2: negative control. **Figure 2.** RPA amplicon of the partial IGS rRNA gene on agarose gel. Lanes: M: 100 bp DNA marker (Promega, Madison, WI, USA); 1: RPA amplicon obtained after 24 min at 39 ◦C with F3-IGS-Hapl and R3-IGS-Hapl primers using TwistAmp® Basic kit; 2: negative control.

**Figure 3.** RPA assays using real-time fluorescent detection with examples of amplification plots. (**A**) Specificity assay with DNA samples of *Meloidogyne* spp. and crude second-stage juvenile (J2) extracts of *M. hapla.* Line: 1: *M. hapla* (CD2461); 2: *M. hapla* (VW9); 3: *M. hapla* (C44); 4: *M. incognita* (CD3038); 5: *M. arenaria* (CD3100); 6: *Meloidogyne naasi* (CD3381); 7: *M. javanica* (isolate 40); 8 and 9: negative control; (**B**) sensitivity assay with a dilution series of a crude J2 extract of *M. hapla*, line: 1: 1 J2 per tube; 2: 1/10 J2 per tube; 3: 1/100 J2 per tube; 4: 1/1000 J2 per tube; 5: negative control; (**C**) crude extract of *M. hapla* with or without crude extracts of non-target nematodes. Line: 1 and 4: 1 J2 per tube; 2, 5L 1 J2 with 20 non-target nematodes per tube; 3 and 6: 1 J2 with 10 non-target nematodes per tube; 7 and 8: 1 J2 per tube containing half of a reaction mixture; 9: negative control; (**D**) testing of crude extracts of *M. hapla.* Line: 1: 1 J2 per tube; 2 and 4: extracts from infected plant roots containing females with egg-masses; 3 and 6: extracts from infected plant roots containing old females without egg-masses; 5: negative control. The vertical line on a graph: fluorescence ∆Rn. ∆Rn is calculated at each cycle as DRn (cycle) = Rn (cycle)—Rn (baseline), where Rn = normalized reporter. The horizontal line on a graph: cycles, each cycle = 20 s. The RPA assay was tested for specificity using DNA extracted from several root-knot **Figure 3.** RPA assays using real-time fluorescent detection with examples of amplification plots. (**A**) Specificity assay with DNA samples of *Meloidogyne* spp. and crude second-stage juvenile (J2) extracts of *M. hapla.* Line: 1: *M. hapla* (CD2461); 2: *M. hapla* (VW9); 3: *M. hapla* (C44); 4: *M. incognita* (CD3038); 5: *M. arenaria* (CD3100); 6: *Meloidogyne naasi* (CD3381); 7: *M. javanica* (isolate 40); 8 and 9: negative control; (**B**) sensitivity assay with a dilution series of a crude J2 extract of *M. hapla*, line: 1: 1 J2 per tube; 2: 1/10 J2 per tube; 3: 1/100 J2 per tube; 4: 1/1000 J2 per tube; 5: negative control; (**C**) crude extract of *M. hapla* with or without crude extracts of non-target nematodes. Line: 1 and 4: 1 J2 per tube; 2, 5L 1 J2 with 20 non-target nematodes per tube; 3 and 6: 1 J2 with 10 non-target nematodes per tube; 7 and 8: 1 J2 per tube containing half of a reaction mixture; 9: negative control; (**D**) testing of crude extracts of *M. hapla.* Line: 1: 1 J2 per tube; 2 and 4: extracts from infected plant roots containing females with egg-masses; 3 and 6: extracts from infected plant roots containing old females without egg-masses; 5: negative control. The vertical line on a graph: fluorescence ∆Rn. ∆Rn is calculated at each cycle as DRn (cycle) = Rn (cycle)—Rn (baseline), where Rn = normalized reporter. The horizontal line on a graph: cycles, each cycle = 20 s.

nematodes. These nematodes include: *Meloidogyne arenaria*, *M. baetica*, *M. christiei*, *M. enterelobii*, *M. floridensis*, *M. incognita*, *M. javanica*, *M. naasi* and *M. nataliei.* The RPA results using real-time fluorescent detection showed high specificity to *M. hapla* only and no cross-reactions were observed against other root-knot nematode species (Figure 3A).

The sensitivity assay was designed for evaluation of the detection limit. Variants with serial dilutions (1, 1/10, 1/100, 1/1000 and 1/10,000 per reaction tube) of crude nematode extractions were obtained from second-stage juveniles (J2s) or females without eggmasses. The reliable detection level of *M. hapla* was estimated at 1/100 of one J2 per a RPA reaction tube (Figure 3B). The detection level of *M. hapla* females varied among replicates and reached 1/100, 1/1000 and 1/10,000 of a female for three, two and one replicates, re-

spectively (Figure 4A).

*Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 12

**Figure 4.** RPA sensitivity assays using (**A**) real-time fluorescent detection and (**B**) lateral flow strips. A dilution series of three crude young females (without egg-masses) extracts of *M. hapla*. Line (Strip): 1, 5, 9: 1/10 female per tube; 2, 6, 10: 1/100 female per tube; 3, 7, 11: 1/1000 female per tube; 4, 8, 12: 1/10,000 female per tube; 13: positive control; 14: negative control. Control (upper) and test (lower) lines are indicated by arrows. The detection of J2 for *M. hapla* was confirmed in the presence of background crude **Figure 4.** RPA sensitivity assays using (**A**) real-time fluorescent detection and (**B**) lateral flow strips. A dilution series of three crude young females (without egg-masses) extracts of *M. hapla*. Line (Strip): 1, 5, 9: 1/10 female per tube; 2, 6, 10: 1/100 female per tube; 3, 7, 11: 1/1000 female per tube; 4, 8, 12: 1/10,000 female per tube; 13: positive control; 14: negative control. Control (upper) and test (lower) lines are indicated by arrows.

observed between the variant of 1 J2 without other nematodes and the variants with 1 J2 with 10 and 20 non-target nematodes (Figure 3C). Lowering in half, a single reaction assay The RPA assay was tested for specificity using DNA extracted from several rootknot nematodes. These nematodes include: *Meloidogyne arenaria*, *M. baetica*, *M. christiei*, *M. enterelobii*, *M. floridensis*, *M. incognita*, *M. javanica*, *M. naasi* and *M. nataliei.* The RPA results using real-time fluorescent detection showed high specificity to *M. hapla* only and no cross-reactions were observed against other root-knot nematode species (Figure 3A).

extracts from at least 20 non-target nematodes. No decrease in fluorescent signals was

The sensitivity assay was designed for evaluation of the detection limit. Variants with serial dilutions (1, 1/10, 1/100, 1/1000 and 1/10,000 per reaction tube) of crude nematode extractions were obtained from second-stage juveniles (J2s) or females without egg-masses. The reliable detection level of *M. hapla* was estimated at 1/100 of one J2 per a RPA reaction tube (Figure 3B). The detection level of *M. hapla* females varied among replicates and reached 1/100, 1/1000 and 1/10,000 of a female for three, two and one replicates, respectively (Figure 4A).

The detection of J2 for *M. hapla* was confirmed in the presence of background crude extracts from at least 20 non-target nematodes. No decrease in fluorescent signals was

observed between the variant of 1 J2 without other nematodes and the variants with 1 J2 with 10 and 20 non-target nematodes (Figure 3C). Lowering in half, a single reaction assay volume showed a decrease in fluorescence signal and reaction rate (Figure 3C). These samples could be considered as positive with threshold level of 12 cycles (~6 min). *Plants* **2021**, *10*, x FOR PEER REVIEW 6 of 12

> *Meloidogyne hapla* detection was also confirmed using extracts obtained from infected tomato and pepper plant roots containing females with egg-masses. Although most replicates from extracts obtained from infected plant roots containing old females without egg-masses gave strong signals, one replicate showed no fluorescence signal (Figure 3D). volume showed a decrease in fluorescence signal and reaction rate (Figure 3C). These samples could be considered as positive with threshold level of 12 cycles (~6 min). *Meloidogyne hapla* detection was also confirmed using extracts obtained from infected tomato and pepper plant roots containing females with egg-masses. Although most rep-

#### *2.4. LF-RPA Assay* licates from extracts obtained from infected plant roots containing old females without egg-masses gave strong signals, one replicate showed no fluorescence signal (Figure 3D).

Lateral flow detection of RPA products also showed specific and sensitive results. Positive test lines on the LF strips were observed for all *M. hapla* samples, whereas samples with other nematode species showed only a control line (Figure 5A). The detection of J2 for *M. hapla* was confirmed from extracts of infected pepper roots with *M. hapla* (Figure 5B) as well as in the presence of background crude extract from 10 to 20 non-target nematodes (Figure 5C). Lowering in half, a single reaction assay volume still detected *M. hapla* samples (Figure 5C). The results of RPA assays showed reliable detection with a sensitivity of 1/10 of a J2 (Figure 5D) and 1/1000 of a female (Figure 4B) in reaction tubes. *2.4. LF-RPA Assay*  Lateral flow detection of RPA products also showed specific and sensitive results. Positive test lines on the LF strips were observed for all *M. hapla* samples, whereas samples with other nematode species showed only a control line (Figure 5A). The detection of J2 for *M. hapla* was confirmed from extracts of infected pepper roots with *M. hapla* (Figure 5B) as well as in the presence of background crude extract from 10 to 20 non-target nematodes (Figure 5C). Lowering in half, a single reaction assay volume still detected *M. hapla* samples (Figure 5C). The results of RPA assays showed reliable detection with a sensitiv-

ity of 1/10 of a J2 (Figure 5D) and 1/1000 of a female (Figure 4B) in reaction tubes.

**Figure 5.** Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. (**A**) Specificity assay with DNA samples of *Meloidogyne* spp. and crude J2 extracts of *M. hapla.* Strip: 1 and 2: *Meloidogyne* sp.1 (CD3380); 3: *M. naasi* (CD3381); 4: *M. baetica* (CD3382); 5: *Meloidogyne* sp.2 (CD3383); 6 and 7: *M. hapla*, (CD3384); 8: *M. arenaria* (CD33093); 9: *M. hapla* (C44); 10: negative control; (**B**) crude extracts of *M. hapla.* Strip: 1 and 2: extracts from infected tomato roots containing plant materials and females with egg-masses; 3: 1 j2 per tube; 4: negative control; (**C**) testing of crude extract of *M. hapla* with or without crude extracts of non-target nematodes. Strip: 1 and 2: 1 J2 per tube; 3 and 4: 1 J2 with 10 non-target nematodes per tube; 5 and 6: 1 J2 with 20 non-target nematodes per tube; 7 and 8: 1 J2 per tube containing half of a reaction mixture; 9: negative control; (**D**) sensitivity assay with a dilution series of a crude j2 extract of *M. hapla*, Strip: 1: 1 J2 per tube; 2: 1/10 J2 per tube; 3: 1/100 J2 per tube; 4: 1/1000 J2 per tube; 5: negative control (upper) and test (lower) lines are indicated by arrows. **Figure 5.** Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. (**A**) Specificity assay with DNA samples of *Meloidogyne* spp. and crude J2 extracts of *M. hapla.* Strip: 1 and 2: *Meloidogyne* sp.1 (CD3380); 3: *M. naasi* (CD3381); 4: *M. baetica* (CD3382); 5: *Meloidogyne* sp.2 (CD3383); 6 and 7: *M. hapla*, (CD3384); 8: *M. arenaria* (CD33093); 9: *M. hapla* (C44); 10: negative control; (**B**) crude extracts of *M. hapla.* Strip: 1 and 2: extracts from infected tomato roots containing plant materials and females with egg-masses; 3: 1 j2 per tube; 4: negative control; (**C**) testing of crude extract of *M. hapla* with or without crude extracts of non-target nematodes. Strip: 1 and 2: 1 J2 per tube; 3 and 4: 1 J2 with 10 non-target nematodes per tube; 5 and 6: 1 J2 with 20 non-target nematodes per tube; 7 and 8: 1 J2 per tube containing half of a reaction mixture; 9: negative control; (**D**) sensitivity assay with a dilution series of a crude j2 extract of *M. hapla*, Strip: 1: 1 J2 per tube; 2: 1/10 J2 per tube; 3: 1/100 J2 per tube; 4: 1/1000 J2 per tube; 5: negative control (upper) and test (lower) lines are indicated by arrows.

#### **3. Discussion**

Polymerase chain reaction is considered the gold standard of molecular detection, however, this method is only available in a laboratory with thermal cycling equipment. In this work, we have developed an affordable, simple, fast and sensitive real-time RPA and LF-RPA assays to detect *M. hapla* from nematode specimens extracted from plant and soil samples. An LF-RPA assay can be performed in a field condition without any special equipment or in areas with a minimal laboratory infrastructure.

Song et al. [28] described the LF-RPA diagnostic assay of *M. hapla* using species-specific primers and a probe designed using the effector gene *16D10* sequence. Authors stated that the entire detection process can be completed within 1.5 h, including 30–60 min for DNA extraction, 20 min for the RPA reaction, and 3–5 min for visual detection on the LF strips [28]. In our assay, the species-specific primers and probes were designed using the IGS rRNA gene sequence. The entire detection process for the LF-RPA assay can be completed within approximately 30 min, including 4 min for crude nematode extract preparation, 20 min (4 + 16) for the RPA reaction, 1 min for mixing and centrifugation of tubes, and 5 min for visual detection on the LF strips. The entire detection process for real-time RPA assay can be completed within approximately 13 min, including 4 min for crude nematode extract preparation, 8 min (5 + 3) for the RPA reaction with 1 min for mixing and centrifugation of tubes. This calculation does not include the time for preparation of the RPA reaction mixture.

In our study the IGS rRNA gene was selected for the RPA assays because of its high copy number and because previously published PCR studies have demonstrated its usefulness to distinguish *Meloidogyne* species [7,29,30]. Zhang et al. [16] noticed that different DNA targets are likely to have extremely different amplification efficiencies, even sharing a series of common characteristics including GC content, primer melting temperature and RPA product length. These authors also concluded that primers are the most important determinant for RPA performance including sensitivity, specificity and reaction rate. Although amplicons obtained from fragments of effector gene *16D10* and the IGS rRNA gene are comparable (148 vs. 164 bp) in a length, it seems that the RPA reaction rate is higher for the IGS rRNA gene than the effector gene fragment. The RPA assays developed based on the IGS rRNA gene are more sensitive for detection than assays based on the effector gene fragment.

Song et al. [28] reported about 1/1000 female (after DNA extraction with proteinase K) as the detection limit of the LF-RPA assay, whereas in our LF-RPA assay, the detection limit can reach up to 1/10,000 (without a special DNA extraction step). Our RPA assays for *M. hapla* also showed higher amplification rates compared with similar assays developed for *M. enterolobii*, in which species-specific primers were also designed based on the IGS rRNA gene [23]. The threshold level for the reliable *M. enterolobii* detection was established as equal to 30 cycles (=10 min) and a baseline of 500,000 (∆Rn) fluorescence level with the TwistAmp® exo kit and Applied Biosystems™ QuantStudio™ Flex Real-Time PCR System, whereas in our present study for *M. hapla*, the threshold level was estimated equal to 8 cycles (~3 min) and a baseline of 250,000.

The real-time RPA and LF-RPA assays developed in our study allowed the detection of J2, young females without and with eggs-masses. The old and dead females without body contents might not be always detectable using this method. The results of our study estimated that the reliable detection of RPA assays using real-time fluorescence were 1/100 of J2 or female and using lateral flow dipsticks were 1/10 of a J2 and 1/1000 of a female. However, in some replicates the detection limit can reach up to 1/10,000 of a female in reaction tube. Reproducibility of the assays in low concentrations of nematode extracts, extracts from old females or directly from soil samples should be carefully tested further to understand factors, which might have an influence on the performing stability of RPA reactions. RPA diagnostics of root-knot nematodes has several other important advantages over PCR methods. The first advantage is that crude nematode extracts or crude extracts from nematode-infected tomato and pepper plant tissues can be directly used for RPA

assays, whereas PCR assays require a DNA extraction step with special treatment of these extracts before use. The second advantage of RPA assays is that results are available in 8–20 min, whereas the results of PCR assays can be observed in 1.5–3 h. The third advantage is higher sensitivity levels of RPA detection over PCR methods; the RPA assay is 10 or 100 times more sensitive than PCR.

However, the application of RPA assays for nematode diagnostics may still face several problems, with cost being a major consideration. Factors affecting the expense of assays depends on the pest, reagent costs, requirement for equipment, infrastructure capacity, employee wages and numbers of samples for testing, among others. The RPA reagents and kits are presently manufactured by only one company, TwistDx, Inc., making the cost of the RPA assay relatively higher than other PCR assays. Reagent costs for RPA assays are currently in range of USD 4.3–5.5 per sample [15] which is higher than for conventional and real-time PCR. Lillis et al. [31] showed that lowering the assay volume from 50 µL, which is the recommended in the manufacturer's protocol, to 5 µL showed similar sensitivity. Our experiments also revealed acceptable diagnostic performance when reducing the reaction volume by half. This approach could be used in cases of resource limitations. It has been noticed that special attention should be paid to the potential of cross-contamination due to the high sensitivity of this reaction. The requirement for use of clean gloves, tubes, and pipets should be carefully considered during the use of RPA assays in a field condition. Thus, RPA has the potential to be a promising alternative to PCR and other methods for rapid detection of nematodes. This assay requires minimal sample preparation, making it ideal for use in the lab, the field, or minimal laboratory infrastructure.

#### **4. Materials and Methods**

#### *4.1. Nematode Samples*

Five isolates of *Meloidogyne hapla* were obtained for RPA assay development. Secondstage juveniles (J2s) and females were extracted from the root or soil samples. The D2–D3 expansion segments of the 28S rRNA gene were sequenced from each isolate to confirm its identity. DNA of several root-knot nematodes, *M. arenaria*, *M. baetica*, *M. christiei*, *M. enterelobii*, *M. floridensis*, *M. incognita*, *M. javanica*, *M. naasi* and *M. nataliei* were also used in specificity experiments (Table 1). These species were also identified by molecular methods. Free-living and plant parasitic nematodes from several field samples collected in California were extracted using the centrifugal flotation method and their extracts were used as background non-target DNA.

#### *4.2. Nematode Extracts*

Second-stage juveniles or females of *M. hapla* were placed in distilled water on a microscope slide. The nematodes were cut using a dental needle under a stereo microscope and put into a 0.2 mL PCR tube with a total volume of 10 µL. This stock crude extract was used to make a series of dilutions sequentially: 1:2, 1:4, 1:8, 1:10, 1:16, 1:100, 1:1000 and 1:10,000 in water. Several extracts were prepared: (i) J2s; (ii) J2s with other non-target nematodes; (iii) female and (iv) plant gall tissue with one or more females and egg-masses. Crude extract of plant gall tissue with nematodes and crude extract of several hundred non-target nematodes soil free-living and plant parasitic nematodes were also obtained by crushing the samples on a microscope slide using a plastic pipe tip or dental needle.

#### *4.3. RPA Primer Design and Testing*

A total of three forward and three reverse RPA primers specific to *M. hapla* were manually designed based on species sequence polymorphisms in the IGS rRNA gene. Primers were synthesized by Integrated DNA Technologies, Inc. (Redwood City, CA, USA). Nine primer sets were screened in different combinations using the TwistAmp® Basic kit (TwistDx, Cambridge, UK). Reactions were prepared according to the manufacturer's instructions. The lyophilized reaction pellets were suspended in 29.5 µL of the rehydration buffer, 2.4 µL of each forward and reverse primers (10 µM) (Table 2), 1 µL of

the DNA template or nematode extract and 12.2 µL of distilled water. For each sample, 2.5 µL of 280 mM magnesium acetate was added to the lid of the tube and the lids were closed carefully. The tubes were inverted 10–15 times and briefly centrifuged to initiate reactions simultaneously. Tubes were incubated at 39 ◦C (4 min) in a MyBlock Mini Dry Bath (Benchmark Scientific, Edison, NJ, USA) and then they were inverted 10–15 times, briefly centrifuged and returned to the incubator block (39 ◦C) for 20 min. Sample tubes were then placed in a freezer to stop the reaction. Amplification products were purified with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Five µL of purified product were run in a 1% TAE (Tris-acetate-EDTA)-buffered agarose gel (100 V, 60 min) and visualized with Gel Green stain. Amplification products were directly sequenced by Genewiz (San Francisco, CA, USA) using amplification primers.

#### *4.4. Real-Time RPA Assay*

Two TwistAmp® exo probes were designed according to the manufacturer's instructions and were synthesized by Biosearch Technologies, Inc. (Petaluma, CA, USA). Two probes were tested and only one probe (Probe-hapla-exo1) was selected for the assay (Table 2) based on best amplification performance. The real time detection of RPA assay products was accomplished using the TwistAmp® exo kit (TwistDx, Cambridge, UK). The lyophilized reaction pellets were suspended in 29.5 µL of the rehydration buffer, 2.1 µL of each forward and reverse primers (10 µM) (Table 2), 0.6 µL of the probe (10 µM), 1 µL of the DNA template or nematode extract and 12.2 µL of distilled water. Magnesium acetate in a volume of 2.5 µL was added to the lid of each tube, the lids were carefully closed, tubes were inverted 10–15 times and briefly centrifuged. The reaction tubes were incubated at 39 ◦C for 5 min, then inverted 10–15 times to mix, and briefly centrifuged. The tubes were immediately placed in Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System to incubate at 39 ◦C for 15 min. The fluorescence signal was monitored in real time and measured every 20 s (cycle) using the fluorophore (FAM) channel. A positive control using *M. hapla* extract (one J2 per reaction tube) and negative control without any nematode DNA were included in each run. Two or three replicates of each variant across several runs were performed for sensitivity and specificity experiments.

#### *4.5. LF-RPA Assay*

Two TwistAmp® nfo probes were designed according to the manufacturer's instructions and tested in the same conditions. Only one probe (Probe-hapla-nfo1) was selected for the assay based on the best visualization results. The LF-RPA assay products were accomplished using the TwistAmp® nfo kit (TwistDx, Cambridge, UK). The reaction mixture for each RPA assay was prepared according to the manufacturer's instructions: the lyophilized reaction pellets were suspended in 29.5 µL of the rehydration buffer, 2.1 µL of each forward and reverse primers (10 µM), 0.6 µL of the probe (10 µM), 1 µL of the DNA template or nematode extract and 12.2 µL of distilled water. Magnesium acetate in a volume of 2.5 µL was added to the lid of each tube, the lids were carefully closed, and the tubes were inverted 10–15 times and briefly centrifuged. The reaction tubes were incubated at 39 ◦C for 4 min, then inverted 10–15 times to mix, briefly centrifuged and returned to the incubator block at 39 ◦C for 16 min. The tubes were placed in the freezer to stop the reaction. For visual analysis with Milenia® Genline Hybridetect-1 strips (Milenia Biotec GmbH, Giessen, Germany), testing solution containing 48 µL of HybriDetect assay buffer and 12 µL of the sample RPA product was prepared in a 0.5 mL PCR tube. Ten µL of the testing solution was placed directly onto the sample area of the dipstick. Dipsticks were placed upright into 100 µL of the assay buffer and visual results were observed within 5 min. The amplification product was indicated by the development of a colored test line, and/or a separate control line to confirm that the system worked properly (Figures 4B and 5). Two or three replicates of each variant were performed for sensitivity and specificity experiments. **Author Contributions:** Conceptualization, S.A.S.; methodology, S.A.S.; software, J.B. and S.A.S.; validation, J.B. and S.A.S.; formal Analysis, J.B. and S.A.S.; investigation, J.B. and S.A.S.; resources, J.B. and S.A.S.; data Curation, J.B. and S.A.S.; writing—original draft preparation, S.A.S.; writing—review and editing, J.B. and S.A.S.; visualization, J.B. and S.A.S.; supervision, S.A.S.; project administration, S.A.S.; funding acquisition, S.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was sponsored by the Specialty Crop Block Grant Program (USDA Project Number: AM190100XXXXG008; CDFA Grant Number: 19-0001-035-MU).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated during and/or analyzed during the currentstudy are available from the corresponding author on reasonable request.

**Acknowledgments:** The authors thank Álvarez-Ortega, S., Brito, J., Chizhov, V.N., Castillo, P., Inserra, R.N., Roberts, P. and Williamson V. for providing the root-knot nematode samples.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Species Diversity of Pin Nematodes (***Paratylenchus* **spp.) from Potato Growing Regions of Southern Alberta, Canada**

**Maria Munawar <sup>1</sup> , Dmytro P. Yevtushenko 1,\*, Juan E. Palomares-Rius <sup>2</sup> and Pablo Castillo <sup>2</sup>**

> <sup>1</sup> Department of Biological Sciences, University of Lethbridge, 4401 University Drive W, Lethbridge, AB T1K 3M4, Canada; maria.munawar@uleth.ca


http://zoobank.org/urn:lsid:pub:39C84EDC-15ED-491E-9373-8876D34C35ED

**Abstract:** Pin nematodes (*Paratylenchus* spp.) are polyphagous parasitic species with a wide host range and geographical distribution; their diversity is unknown in the potato growing region of Alberta, Canada. The present study aims to provide morphological and molecular characterization of three pin nematode species, namely *P. neoprojectus*, *P. tateae*, and a new species, *Paratylenchus enigmaticus* sp. nov. All of them were recovered from the potato growing region of southern Alberta. The nematodes were isolated using the sieving and flotation-centrifugation method, and their morphology was assessed by light microscopy. Molecular characterization was performed using partial 18S, D2–D3 expansion domains of the 28S and ITS ribosomal genes. This study is the first report of molecular characterization of *P. tateae* and *P. neoprojectus*, being new records from southern Alberta, and two Spanish populations of *P. tateae* comprising the first report of this species in Europe. The phylogenetic analysis of the 18S, D2–D3 expansion domains of the 28S and ITS ribosomal DNA regions underscores the importance of using molecular data for accurate species identification and clarifies the status of *P. nanus* type B and *P. sheri*. Moreover, our findings will be useful to determine the impact of pin nematodes on potato production in future field research.

**Keywords:** *Paratylenchus tateae*; *Paratylenchus neoprojectus*; plant-parasitic nematode; integrative taxonomy; morphology; DNA sequencing; phylogeny; new record; new species

#### **1. Introduction**

Potato is one of the most important crops in Canada, with Alberta ranking among the top provinces producing superior quality potatoes with the highest marketable yields [1]. To maintain high standards of potato production, Alberta's farmed fields are regularly surveyed and examined for the presence of pest species. Recent reports have described the incidence of plant-parasitic nematodes (PPN) in cultivated soils of Canada [2–4].

*Paratylenchus* species are commonly known as pin nematodes. The short stylet species feed ecto-parasitically; however, some species feed endo-parasitically by gaining entry into lateral roots [5–7]. Pin nematodes are amongst the most frequently occurring PPN in Canada [8], and previous studies have reported the association of pin nematodes with forages, turf grasses, legumes, and cereal crops of Eastern and Central Canada [2,9–13]. Biological studies have indicated that females of *P. projectus* Jenkins [14] lay 1–2 eggs/day, with an average life cycle of 30–38 days at 20–28 ◦C. Additionally, several *Paratylenchus* species have a persistent survival stage (mainly the fourth stage), which helps them to maintain inoculum levels during periods of adversity [15].

*Paratylenchus* species have a wide host-range, and several short stylet species, such as *P. bukowinensis* Micoletzky [16], *P. dianthus* Jenkins and Taylor [17], *P. hamatus* Thorne and Allen [18], *P. microdorus* Andrassy [19], *P. neoamblycephalus* Geraert [20], *P. shenzhenensis*

**Citation:** Munawar, M.;

Yevtushenko, D.P; Palomares-Rius, J.E; Castillo, P. Species Diversity of Pin Nematodes (*Paratylenchus* spp.) from Potato Growing Regions of Southern Alberta, Canada. *Plants* **2021**, *10*, 188. https://doi.org/ 10.3390/plants10020188

Received: 26 December 2020 Accepted: 18 January 2021 Published: 20 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Wang, Xie, Li, Xu, Yu, and Wang [21] and *P. projectus*, cause varying degrees of damage to their hosts, including root injury and poor plant development, consequently decreasing yield and plant longevity [7,22].

Currently, the genus contains over 100 species, with only 11 reported in Canada [23,24]. *Paratylenchus* species are among the smallest PPN and this, together with their apparent similarities with other related species, makes them challenging to study and identify [25]. During a survey of potato fields, we isolated three *Paratylenchus* species. Preliminary examination revealed that all the species have advulval flaps, 4 lateral lines, and short stylets (<40 µm).

As several short stylet pin nematodes species are considered to be plant-pathogenic [22], we performed morphological/morphometrical and molecular studies on these *Paratylenchus* populations and identified them as *P. neoprojectus* Wu and Hawn [26], *P. tateae* Wu and Townshend [27], and a new *Paratylenchus* sp. that we named *P. enigmaticus* sp. nov. As the diversity of pin nematode species associated with potato growing areas of Alberta is largely unknown, the aims of the present work were to: (i) characterize the populations of *P. tateae*, *P. neoprojectus*, and *P. enigmaticus* sp. nov. found in potato growing areas of southern Alberta; (ii) update the pin nematode diversity record from Canada; (iii) study the phylogenetic relationship of these species with other pin nematodes. The results of this study will aid in distinguishing pathogenic forms from non-pathogenic species, and our findings will be useful in future field experiments to determine the impact of these PPN on potato production.

#### **2. Results**

#### *2.1. Description of Female Paratylenchus neoprojectus Wu and Hawn*

(Figures 1 and 2; Table 1) [26].

Body slender, ventrally arcuate with a bend in the middle of the body when heat relaxed; cuticle finely annulated; lateral field equidistant with four distinct lines; lip region rounded narrow, with anterior end flattened, continuous with the rest of the body; labial framework sclerotization weak; pharyngeal region typical paratylenchoid type; stylet rigid, straight; rounded stylet knobs; dorsal pharyngeal gland opening 5.0–6.0 µm behind stylet knobs; median pharyngeal bulb large elongate, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve bilobed; excretory pore situated at the level or middle of pharyngeal basal bulb. Hemizonid 1–2 annuli long situated just posterior to the excretory pore. The body slightly narrower posterior to vulva; ovary outstretched, well developed, in some specimens it reaches to the level of pharynx; spermatheca and crustaformeria well developed, the columnar arrangement of crustaformeria usually not discernable; spermatheca rounded; the vulva a transverse slit occupying half of the corresponding body width; vulval lips prominent, the anterior lip protrudes further than the posterior lip; vulval flaps present, but not prominent in fresh specimens; a small, rudimentary post uterine branch present along the ventral body wall; anus indistinct; tail slender, conoid, finely annulated, and gradually tapers to form a finely rounded terminus.

**Figure 1.** Light photomicrographs of *Paratylenchus neoprojectus* females. (**A**) Entire body; (**B**–**D**) pharyngeal regions; (**E**) posterior region with gonad; (**F**) lateral lines; (**G**–**K**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm; (**F**) 5 µm; (**G**–**K**) 20 µm. Arrowheads: (a) Anus; (exp) excretory pore; (v) vulva. **Figure 1.** Light photomicrographs of *Paratylenchus neoprojectus* females. (**A**) Entire body; (**B**–**D**) pharyngeal regions; (**E**) posterior region with gonad; (**F**) lateral lines; (**G**–**K**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm; (**F**) 5 µm; (**G**–**K**) 20 µm. Arrowheads: (a) Anus; (exp) excretory pore; (v) vulva.


**Table 1.** Morphometrics of *Paratylenchus neoprojectus* females and juveniles. All measurements are in µm and presented as mean ± standard deviation (range).

\* Van den Berg et al. [28] represent the measurements of *P. nanus* type B. In this study, we refer this population as *P. neoprojectus*.

**Figure 2.** Light photomicrographs of *Paratylenchus neoprojectus* juvenile. (**A**) Entire body; (**B**,**C**) pharyngeal regions; (**D**,**E**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm. **Figure 2.** Light photomicrographs of *Paratylenchus neoprojectus* juvenile. (**A**) Entire body; (**B**,**C**) pharyngeal regions; (**D**,**E**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm.

#### **Table 1.** Morphometrics of *Paratylenchus neoprojectus* females and juveniles. All measurements are in µm and presented as mean ± standard deviation (range). 2.1.1. Juveniles

 **Present Study Wu & Hawn [26] \* Van den Berg et al. [28] Characters Females Juveniles Females Females Juveniles**  n 11 4 76 17 4 342.0 ± 19.6 Only one juvenile form was detected. Individuals in this stage were similar in morphology to the adult females. However, they were characterized by the presence of weak stylet; pharynx components under-developed; genital primordium under-developed; anus indistinct; and a posterior body with a finely rounded terminus.

(322.0–365.0) 327–405 359 (300–415) 339.5 (299–390)

#### Body length 383.5 ± 36.7 (330.0–434.0) 2.1.2. Remarks

a

24.0 ± 1.7 (21.0–26.0) 22.3 ± 1.9 (20.5–24.3) 18–26 22.1 (19.5–24.6) 20.4 (17.7–22.9) <sup>b</sup>3.8 ± 0.3 (3.3–4.3) 3.9 ± 0.3 (3.5–4.1) 3.8–4.6 3.9 (3.5–4.4) 4.1 (3.7–4.7) <sup>c</sup>14.6 ± 1.8 (12.1–18.5) 12.8 ± 1.6 (11.0–15.0) 14–16 15.3 (14–18.5) 13.8 (12.3–18.9) *Paratylenchus neoprojectus* was originally described from Central Alberta, Canada in the rhizosphere of alfalfa [26]. Following the formal description, the species has appeared twice in the literature [23]. The first population was reported from India [29] without morphological characterization or illustrations; only morphometrics of adult females were provided. Since overlapping morphometrical characters are common in pin nematode species [25,28], the identification of this Indian population needs to be confirmed.

c' 2.7 ± 0.2 (2.3–3.0) 2.3 ± 0.3 (1.9–2.6) - 2.4 (2.0–2.8) 2.2 (1.7–2.5) <sup>V</sup>84.4 ± 1.3 (82.0–85.8) - 82–85.7 84 (82.5–85) - Stylet percentage 7.0 ± 0.8 (5.8–8.3) - - 8 (6.8–9.3) - Lip height 3.3 ± 0.4 (3.0–4.0) - - 3.5 (3–4) - The second population was reported from Iran [30], and the illustrations showed the absence of a post uterine sac (vs. present in the original description), a broadly rounded tail terminus (vs. conically or finely rounded in the original description), and a short ovary (vs. an ovary that reaches to the pharyngeal basal bulb level in the original description). All these characters are not in agreement with the original description of *P. neoprojectus*, therefore a detailed re-evaluation based on integrative taxonomy is required to determine the exact status of this population.

Lip width 6.4 ± 0.4 (6.0–7.0) - - 7 (6.5–7.5) - Stylet length 25.3 ± 1.3 (25.0–29) 13.3 ± 1.0 (12.0–14.0) 28–31 28.5 (26–31) 10 (3.5–14.5) Median bulb length 23.4 ± 1.6 (21.0–25.0) - - - - Median bulb width 9.3 ± 0.8 (8.0–11.0) - - - - Anterior end to excretory pore 79.1 ± 4.8 (70.0–85.0) 75.0 ± 5.2 (70.0–80.0) - 77.5 (71–85) 71 (65–78.5) Pharynx length 99.0 ± 4.2 89.0 ± 6.2 82–94 92 (85–110) 83.5 (72.5–94.5) In 2014, Van den Berg et al. [28] reported a detailed morphological and molecular characterization of several pin nematode species from the USA and South Africa. Based on their molecular data, the authors demonstrated that *P. nanus* has two sibling species type A and type B. Comparing the morphological, molecular, and morphometrical characteristics (Figures 1 and 2; Table 1), we conclude that *P. nanus* type B should be considered as *P. neoprojectus*. *Paratylenchus neoprojectus* and *P. nanus* are closely related species, but can be differentiated by the body shape (ventrally bent vs. open C-shape of *P. nanus*), position of the excretory pore (at the level or posterior to pharyngeal bulb vs. at level or anterior to pharyngeal bulb), ovary development (reaches the level of the pharyngeal basal bulb vs. short), presence of post uterine branch (vs. absent), and tail terminus morphology (conically or narrowly rounded vs. subacute to rounded, slightly indented). *Paratylenchus*

*neoprojectus* is also close to *P. projectus* and can be differentiated from it by the lip region morphology (conical rounded vs. trapezoid), more posterior position of the excretory pore (vs. anterior), and tail terminus morphology (conically or narrowly rounded vs. often digitate terminus).

In the present study, the *P. neoprojectus* population from southern Alberta matches with the species' original description, except for minor differences in the body length; the southern Alberta population is slightly longer than the original one (330–434 vs. 327–405 µm).

#### 2.1.3. Habitat and Locality

This population was found in the rhizosphere of *Chenopodium* sp. growing on the headland (uncultivated field margin) of a potato field, (latitude 49◦48040.5" N; longitude— 111◦23055.4" W); Municipal District of Forty Mile County No. 8, Alberta, Canada.

#### *2.2. Description of Female Paratylenchus tateae Wu and Townshend*

#### (Figures 3–5; Table 2) [27].

Body slender, ventrally arcuate when heat relaxed; cuticle finely annulated; lateral field equidistant with four distinct lines; lip region conoid narrow, with anterior end flattened, continuous with the rest of the body; labial framework sclerotization weak; pharyngeal region, typical paratylenchoid type; stylet rigid, straight; stylet knobs, rounded; dorsal pharyngeal gland opening 4.5–6.0 µm behind stylet knobs; median pharyngeal bulb elongated, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve inconspicuous; excretory pore situated at the level of pharyngeal basal bulb or slightly anterior to it. Hemizonid 2–3 annuli long situated just anterior to excretory pore; body slightly narrower posterior to vulva; ovary outstretched, occasionally reflexed; spermatheca and crustaformeria not distinguishable in most of the specimens; in mature females, the spermatheca irregularly rounded without sperm; vulva a transverse slit occupying half of the corresponding body width; vulval lips prominent, the anterior lip protrudes further than the posterior lip; vulval flaps present, but not readily distinct in fresh specimens, observable in preserved specimens; a small, rudimentary post uterine branch present along the ventral body wall; anus indistinct; tail slender, conoid, finely annulated, and gradually tapers to form a finely pointed to rounded terminus, bluntly rounded terminus and tip with peg was observed in Spanish populations.

**Figure 3.** Light photomicrographs of *Paratylenchus tateae* female, Canadian population. (**A**) Entire body; (**B**–**E**) pharyngeal regions; (**F**) lip region; (**G**) lateral lines; (**H**) posterior region with gonad; (**I**–**M**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm; (**F**,**G**) 5 µm; (**H**–**M**) 20 µm. Arrowheads: (a) Anus; (exp) excretory pore; (sp) spermatheca; (v) vulva. **Figure 3.** Light photomicrographs of *Paratylenchus tateae* female, Canadian population. (**A**) Entire body; (**B**–**E**) pharyngeal regions; (**F**) lip region; (**G**) lateral lines; (**H**) posterior region with gonad; (**I**–**M**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm; (**F**,**G**) 5 µm; (**H**–**M**) 20 µm. Arrowheads: (a) Anus; (exp) excretory pore; (sp) spermatheca; (v) vulva.


**Table 2.** Morphometrics of Canadian and Spanish populations of *Paratylenchus tateae*. All measurements are in µm and presented as mean ± standard deviation (range).

a

width

to anus

Distance from vulva

58.8 ± 5.6

(24.0–35.0)

59.6 ± 4.8

(21.0–30.0)

c

Anterior end to excretory pore

*Plants* **2021**, *10*, x FOR PEER REVIEW 8 of 25

**Figure 4.** Light photomicrographs of *Paratylenchus tateae* juvenile, Canadian population. (**A**) Entire body; (**B**,**C**) pharyngeal regions; (**D**,**E**) tails. Scale bars: (**A**) 50 µm; (**B***–***E**) 20 µm. **Figure 4.** Light photomicrographs of *Paratylenchus tateae* juvenile, Canadian population. (**A**) Entire body; (**B**,**C**) pharyngeal regions; (**D**,**E**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm. to tail terminus (52.0–70.6) (51.0–67.0) - - - - Tail length 28.2 ± 3.0 26.1 ± 2.5 20.7 ± 1.7 25.3 ± 3.3 25.6 ± 2.9 (22.5–30.0) 22–33

**Table 2.** Morphometrics of Canadian and Spanish populations of *Paratylenchus tateae*. All measurements are in µm and

(21.5–32.5)

(18.0–23.0)

**Townshend [27]** 

Albacete type population

(3.3–4.1) 3.8–5.9

(2.6–3.1) -

(4.5–6.0) -

(8.0–9.5) -

Alpera,

3.6 ± 0.2

2.9 ± 0.2

5.2 ± 0.4

8.6 ± 0.5

**Figure 5.** Light photomicrographs of *Paratylenchus tateae* female, Spanish population. (**A**) Entire body; (**B**) pharyngeal regions; (**C**,**D**,**F**) tails; (**E**) vulval region. Scale bars: (**A**) 50 µm; (**B***–***F**) 10 µm. Arrowheads: (dgo) Dorsal pharyngeal gland orifice; (exp) excretory pore; (v) vulva. **Figure 5.** Light photomicrographs of *Paratylenchus tateae* female, Spanish population. (**A**) Entire body; (**B**) pharyngeal regions; (**C**,**D**,**F**) tails; (**E**) vulval region. Scale bars: (**A**) 50 µm; (**B**–**F**) 10 µm. Arrowheads: (dgo) Dorsal pharyngeal gland orifice; (exp) excretory pore; (v) vulva.

#### 2.2.1. Juveniles

Only one juvenile form was detected. This stage of individuals was similar in morphology to the adult females. However, they were characterized by the presence of weak stylet; pharynx components under-developed; genital primordium under-developed; anus indistinct; posterior body with a finely pointed terminus.

#### 2.2.2. Remarks

*Paratylenchus tateae* was originally described from Ontario, Canada, in the rhizosphere of several crops, such as corn, alfalfa, timothy, and white and red clover [27]. After the formal description, the species was reported twice in the literature [23], one of them reported in Saskatchewan [31], however Anderson and Kimpinski [32] collected samples from the same location and considered the Saskatchewan population as *P. labiosus*. The other population was described in India [29], and the author suggests that the Indian population differs from the Canadian population by smaller body length and a more posterior position of the vulva. Additionally, the description of the Indian population includes a rounded head, a disc-like lip region with prominent projecting submedian lobes, and the absence of a post uterine sac. All of these characteristics are contrary to the original description of *P. tateae*, which states the presence of a distinctive truncated lip region, weakly developed spermatheca, and a short, rudimentary post-uterine branch. Based on our current knowledge, we conclude that the Indian population presented by Bajaj [29] might not be *P. tateae*.

Morphologically and morphometrically, *P. tateae* is similar to *P. brevihastus* Wu [33]; the later species was also described in Ontario in the rhizosphere of alfalfa, blue violets, oats, red clover, and grasses. The only characters differentiating *P. tateae* from *P. brevihastus* are the absence of males and weakly developed spermatheca. We do not suggest synonymization here; we are in agreement with Van den Berg et al. [28], who stated that such actions should only be performed after careful molecular and morphological comparisons.

In the present study, we found two populations of *P. tateae* from southern Alberta, and two from Spain. All the populations match with the original description, except for minor differences in body length, as the Alberta population is slightly shorter than the original description (269–380 vs. 315–401 µm), while other characteristics are in the species variability range.

#### 2.2.3. Habitat and Locality

Two *P. tateae* populations were found in the potato growing fields of the Municipal District of Taber, Alberta, Canada. The first field was located at latitude 49◦46055.8" N, longitude—112◦21030.8" W, whereas the second was located at latitude 49◦47048.5" N, longitude—112◦20049.6" W. Two *P. tateae* populations were found in Spain, in the rhizosphere of almond and wheat, at Ariza, Zaragoza province and Alpera, Albacete province, respectively.

#### *2.3. Description of Female Paratylenchus enigmaticus sp. nov.*

(Figures 6–8; Table 3).

http://zoobank.org/urn:lsid:zoobank.org:act:39C84EDC-15ED-491E-9373-8876D34C3 5ED.

Body slender, ventrally arcuate to form an open, C-shaped body habitus when heat relaxed; cuticle finely annulated; lateral field equidistant with four distinct lines, outer lines are more prominent than the inner ones; lip region conoid rounded, with anterior end flattened, continuous with the rest of the body; labial framework sclerotization weak; pharyngeal region typical paratylenchoid type; stylet rigid, straight; stylet knobs rounded; dorsal pharyngeal gland opening 4.0–6.0 µm behind stylet knobs; median pharyngeal bulb slender elongate, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve rounded; excretory pore situated at the level or anterior to pharyngeal basal bulb; hemizonid 1–2 annuli long

situated immediately posterior to excretory pore; body slightly narrower posterior to vulva; ovary outstretched, well developed; spermatheca and crustaformeria well developed; spermatheca rounded; vulva a transverse slit occupying half of the corresponding body width; vulval lips prominent, the anterior lip is protruding further than the posterior lip; vulval flaps present, but not prominent in fresh specimens; a small rudimentary post uterine branch present along the ventral body wall; anus indistinct; the tail slender, conoid, finely annulated, and gradually tapers to form a rounded terminus. immediately posterior to excretory pore; body slightly narrower posterior to vulva; ovary outstretched, well developed; spermatheca and crustaformeria well developed; spermatheca rounded; vulva a transverse slit occupying half of the corresponding body width; vulval lips prominent, the anterior lip is protruding further than the posterior lip; vulval flaps present, but not prominent in fresh specimens; a small rudimentary post uterine branch present along the ventral body wall; anus indistinct; the tail slender, conoid, finely annulated, and gradually tapers to form a rounded terminus.

nerve ring; basal bulb pyriform, pharyngeal-intestinal valve rounded; excretory pore situated at the level or anterior to pharyngeal basal bulb; hemizonid 1–2 annuli long situated

*Plants* **2021**, *10*, x FOR PEER REVIEW 11 of 25

**Figure 6.** Line drawings of *Paratylenchus enigmaticus* sp. nov. (**A**) Pharyngeal region female; (**B**) pharyngeal region juvenile; (**C**) lateral field lines; (**D**,**E**) juvenile tails; (**F**) posterior region with genital branch; (**G**–**J**) female tails. Scale bars: (**A**,**B**) 20 µm; (**C**) 5 µm; (**D**–**J**) 20 µm. **Figure 6.** Line drawings of *Paratylenchus enigmaticus* sp. nov. (**A**) Pharyngeal region female; (**B**) pharyngeal region juvenile; (**C**) lateral field lines; (**D**,**E**) juvenile tails; (**F**) posterior region with genital branch; (**G**–**J**) female tails. Scale bars: (**A**,**B**) 20 µm; (**C**) 5 µm; (**D**–**J**) 20 µm.

vulva.

**Figure 7.** Light photomicrographs of *Paratylenchus enigmaticus* sp. nov. female. (**A**) Entire body; (**B**–**E**) pharyngeal regions; (**F**) posterior region with gonad; (**G**) lateral lines; (**H**–**L**) tails. Scale bars: (**A**) 50 µm; (**B**–**F**) 20 µm; (**G**) 5 µm; (**H**–**L**) 20 µm. Arrowheads: (a) Anus; (exp) excretory pore; (v) **Figure 7.** Light photomicrographs of *Paratylenchus enigmaticus* sp. nov. female. (**A**) Entire body; (**B**–**E**) pharyngeal regions; (**F**) posterior region with gonad; (**G**) lateral lines; (**H**–**L**) tails. Scale bars: (**A**) 50 µm; (**B**–**F**) 20 µm; (**G**) 5 µm; (**H**–**L**) 20 µm. Arrowheads: (a) Anus; (exp) excretory pore; (v) vulva.


**Table 3.** Morphometrics of Canadian and Belgian populations of *Paratylenchus enigmaticus* sp. nov. All measurements are in µm and presented as mean ± standard deviation (range).

\* Belgian populations (T1–T5) represent measurement of females.

**Figure 8.** Light photomicrographs of *Paratylenchus enigmaticus* sp. nov. juvenile. (**A**) Entire body; (**B**,**C**) pharyngeal regions; (**D**,**E**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm. **Figure 8.** Light photomicrographs of *Paratylenchus enigmaticus* sp. nov. juvenile. (**A**) Entire body; (**B**,**C**) pharyngeal regions; (**D**,**E**) tails. Scale bars: (**A**) 50 µm; (**B**–**E**) 20 µm.

n 11 5 10 10 10 10 10

#### **Table 3.** Morphometrics of Canadian and Belgian populations of *Paratylenchus enigmaticus* sp. nov. All measurements are in µm and presented as mean ± standard deviation (range). 2.3.1. Juvenile

 **Canadian Population \* Belgian Population Claerbout et al. [34] Holotype Paratype Characters Female Females Juveniles T1 T2 T3 T4 T5**  Only one form was detected. This stage of individuals was similar in morphology to the adult females. However, they were characterized by the presence of weak stylet; underdeveloped pharynx components; underdeveloped genital primordium; indistinct anus; and posterior body with a rounded terminus.

335 ± 20

365 ± 39

358 ± 43

328 ± 31

#### Body length 372 382.7 ± 30.9 344.3 ± 9.5 365 ± 40 2.3.2. Diagnosis and Relationship

(343.0–431.0) (331.0–357.0) (308–465) (302–360) (313–422) (300–411) (293–368) a 24.6 25.7 ± 2.1 (21.7–28.7) 23.8 ± 0.4 (23.1–24.4) 24.2 ± 3.8 (14.9–27.6) 24.3 ± 3.4 (19.3–27.2) 26.7 ± 2.3 (22–29) 23.7 ± 2.6 (18.5–27.5) 23.2 ± 3.3 (18.1–28.1) b 3.9 4.1 ± 0.3 (3.7–4.7) 4.2 ± 0.2 (3.9–4.4) 3.7 ± 0.7 (2.7–4.6) - 3.4 ± 0.7 (2.5–4.9) 3.2 ± 0.5 (2.8–4.2) c 15.7 15.4 ± 1.3 14.9 ± 0.5 15.0 ± 1.5 14.9 ± 1.5 14.9 ± 1.9 14.8 ± 2.3 13.0 ± 1.5 The new species is characterized by the presence of 4 lateral lines, advulval flaps, and a moderate stylet length of 28.8 (27.3–30.8) µm. The lip region is conoid rounded, with the anterior end flattened, continuous with the rest of the body. The excretory pore is situated at the level or anterior to the pharyngeal basal bulb. The spermatheca is rounded, and a small rudimentary post uterine branch is present. The tail conoid gradually tapers to form a rounded terminus.

(12.9–17.5) (14.4–15.7) (12.3–17.2) (13.2–17) (12.7–17.8) (13.7–19.8) (10.1–15.7) c' 2.5 2.6 ± 0.3 (2.3–3.1) 2.3 ± 0.3 (1.9–2.6) - - - - - V 84.1 85 ± 0.9 (83.0–86.3) - 83.2 ± 2.1 (80.4–87.8) 83.2 ± 2.1 (80–87) 83.0 ± 1.5 (80–84) 83.5 ± 0.9 (82.8–84.9) 83.1 ± 2.1 (80.1–88) Lip height 3.1 3.0 ± 0.3 (2.6–3.6) - - - - - - Lip width 7.5 7.1 ± 0.4 (6.5–7.7) - - - - - - Stylet length 28.9 28.8 ± 1.1 (27.3–30.8) 12.5 ± 0.9 (11.2–13.5) 27.3 ± 1.3 (23.5–28.4) 25.5 ± 1.6 (22.3–26.5) 26.6 ± 1.5 (25.2–30.5) 26.8 ± 1.3 (24.6–27.9) 27.0 ± 1.5 (24.6–28.6) Stylet percentage 7.7 7.6 ± 0.5 (6.8–8.2) - 7.5 ± 0.9 (6.0–8.8) 7.6 ± 0.7 (7.2–8.8) 7.3 ± 0.7 (6.2–7.9) 7.6 ± 0.8 (6.6–8.4) 8.3 ± 0.5 (7.3–8.9) Median bulb length 21.2 20.4 ± 1.0 (18.5–21.3) - - - - - - Median bulb width 9.8 9.6 ± 1.1 Morphologically, the new species is close to *P. dianthus*, *P. neoprojectus*, *P. nanus* Cobb, [35] and *P. projectus*. The new species can be differentiated from *P. dianthus* by lip region morphology (conoid rounded vs. truncate), presence of small post uterine sac (vs. absent), tail terminus morphology (broadly rounded vs. finely rounded, rarely clavate, or sometimes digitate), and higher c' value (3.5 (3.0–4.5) vs. 2.5). From *P. neoprojectus*, the new species can be differentiated by lip region morphology (conoid rounded vs. rounded), tail terminus morphology (broadly rounded vs. conically rounded), and position of excretory pore (at the level or anterior to pharyngeal bulb vs. at the level or middle of pharyngeal bulb). From *P. nanus* it differs by lip region morphology (conoid rounded vs. rounded), tail terminus morphology (broadly rounded vs. subacute to rounded, slightly indented), and shorter stylet length (28.8 (27.3–30.8) µm vs. 32–34 µm). From *P. projectus*, the new species differs by lip region morphology (conoid rounded vs. offset, conoid truncate, or trapezoid), presence of small post uterine sac (vs. absent), tail terminus morphology (broadly rounded vs. rounded dorsally sinuate), shorter stylet length (28.8 (27.3–30.8) µm vs. 25–37 µm), and higher c' value (3.5 (3.0–4.5) vs. 2.7).

#### (8.0–11.4) - - - - - - 2.3.3. Remarks

Anterior end to excretory pore

<sup>79</sup>76.0 ± 4.2 (70.0–82.0) 65.2 ± 2.8 (63.0–70.0) - - - - - Pharynx length 95 93.8 ± 5.2 (83.0–100.0) 81.6 ± 4.3 (76.0–88.0) 100.7 ± 19.7 (75.2–137.7) 88.0 ± 23.3 (42.9–105.8) 109.9 ± 16.9 (83.3–123.5) 114.7 ± 18.4 (84.6–125.7) 120.4 ± 14.6 (95.0–144.0) The species was first found (but not described) in the glasshouse-grown lettuce from Belgium. The species causes damage to the root system, but this was not related to significant yield reduction in lettuce heads [34]. In the present study, same species was found in the potato growing region of southern Alberta. In the Belgian population, the

authors noted the presence of a large proportion of pre-adults 51–96% and stated this might be due to soil disturbance [34]. The Canadian population also exhibits the same feature; the juveniles were observed in higher numbers than females. Morphological, molecular, and morphometrical comparisons indicate that the Canadian and the Belgian populations are conspecific, and in this study are described as *P. enigmaticus* sp. nov.

#### 2.3.4. Type Habitat and Locality

*Paratylenchus enigmaticus* sp. nov. was found in a potato field (latitude 49◦42034.3" N; longitude—112◦3 054.1" W); the municipal district of Taber, Alberta, Canada.

#### 2.3.5. Etymology

The species name, *enigmaticus*, refers to the species identity remaining unresolved for several months.

#### 2.3.6. Type Material

Holotype female, 9 paratypes females, and 2 juveniles (7 slides, numbers UL-DY1-01 to UL-DY1-07) and additional 5 slides containing females were deposited in the Nematode Collection of the University of Lethbridge, Alberta, Canada. Two females and three juveniles were deposited in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain.

#### *2.4. Molecular Characterization and Phylogenetic Analysis of Paratylenchus Populations from Canada and Spain*

The amplification of the D2–D3 expansion domains of the 28S rRNA, ITS region, and 18S rRNA genes of *Paratylenchus* populations yielded single fragments of ~1000 bp, 800 bp, and 800 bp, respectively. Ten new sequences from the D2–D3 expansion domains of the 28S rRNA gene, 11 from ITS, and two new sequences from the 18S rRNA gene were obtained in this study.

The D2–D3 expansion domains of the 28S rRNA sequences of *P. enigmaticus* sp. nov. (MW282760–MW282761) and *Paratylenchus* sp. T1–T5 (MN535542–MN535545) from Belgium showed no intraspecific variability (100% similarity) from each other. The sequence identities of *P. enigmaticus* sp. nov. with *Paratylenchus* sp. T1–T5 from Belgium, *P. tenuicaudatus* Wu [36] (KU291239, from Iran), and *P. tateae* (MW282754–MW282759) were 99% (1 bp difference and 0 indels), 95% (38 bp difference and 1 indel), and 99% (3–4 different nucleotides and 0 indels), respectively. Similarly, the D2–D3 sequences of *P. tateae* from Canada and Spain showed low intraspecific variability (99% similarity). The sequence identities of *P. tateae* with *P. sheri* Raski [37] (MN088374, from Iran), and *P. similis* Khan, Prasad, Mathur [38] (MN088375, from Iran) were 99% (differed in 5 nucleotides and 0 indels) and 98% (differed in 16 bp and 0 indels). *Paratylenchus neoprojectus* (MW282762–MW282763) sequences obtained in this study differs in 0–7 nucleotides and 0 indels (99–100% similarity) from sequences of *P. neoprojectus* (=*P. nanus* type B) from USA (KF242201, MH790252, MH6722687, MH237651), South Korea (KY468900, KY468899, KF242199, KY468901) and South Africa (KF242200, KF242198). Finally, Canadian *P. neoprojectus* sequence differs in 10 nucleotides and 0 indels (98% similarity) from a short 542 bp sequence of *P. coronatus* Colbran [39] (MK506808) from Iran.

The ITS sequences of Canadian and Spanish populations of *P. tateae* MW282766– MW282771) showed lower intraspecific variability at 99% similarity with 3 different nucleotides and 1–2 indels. The ITS sequences of *P. neoprojectus* (MW282775–MW282776) and *P. enigmaticus* sp. nov. showed low intraspecific variability with 4 and 1–11 different nucleotides, respectively, and 0–3 indels (98–99% similarity). The ITS sequences of *P. enigmaticus* sp. nov. (MW282772–MW282774) and *Paratylenchus* sp. T1–T5 from Belgium (MN535542–MN535545) are very similar, with 97% similarity (16–17 nucleotides difference, 4 indels), whereas the other close species, i.e., *P. hamatus* (KF242253, KF242246), *P. tenuicaudatus* (KF24226, KF242261), and *Paratylenchus* sp. SAS (KF242243) from the USA showed 90–91% (60–71 nucleotides difference, 13–18 indels) similarity with *P. enigmaticus* sp. nov. The *P. neoprojectus* sequence of the Canadian population differs in 4–25 nucleotides and 0–7 indels (97–99% similarity) from sequences of *P. neoprojectus* (=*P. nanus* type B) from USA (MH236098), South Korea (MN710514, MN710515, KY468905, KY468904), and South Africa (KF242264, KF242263). The molecular information in the NCBI database regarding the 18S rRNA gene of pin nematode species is insufficient to calculate the sequence identities for this marker because few sequences have been deposited and there are not many molecular differences between species.

Phylogenetic relationships among *Paratylenchus* species inferred from analyses of the D2–D3 expansion domains of 28S rRNA, ITS region, and partial 18S rRNA sequences using BI are shown in Figures 9–11, respectively. The phylogenetic trees generated from the three nuclear markers, included 89, 81, and 50 sequences, with 680, 875, and 1610 nucleotides, respectively.

The D2–D3 expansion domains of the 28S rRNA phylogenetic tree of *Paratylenchus* spp. showed two main clades, one highly supported (PP = 1.00), including the three species described in this study, and another weakly supported (PP = 0.51), including several *Paratylenchus* spp.; most of them with a longer stylet (>40 µm; Figure 9). The *P. enigmaticus* sp. nov. clustered together in a highly supported subclade (PP = 1.00) with sequences of *Paratylenchus* sp. T1–T5 from Belgium, and was well separated (PP = 0.98) from *Paratylenchus* sp. A (AY780945) from California, USA (Figure 9). Moreover, *P. neoprojectus* clustered together in a highly supported subclade (PP = 1.00) with sequences of *P. neoprojectus* (=*P. nanus* type B) and *P. coronatus* (MK506808). It is also noted that the sequence of *P. sheri* (MN088374) provided by Mirbabaei et al. [40] grouped with the Canadian and Spanish populations of *P. tateae*. The molecular identities suggest that this sequence belongs to *P. tateae* instead of *P. sheri*. The morphological and molecular details associated with the *P. sheri* sequence suggest a possible error in the sequencing. It is therefore recommended to use the same specimen for morphological and molecular studies. Consequently, we consider MN088374 as *P. tateae* in our study.

The 50% majority rule consensus ITS BI tree also shows 2 clades, one representing short stylet species, including the three species described in this study, and the second containing mostly long stylet species (Figure 10). Likewise, the D2–D3 expansion domains of the 28S rRNA tree, *P. enigmaticus* sp. nov. grouped with *Paratylenchus* sp. T1–T5 from Belgium (PP = 1.00), and shares a clade with *P. hamatus*, *P. tenuicaudatus*, and *Paratylenchus* sp. SAS. Canadian and Spanish populations of *P. tateae* grouped with several populations of *P. neoprojectus* (PP = 0.91).

Finally, the phylogenetic relationships of *Paratylenchus* species inferred from analysis of partial 18S rRNA gene sequences shows two clades that are well defined (Figure 11), but several subclades that do not resolve well in the clade include *P. enigmaticus* sp. nov. (MW282764) and *P. neoprojectus* (MW282765).

*Plants* **2021**, *10*, x FOR PEER REVIEW 17 of 25

**Figure 9.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from the D2–D3 expansion domains of the 28S rRNA sequence alignment under the general, time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G). Posterior probabilities of more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site. The 50% majority rule consensus ITS BI tree also shows 2 clades, one representing **Figure 9.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from the D2–D3 expansion domains of the 28S rRNA sequence alignment under the general, time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G). Posterior probabilities of more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site.

short stylet species, including the three species described in this study, and the second containing mostly long stylet species (Figure 10). Likewise, the D2–D3 expansion domains

of *P. neoprojectus* (PP = 0.91).

of the 28S rRNA tree, *P. enigmaticus* sp. nov. grouped with *Paratylenchus* sp. T1–T5 from Belgium (PP = 1.00), and shares a clade with *P. hamatus*, *P. tenuicaudatus*, and *Paratylenchus* sp. SAS. Canadian and Spanish populations of *P. tateae* grouped with several populations

**Figure 10.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under the general, time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G). Posterior probabilities greater than 0.70 are given for the corresponding clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site. **Figure 10.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under the general, time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G). Posterior probabilities greater than 0.70 are given for the corresponding clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site.

(MW282764) and *P. neoprojectus* (MW282765).

**Figure 11.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from the partial 18S rRNA sequence alignment under the general, time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site. **Figure 11.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from the partial 18S rRNA sequence alignment under the general, time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site.

> **3. Discussion**  *Paratylenchus* is a large genus that comprises short and long stylet species [23]. The majority of short stylet species are considered pathogenic and cause significant damage to their host plants [22]. So far, six short stylet species from Canada have been reported, namely *P. brevihastus*, *P. labiosus*, *P. neoprojectus*, *P. projectus*, *P. tateae*, and *P. tenuicaudatus*. *Paratylenchus* is a large genus that comprises short and long stylet species [23]. The majority of short stylet species are considered pathogenic and cause significant damage to their host plants [22]. So far, six short stylet species from Canada have been reported, namely *P. brevihastus*, *P. labiosus*, *P. neoprojectus*, *P. projectus*, *P. tateae*, and *P. tenuicaudatus*. All of these are Canadian native species except *P. projectus,* which is a cosmopolitan species known to have a global distribution [23].

> Finally, the phylogenetic relationships of *Paratylenchus* species inferred from analysis of partial 18S rRNA gene sequences shows two clades that are well defined (Figure 11), but several subclades that do not resolve well in the clade include *P. enigmaticus* sp. nov.

> All of these are Canadian native species except *P. projectus,* which is a cosmopolitan species known to have a global distribution [23]. Morphological identification of *Paratylenchus* species is difficult because of their variable characters and overlapping morphometrical values. Stylet length, number of lateral lines, and presence/absence of vulva flaps are considered to be robust characters for species differentiation; however, body length, tail length and shape, position of excretory pore, and ratios of c, c' were concluded to be unreliable for species separation [25,41,42]. As the majority of *Paratylenchus* species presents a limited selection of differences in morphology, several nematologists have attempted to synonymize morphologically similar species. For example, Brzeski [43] synonymized *P. tateae*, *P. labiosus*, and *P. italiensis* with *P. similis,* because of their similar morphology and overlapped morphometrical values. Ghaderi et al. [25] accepted the synonymization of *P. similis* and *P. tateae*; however, with the availability of molecular data, the same authors [23] rejected the change and referred Morphological identification of *Paratylenchus* species is difficult because of their variable characters and overlapping morphometrical values. Stylet length, number of lateral lines, and presence/absence of vulva flaps are considered to be robust characters for species differentiation; however, body length, tail length and shape, position of excretory pore, and ratios of c, c' were concluded to be unreliable for species separation [25,41,42]. As the majority of *Paratylenchus* species presents a limited selection of differences in morphology, several nematologists have attempted to synonymize morphologically similar species. For example, Brzeski [43] synonymized *P. tateae*, *P. labiosus*, and *P. italiensis* with *P. similis,* because of their similar morphology and overlapped morphometrical values. Ghaderi et al. [25] accepted the synonymization of *P. similis* and *P. tateae*; however, with the availability of molecular data, the same authors [23] rejected the change and referred to both species as valid taxa, and also commented that several populations of *P. similis* may indeed be *P. tateae*. Bahmani et al. [44] also presented a detailed argument on the validity of *P. labiosus,* which was supported by molecular data in Mirbabaei et al. [40].

> to both species as valid taxa, and also commented that several populations of *P. similis* The possible presence of species complexes in pin nematodes was highlighted by Van den Berg et al. [28] and Mirbabaei et al. [40]. We are in agreement with the authors that

similar appearances and overlapping morphometrical characters may present difficulties in ascertaining species status. Nevertheless, such morphological complexes can be resolved using molecular data. Several taxonomic issues have been successfully addressed with molecular studies, such as the validity and differentiation of *Radopholoides* from *Hoplotylus* and *Radopholus* [45], the transfer of *Tylaphelenchus jiaae* to the genus *Pseudaphelenchus* as *P. jiaae* [46], the revision and species synonymization in *Laimaphelenchus* [47], the species delimitation in members of Criconematoidea [48–51], and the resolution of the cryptic diversity and species complexes in Longidoridae [52–54].

Our phylogenetic analysis of D2–D3 expansion domains of the 28S rRNA also indicates that the status of *P. nanus* type B [28] and *P. sheri* [40] need detailed revision. By comparing all the available molecular and morphometric data from both species, it is evident that *P. nanus* type B is a population of *P. neoprojectus* and *P. sheri* is a population of *P. tateae*. Additionally, our *P. enigmaticus* sp. nov. appears conspecific with the Belgian population (T1–T5). It is notable that molecular data not only resolve the taxonomic issues, but also aids in eliminating the propagation of redundant data.

In the literature, several studies have outlined a wide host range [55–57] and survival abilities of pin nematodes [58,59]. Biologically, the final juvenile stage of certain species of pin nematode constitutes the highest portion of the total population. Rhoades and Linford [58] and Wood [15] refer to this stage as a resistant non-feeding stage which is more capable of withstanding desiccation and sudden freezing than the younger and adult stages.

The Canadian and Belgian populations of *P. enigmaticus* sp. nov. have a higher proportion of juveniles than adults, whereas *P. tateae* and *P. neoprojectus* have higher quantities of females than juveniles. It appears that *P. enigmaticus* sp. nov. has a resistant stage; however, the presence of such a stage needs confirmation through further study.

There are limited data regarding the prevalence of pin nematodes in the potato growing areas of southern Alberta and other parts of Canada. Thus far, *P. labiosus* and *P. projectus* are the only species detected in the potato growing areas of Prince Edward Island and New Brunswick [13,32,60,61]. In the present study, we identified *P. neoprojectus*, *P. tateae*, and *P. enigmaticus* sp. nov. in southern Alberta, along with *P. tateae* populations from Spain, using an integrative taxonomical approach. Our study also underscores the importance of using molecular data for accurate species identification and clarifying the status of *P. nanus* type B and *P. sheri*.

Lower densities of identified species in the samples suggest that these are mild parasitic species and, as of yet, do not behave as potential pests. However, pin nematodes have a reputation of building high population densities in short periods, and, under favorable circumstances, can be a threat to their hosts [22,34]. Indeed, a higher incidence of root lesion nematodes (*Pratylenchus* spp.) in southern Alberta has been reported by Forge et al. [4]. Having that in mind, the densities of pin nematodes are worth monitoring as some species can penetrate roots through existing entry points and may aggravate the plant damage. Therefore, further studies are required to assess species-specific yield losses and thresholds.

#### **3. Materials and Methods**

#### *3.1. Isolation and Morphological/Morphometrical Studies*

Nematodes were extracted from soil samples using the modified Cobb sieving and flotation-centrifugation method [62]. For preliminary examinations, fresh nematodes were transferred to the drop of distilled water, heat relaxed at 60 ◦C for 30–45 s, and observed under the Zeiss Axioskope 40 microscope. Permanent mounts were prepared as described in Seinhorst [63] and De Grisse [64]. Light micrographs of the mounted specimens were acquired using a Zeiss Axioskope 40 microscope equipped with a Zeiss Axiocam 208 camera (Carl Zeiss Microscopy, Jena, Germany). Standard morphometrical characters were selected based on previously published studies [25,28,57,65]. Measurements were made using ZEN blue 3.1 imaging software (Carl Zeiss Microscopy).

#### *3.2. DNA Extraction, PCR and Sequencing*

Nematode DNA was prepared according to Maria et al. [65]. Three sets of DNA primers (Integrated DNA Technologies, Coralville, IA, USA) were used in the PCR analyses to amplify the nucleotide sequences of the partial 18S, D2–D3 expansion domains of the 28S rRNA and ITS of ribosomal genes, including 5.8S rRNA and both ITS regions (ITS1 and ITS2) (rRNA). The partial 18S rRNA region was amplified with 1813F and 2646R primers [66]. The D2–D3 expansion domains of the 28S rRNA regions were amplified using 28–81F and 28–1006rev primers [67], and the ITS region was amplified using F194 [68] and AB28 primers [69]. The ribosomal gene cluster (whole rDNA cistron) is a multicopy, tandem repeated array in the genome. Each repeat is transcribed as a single rRNA precursor and cleaved, leading to the mature small subunit rRNA (SSU), the mature 5.8S rRNA, and the mature large subunit rRNA (LSU). The SSU is separated from the 5.8S rRNA by the first internal transcribed spacer (ITS1), and the second internal transcribed spacer (ITS2) is located between the 5.8S rRNA and the LSU [70]. A nice scheme of these repeats and the position of many of the primers used by nematologists could be found in Carta and Li [71]. The PCR conditions were as described in Holterman et al. [66,67] and in Ferris et al., [68]. Amplified PCR products were resolved by electrophoresis in 1% agarose gels and visualized by staining with GelRed (Biotium, Fremont, CA, USA). Amplified DNA fragments were purified using an E.Z.N.A Gel Extraction kit (Omega Biotek, Norcross, GA, USA), following manufacturer's instructions, ligated into the pJET1.2 vector (Thermo Fisher Scientific, Mississauga, ON, Canada), and introduced into *Escherichia coli* DH5α competent cells (Thermo Fisher Scientific). The presence of the PCR-derived inserts in the plasmids from transformed *E. coli* cells was confirmed by PCR. Plasmid DNA was isolated and purified using E.Z.N.A Plasmid DNA minikit I (Omega Biotek), according to the manufacturer's instructions, and sent to Genewiz, Inc for DNA sequencing (South Plainfield, NJ, USA). DNA sequences were aligned using the Bioedit sequence alignment tool and compared for similarities with all known nematode species sequences in the GenBank database.

#### *3.3. Phylogenetic Analyses*

Sequenced genetic markers from the nematodes examined in the present study (after discarding primer sequences and ambiguously aligned regions) and several pin nematode sequences obtained from GenBank were used in the phylogenetic reconstruction. Outgroup taxa for each dataset were selected based on previously published studies [57]. Multiple sequence alignments of the newly obtained and published sequences were made using the FFT-NS-2 algorithm of MAFFT V.7.450 [72]. Sequence alignments were visualized with BioEdit [73] and manually edited using Gblocks ver. 0.91b [74] in the Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks\_server.html) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences +1; maximum number of contiguous nonconserved positions: 8; minimum length of a block: 5; allowed gap positions: With half).

Phylogenetic analyses of the sequence datasets were conducted based on Bayesian inference (BI) using MRBAYES 3.2.7a [75]. The best-fit model of DNA evolution was calculated with the Akaike information (AIC) of JMODELTEST V.2.1.7 [76]. The bestfit model, base frequency, proportion of invariable sites, substitution rates and gamma distribution shape parameters in the AIC were used for phylogenetic analyses. BI analyses were performed under a general time reversible model, with a proportion of invariable sites and a rate of variation across sites (GTR + I + G) for the partial 18S rRNA, D2–D3 expansion domains of the 28S rRNA, and ITS region sequences. These BI analyses were run separately per dataset with four chains for 2 <sup>×</sup> <sup>10</sup><sup>6</sup> generations. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding burn-in samples of 20% and evaluating convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees from all analyses were edited using FigTree software V.1.4.4 (http://tree.bio.ed.ac. uk/software/figtree/).

**Author Contributions:** Conceptualization, M.M. and D.P.Y.; methodology, M.M., D.P.Y., J.E.P.-R., and P.C.; software, M.M., J.E.P.-R., and P.C.; validation, M.M., D.P.Y., P.C. and J.E.P.-R.; formal analysis, M.M. and J.E.P.-R.; investigation, M.M., D.P.Y. and J.E.P.-R.; resources, D.P.Y., P.C.; data curation, M.M. and J.E.P.-R.; writing—original draft preparation, M.M., D.P.Y. and J.E.P.-R., writing—review and editing, M.M., D.P.Y., P.C. and J.E.P.-R.; visualization, M.M. and J.E.P.-R.; supervision and project administration, D.P.Y.; funding acquisition, D.P.Y. and P.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Potato Early Dying Complex project funded by the University of Lethbridge Research Operating Fund, and the Canadian Potato Early Dying Network project funded by the Canadian Agri-Science Cluster for Horticulture 3 grant to D.P.Y., in collaboration with the Potato Growers of Alberta, McCain Foods Canada Ltd., Cavendish Farms Corp. and Lamb Weston Inc.; and by the Spanish Ministry of Science, Innovation and Universities, grant number "RTI2018-095925-A-I00" to P.C. and J.E.P.-R.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** We thank potato growers in Alberta, Canada, for providing access to their fields, and Mariana Vetrici (University of Lethbridge, AB, Canada) for the collection of soil samples. We also thank Carolina Cantalapiedra-Navarrete (Institute for Sustainable Agriculture (IAS), CSIC, Spain), for the excellent technical assistance in molecular analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

