Pratylenchus vovlasi sp. Nov. (Nematoda: Pratylenchidae) on Raspberries in North Italy with a Morphometrical and Molecular Characterization

Root-lesion nematode species rank third only to root-knot and cyst nematodes as having the greatest economic impact on crops worldwide. A survey of plant-parasitic nematodes associated with decaying raspberries (Rubus sp.) in northern Italy revealed that root-lesion nematodes were the most frequently occurring species among other phytonematodes. Several Pratylenchus species have been associated with Rubus sp. in Canada (Quebec, British Columbia) and USA (North Carolina, Maryland, New Jersey) including P. penetrans and P. crenatus. In the roots and rhizosphere of symptomatic raspberries, nematodes of two Pratylenchus spp. were detected. Detailed morphometrics of the two root-lesion nematode isolates were consistent with Pratylenchus crenatus and with an undescribed Pratylenchus species. The extracted nematodes were observed and measured as live and fixed materials and subsequently identified by integrative taxonomy (morphometrically and molecularly). The latter species is described herein as Pratylenchus vovlasi sp. nov., resulting morphometrically closest to P. mediterraneus and phylogenetically to P. pratensis. The molecular identification of Pratylenchus vovlasi sp. nov. was carried out by sequencing the ITS region, D2-D3 expansion domains of the 28S rRNA gene and a partial region of the nuclear hsp90 gene. ITS-RFLP and sequence analyses revealed that Pratylenchus vovlasi sp. nov. had species-specific restriction profiles with no corresponding sequences present in the database. The phylogenetic relationships with ITS and D2-D3 sequences placed the Pratylenchus vovlasi sp. nov. in a clade with P. pratensis and P. pseudopratensis. This research confirms the occurrence of cryptic biodiversity within the genus Pratylenchus as well as the need for an integrative approach to the identification of Pratylenchus species.


Introduction
Raspberries (Rubus sp.) have a long history of human consumption and cultivation in Europe, with Russia being the leading producer [1]. Raspberries have been eaten fresh for thousands of years. In the last decades, there has been an increase in the demand for fruit and fruit-based products as consumers seek out healthier dietary options. In particular, berries are considered one of the best dietary sources of bioactive compounds that have important antioxidant properties, with associated health effects such as protective effects against several cancers and cardiovascular disorders [2,3] and thus the berry market is expected to expand during the next years. Raspberries are the most productive in areas with mild winters and long, moderate summers. The production of raspberries is greatly influenced by biotic and abiotic factors. Among the biotic factors, plant-parasitic nematodes have an important role in the reduction of the raspberry yield [4,5]. Several genera of plantparasitic nematodes have been found to be associated with the raspberry [4][5][6][7][8][9]. The most frequently observed species in soil and the roots of raspberries belong to the root-lesion nematode genus Pratylenchus, mainly P. crenatus Loof, 1960, (71.7% of soil samples, 53.4% of root samples), P. penetrans (Cobb, 1917) Filipjev and Schuurmans Stekhoven, 1941 (51.6%, 54.2%) and P. scribneri Steiner in Sherbakoff and Stanley 1943 (9.2%, 10.2%; [9]). In Italy, raspberries are grown mainly in the northern areas including Trentino Alto Adige, the main production area, Verona province and Piedmont. Other Italian areas growing raspberries are in Romagna in the north and Calabria, Sicily, Campania and Basilicata in the south. Considering the economic significance of root-lesion nematodes on raspberries and the need to accurately distinguish these damaging species for their practical management in the field, we provide here the morphometrical and molecular characterization of a new root-lesion nematode, Pratylenchus vovlasi sp. nov. parasitizing raspberries in the Piedmont area. The specific objectives of this paper were: (i) to carry out a comprehensive identification with morphological and morphometric approaches of P. vovlasi sp. nov. with a differential diagnosis to closely related species; (ii) to provide a molecular characterization of P. vovlasi sp. nov. and estimate its phylogenetic relationships with other representatives of the Pratylenchus genus using three molecular markers: two ribosomal markers (the D2-D3 expansion segments of the 28S rRNA and the internal transcribed spacer region (ITS rRNA) and the nuclear region of the partial heat shock protein 90 (hsp90) gene.

Results
Two species were identified from raspberries in the Piedmont region. Detailed morphometrics and molecular analyses of the two root-lesion nematode populations were consistent with P. crenatus and with an undescribed Pratylenchus species. As P. crenatus is a well-known and well molecular characterized species, we concentrated our efforts on the integrative taxonomic identification of the undescribed species.
The partial hsp90 gene amplified products of two individual specimens were cloned and four clones for each individual specimen were sequenced (OA984937-OA984944). The sequence analyses revealed the occurrence of two different fragments for each specimen and both fragments coded for hsp90 differed in the length of the intron (52 bp vs. 43 bp) and The sequence analyses revealed the occurrence of two different fragments for each spec men and both fragments coded for hsp90 differed in the length of the intron (52 bp vs 4 bp) and nucleotide variability. The 298 bp fragment showed an 82% similarity (254/31 identities) with the 310 bp fragment at a nucleotide level. At an amino acid level, the tw fragments showed 96% identities (81/84) and 98% positives (83/84). The new hsp90 se quences for P. vovlasi sp. nov. showed a high intraspecific variability by 3-60 nucleotide and 0-13 indels (81-99% similarity). The hsp90 sequences for P. vovlasi sp. nov. differed b 13, 16 and 61 nucleotides and 4, 4 and 24 indels from the most closely related species, P speijeri De Luca, Troccoli, Duncan, Subbotin, Waeyenberge, Coyne, Brentu and Inserra 2012 (HE601547) with a 96% similarity, P. coffeae (Zimmermann, 1898) Filipjev and Schuu mans Stekhoven, 1941 (HE601548) with a 95% similarity and P. hippeastri Inserra, Troccol Gozel, Bernard, Dunn and Duncan, 2007 (HE601549) with an 81% similarity, respectively

Restriction Profiles
PCR-RFLP analyses of the ITS region allowed us to determine the species-specifi patterns for the Italian population of P. vovlasi sp. nov. (Figure 1) that clearly identifie this species.

Phylogenetic Relationships
The phylogenetic relationships among Pratylenchus species inferred from the ana yses of the D2-D3 expansion domains of 28S rRNA, ITS and the partial hsp90 gene se quences using BI are shown in Figures 2-4, respectively. The phylogenetic trees generate with the ribosomal and nuclear markers included 54, 60 and 31 sequences with 693, 54 and 284 positions in length, respectively (Figures 2-4). The D2-D3 tree of Pratylenchus spp showed a well-supported subclade (PP = 1.00) including P. vovlasi sp. nov., P. pratensis an P. pseudopratensis ( Figure 2). All other clades followed the same pattern as previous stud ies on Pratylenchus species.

Phylogenetic Relationships
The phylogenetic relationships among Pratylenchus species inferred from the analyses of the D2-D3 expansion domains of 28S rRNA, ITS and the partial hsp90 gene sequences using BI are shown in Figures 2-4, respectively. The phylogenetic trees generated with the ribosomal and nuclear markers included 54, 60 and 31 sequences with 693, 548 and 284 positions in length, respectively (Figures 2-4). The D2-D3 tree of Pratylenchus spp. showed a well-supported subclade (PP = 1.00) including P. vovlasi sp. nov., P. pratensis and P. pseudopratensis ( Figure 2). All other clades followed the same pattern as previous studies on Pratylenchus species.  The 50% majority-rule consensus ITS BI tree showed several clades that were no well-defined ( Figure 2) but a well-supported subclade (PP = 1.00) including P. vovlasi sp nov. and P. pratensis ( Figure 3). This subclade was phylogenetically related to P. vulnu Allen and Jensen, 1951 and P. kumamotoensis Lal and Khan, 1990 in a moderately sup ported subclade (PP = 0.95) ( Figure 3). Finally, the hsp90 BI tree confirmed the occurrenc of two isoforms within P. vovlasi sp. nov. that were clearly separated in two independen well-supported subclades (PP = 0.98 and PP = 1.00, respectively) ( Figure 4). The 50% majority-rule consensus ITS BI tree showed several clades that were not well-defined ( Figure 2) but a well-supported subclade (PP = 1.00) including P. vovlasi sp. nov. and P. pratensis ( Figure 3). This subclade was phylogenetically related to P. vulnus Allen and Jensen, 1951 and P. kumamotoensis Lal and Khan, 1990 in a moderately supported subclade (PP = 0.95) ( Figure 3). Finally, the hsp90 BI tree confirmed the occurrence of two isoforms within P. vovlasi sp. nov. that were clearly separated in two independent well-supported subclades (PP = 0.98 and PP = 1.00, respectively) ( Figure 4).  A Bayesian 50% majority-rule consensus tree as inferred from the ITS sequence alignment under a transversional model with a proportion of invariable sites and a rate of variation across sites (TVM + I + G). Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are in bold letters. Scale bar = expected changes per site. A Bayesian 50% majority-rule consensus tree as inferred from the ITS sequence alignment under a transversional model with a proportion of invariable sites and a rate of variation across sites (TVM + I + G). Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are in bold letters. Scale bar = expected changes per site.

Description
Female: the body assumes an almost straight to open C posture when heat-killed ( Figure 5A). The lip region is slightly offset from body contour and bears three annuli, which narrow in diameter towards the anterior end ( Figures 5D and 6D). In the en face SEM view lip region, they appear dumb-bell-shaped ( Figure 7A,B,D,I) with an acute pattern (sensu Subbotin et al. [10]) fitting the group II according to the classification scheme of Corbett and Clark [11]. The stylet is relatively small and delicate with conus about 45 ± 2.5 (41-49) % of the entire stylet length (Table 1). The stylet shaft is slender ending with rounded basal knobs, which are slightly anteriorly flattened. The pharyngeal procorpus narrows just anterior to the small, oval metacorpus. The valve of the median bulb is conspicuous. The isthmus is short, encircled by a nerve ring and widening to a pharyngeal lobe with a dorsal nucleus just posterior to the cardia and ventro-sublateral nuclei near the tip of the pharyngeal lobe ( Figure 5B,C) and overlapping the intestine ventro-laterally for almost a two-body diameter at the cardia level. The secretory-excretory pore is level with the cardia just posterior to the hemizonid. The body annulation is distinct; the lateral field usually with four smooth incisures and a few specimens have an additional line ( Figure 6K) or more rarely a few oblique striae ( Figure 6J) in the middle of the central band. The outline of outer bands becomes indented towards the tail end, posterior to phasmid, with the inner lines fusing just posterior to the phasmid. The genital tract is well developed with oocytes arranged in a single row. The spermatheca is large and spherical to oval, usually full of sperm ( Figure 5F,E,G); its posterior margin is 50.0 ± 9.6 (36.5-64.5) µm from the vagina ( Table 1). The vulva is slightly sunken and the vulval lips are not prominent ( Figure 5E,F). The post-uterine sac is about 1.1 vulval body diameter long and is usually undifferentiated. The phasmids are located to the mid-tail, 13.2 ± 2.2 (11)(12)(13)(14)(15)(16)(17) µm from the tail tip. The tail is typically subcylindrical, tapering towards the tip with a rounded-truncate terminus in most specimens; a few specimens have broadly rounded, conical tails with coarsely pointed or indented striated termini ( Figures 5H,I and 6F,H,I,L).   Figure 5A). The lip region is slightly offset from body contour and bears three annuli, which narrow in diameter towards the anterior end ( Figures 5D and 6D). In the en face SEM view lip region, they appear dumb-bell-shaped ( Figure 7A,B,D,I) with an acute pattern (sensu Subbotin et al. [10]) fitting the group II according to the classification scheme of Corbett and Clark [11]. The stylet is relatively small and delicate with conus about 45 ± 2.5 (41-49) % of the entire stylet length (Table 1). The stylet shaft is slender ending with rounded basal knobs, which are slightly anteriorly flattened. The pharyngeal procorpus narrows just anterior to the small, oval metacorpus. The valve of the median bulb is conspicuous. The isthmus is short, encircled by a nerve ring and widening to a pharyngeal lobe with a dorsal nucleus just posterior to the cardia and ventro-sublateral nuclei near the tip of the pharyngeal lobe ( Figure 5B,C) and overlapping the intestine ventro-laterally for almost a two-body diameter at the cardia level. The secretory-excretory pore is level A Bayesian 50% majority-rule consensus tree as inferred from the hsp90 sequence alignment under the general time-reversible model of the sequence evolution with a correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are in bold letters. Scale bar = expected changes per site.
Male: similar to the female except in the posterior end of the body and in a slightly smaller and usually slender body length. The lip region is usually higher and narrower than in the female ( Figure 6B,C). The stylet is slightly smaller than that of female with smaller, more rounded knobs. The pharyngeal bulb is small and round; the isthmus is slender and rather short ending in a long, narrow glandular lobe. The testis is outstretched and filled with round spermatozoa in the vas deferens. The spicules are paired, weakly cephalated and ventrally arcuate. The gubernaculum is simple and slightly curved. The tail is conical and bent on the ventral side with a prominent, crenate bursa.

Type Host and Locality
Pratylenchus vovlasi sp. nov. was found associated with the roots and soil of Rubus sp. with a population density of 650 nematodes/250 cm 3 of soil in the locality of Prarostino, Turin province, Piedmont Region, north Italy.
Holotype, female and male paratypes mounted on glass slides were deposited in the nematode collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), CNR, Bari, Italy (collection numbers IPSP-M-1238-1250). The additional paratypes were distributed to the United States Department of Agriculture Nematode Collection, Beltsville, MD, USA (collection number IPSP-M-1237), WaNeCo Plant Protection Service, Wageningen, The Netherlands, (collection number IPSP-M-1246) and Instituto de Agricultura Sostenible, CSIC, Córdoba, Spain (collection number IPSP-M-1245).

Diagnosis and Relationships
Pratylenchus vovlasi sp. nov. is characterized by a lip region slightly offset with three annuli narrowing towards the anterior end, sub-median sectors fused with the oral disc and separated by a lateral sector to give a dumb-bell -shaped pattern; the stylet is rather small (15.0 µm long) with rounded knobs and a short pharyngeal overlap. The lateral field has four smooth incisures in most specimens, the spermatheca is round to oval and is usually full of sperm, the vulva is located in a relative anterior position and the tail is subcylindrical with a truncate, smooth terminus and with common males. The matrix code of the new species according to Castillo and Vovlas [12] is A2 B2, C2, D2, E2, F3, G1,2, H1, I2, J1, K1 (Table 2).

Diagnosis and Relationships
Pratylenchus vovlasi sp. nov. is characterized by a lip region slightly offset with three annuli narrowing towards the anterior end, sub-median sectors fused with the oral disc and separated by a lateral sector to give a dumb-bell -shaped pattern; the stylet is rather small (15.0 µm long) with rounded knobs and a short pharyngeal overlap. The lateral field has four smooth incisures in most specimens, the spermatheca is round to oval and is usually full of sperm, the vulva is located in a relative anterior position and the tail is subcylindrical with a truncate, smooth terminus and with common males. The matrix code of the new species according to Castillo and Vovlas [12] is A2 B2, C2, D2, E2, F3, G1,2, H1, I2, J1, K1 (Table 2).
Related species sharing with P. vovlasi sp. nov. a three lip annuli, a divided face as seen with SEM (Group 2 according to Corbett and Clark [11]), a functional spermatheca and numerous males include P. bhattii Siddiqi, Dabur and Bajaj, 1991, P. kralli Ryss, 1982, P. mediterraneus Corbett, 1983, P. thornei Sher and Allen, 1953, P. pratensis, P. pseudopratensis Seinhorst, 1968, P. penetrans, P. fallax and P. convallariae Seinhorst, 1959.     P. vovlasi sp. nov. is most closely related to P. mediterraneus, matching 10 out of 11 characteristics according to the matrix code [12] and differing from it just by a slightly shorter pharyngeal overlap (32 ± 6.7 (20-43) vs. 25-55 µm, code I2 vs. I3 in the tabular key), a face pattern showing transverse incisures in the middle of the sub-medial sectors (not present in P. mediterraneus), lateral sectors fused (vs. separated) with the oral disc, smooth lateral fields, not areolated vs. crenate (outer bands), occasionally areolated and with the middle band variously ornamented ( Figure 6J,K and Figure 7K). From P. pratensis, P. fallax, P. penetrans and P. vulnus, it differs in the en face SEM pattern (belonging to Group 2 vs. Group 3, according to Corbett and Clark, [11]) and the tail tip morphology despite a certain degree of overlap (mostly truncate and smooth vs. usually oblique and annulated in P. pratensis, rounded or with a slightly irregular contour in P. fallax, generally rounded in P. penetrans and narrowly rounded to subacute and occasionally irregular in P. vulnus). Furthermore, the new species differs from P. pratensis by a mostly rounded vs. oval to rectangular spermatheca and the number of tail annuli (14-20 vs (Table 2) and a more anterior position of the vulva (78 (74-80) vs. 77-82%). From P. pseudopratensis, it differs in the en face SEM view (not dumb-bell-shaped in P. pseudopratensis), a shorter stylet (range: 14.3-16.3 vs. 16-17 µm), the shape of the spermatheca (rectangular, sometimes empty in P. pseudopratensis) and the tail tip (smooth vs. crenated; code H1 vs. H2 after Castillo and Vovlas, [12]).

Etymology
The species epithet, P. vovlasi, is dedicated to Dr. Nikos Vovlas, an eminent Italian nematologist and taxonomist from the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy. Other Pratylenchus species with three lip annuli, a functional spermatheca and the presence of males and with a matrix code (sensu Castillo and Vovlas, [12]) similar to P. vovlasi sp. nov. have been described without examining their lip patterns. These species include P. convallariae, P. kralli and P. bhattii, from which the new species differs by the following characteristics: from P. convallariae, by a shorter stylet (15 (14.3-16.3), feature C2 in Table 2, vs. 17 (16)(17)(18) µm feature C3), a shorter PUS (1.1, feature F3 vs. more than 1.4-2 vulval body diameter, feature F6 in Table 2) and by a smooth tail tip (H1) vs. coarsely and often irregularly annulated (H2); from P. kralli, in the stylet knob shape (mostly rounded vs. anteriorly directed), the post-uterine sac length (1.1 vs. more than 1.5 vulval body diameter), the tail tip is truncated, smooth and more rarely pointed vs. pointed and showing a slight groove; from P. bhattii by a slightly longer stylet (15 (14.3-16.3) vs. 13.5 (13)(14) µm) and a more posterior vulva (V = 78 vs. 73% mean value) with the vulval lips continuous with the body contour vs. raised in a prominent protuberance. Finally, the new species can be compared with P. rwandae Singh, Nyiragatare, Janssen, Couvreur, Decraemer and Bert, 2018, but differs from it by a labial region in the en face view showing clearly separated sub-median sectors with transverse incisures in the middle (vs. slightly separated from the lateral sectors and without transverse incisures), males present (vs. absent), females with mostly round and full spermatheca (vs. oval to rounded and empty), lateral fields with four incisures (vs. six or more at the mid-body) and a tail with a fewer number of ventral annuli (14)(15)(16)(17)(18)(19)(20) and usually a truncated tip (vs. 18-28 tail annuli and a highly variable tail tip).  [12].

Discussion
The identification of the Pratylenchus species is difficult because many diagnostic characteristics overlap and also due to the increasing number of nominal species. An accurate identification is needed in order to adopt appropriate control strategies. The primary objective of this study was to identify and molecularly characterize root-lesion nematodes parasitizing raspberries cultivated in the Piedmont region. The present study reports on the occurrence of two root-lesion nematodes associated with raspberry fields in the Piedmont region, P. crenatus, along with an abundant species herein identified as P. vovlasi sp. nov. Pratylenchus crenatus was previously reported on raspberries in several European countries [6,9].
Our results demonstrated that the application of rRNA molecular markers integrated with morphological studies could help in the diagnosis and characterization of root-lesion nematode species. Based on the molecular characterization using the D2-D3 expansion domains of the 28S rRNA gene, ITS region and the partial hsp90 gene, the abundant species was clearly identified as P. vovlasi sp. nov. This species proved very similar in morphometry and morphology to P. mediterraneus, differing only by a shorter pharyngeal overlap, as well as with P. thornei, P. penetrans, P. fallax and P. convallariae. By blasting at NCBI the D2-D3 region, it was 96-97% similar to P. pratensis and P. pseudopratensis, respectively, while by using ITS sequences it was 85-87% similar to P. pratensis and P. fallax. These results represented an additional confirmation of the extraordinary cryptic diversity of the nematodes of the genus Pratylenchus, as reported in previous studies [13][14][15][16][17]. Phylogenetic analyses of the ITS and the D2-D3 sequences confirmed a sister relationship between P. vovlasi sp. nov. with P. pratensis and P. pseudopratensis. Furthermore, in both phylogenetic trees, the new species was closely related to P. vulnus and P. kumamotoensis (Figures 2 and 3). Major clades for D2-D3 and ITS phylogenetic trees were highly correlated with previous phylogenetic studies carried out by Subbotin et al. [10], Palomares-Rius et al. [14,18] and Araya et al. [17]. We agreed with De Luca et al. [13] who suggested that the ITS-containing region allowed a better discrimination among the closely related species studied because it evolved faster than the D2-D3 expansion segments of 28S rDNA and accumulated more substitution changes.
The current study confirmed the occurrence of different hsp90 isoforms in the Pratylenchus species as already reported by Fanelli et al. [19]. In P. vovlasi sp. nov. the different isoforms differed from each other in the length of the intron and the nucleotide variability grouping in two well-supported clusters. This finding suggested that the different isoforms of P. vovlasi sp. nov. hsp90 arose by gene duplication events relatively recently because the nucleotide variability was low and the gene structure was still conserved [20,21]. Similarly, these results confirmed that the primers for the amplification of the hsp90 gene could also amplify other paralogous genes in Pratylenchus and could be used for species delimitation within this genus [22,23]. Furthermore, the occurrence of different hsp90 isoforms in P. vovlasi sp. nov. confirmed that this gene family could contribute to the adaptation to different hosts and to different environments.
In this study, P. vovlasi sp. nov. was isolated from raspberries in north Italy together with P. crenatus in the same area. Further research on its pathogenicity and the economic damage on this crop is needed.

Nematode Isolate and Morphological Studies
No specific permits, other than that of the farm owner, were required for the indicated fieldwork studies. The soil samples were obtained in raspberry cultivated areas in Piedmont and did not involve any endangered species or those protected in Italy, nor were the sites protected in any way.
The soil samples recovered from the rhizosphere and roots of the raspberries located in the Piedmont region were sent to the IPSP laboratory, Bari, in the autumn of 2010. The samples were collected with a shovel from the upper 50 cm of the soil of raspberries arbitrarily chosen at random. The nematodes were extracted from 500 cm 3 of the soil by centrifugal flotation [24]. The specimens for light microscopy were killed by gentle heat, fixed in a solution of 4% formaldehyde + 1% propionic acid and processed to pure glycerin using Seinhorst's method [25]. The specimens were examined with a Zeiss III compound microscope with a Nomarski differential interference contrast at powers up to ×1000. The measurements and drawings were made at the camera lucida on glycerin-infiltrated specimens. All measurements were expressed in micrometers (µm) unless otherwise stated.
For the scanning electron microscope (SEM), fixed specimens were dehydrated in a gradient series of ethanol, critical-point dried, sputter-coated with gold according to the protocol of Abolafia et al. [26] and observed with a Zeiss Merlin Scanning Electron Microscope (5 kV; Zeiss, Oberkochen, Germany).

DNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing
DNA was extracted from 20 single individual root-lesion nematode specimens. The specimens were handpicked and placed singly on a glass slide in 3 µL of the lysis buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 15 mM MgCl 2 , 0.1% Triton × 100, 0.01% gelatin with 90 µg/mL proteinase K) and then cut into small pieces by using a sterilized syringe needle under a dissecting microscope. The samples were incubated at 65 • C for 1 h and then at 95 • C for 15 min to deactivate the proteinase K [27]. The following sets of primers were used for the amplification of the gene fragments in the present study: (i) D2-D3 expansion segments of the 28S rRNA gene using forward D2A and reverse D3B primers; (ii) ITS1-5.8-ITS2 rRNA using forward TW81 and reverse AB28 primers [28,29]; (iii) the hsp90 gene using forward U831 and reverse L1110 primers [30]. New sequences were submitted to the GenBank database under the accession numbers indicated on the phylogenetic trees.

PCR-RFLP
Ten µL of each ITS-PCR product from three individual nematodes were digested with five units of the following restriction enzymes: AluI, AvaII, BamHI, DdeI, HinfI and RsaI. The digested products were separated onto a 2.5% agarose gel by electrophoresis, stained with gel red dye, visualized on a UV transilluminator and photographed with a digital system.

Phylogenetic Analysis
Sequenced genetic markers in the present study (after discarding primer sequences and ambiguously aligned regions) and several Pratylenchus spp. sequences obtained from GenBank were used for the phylogenetic reconstruction. The outgroup taxa for each dataset were selected based on previous published studies [17,18,23]. Multiple sequence alignments of the newly obtained and published sequences were made using the FFT-NS-2 algorithm of MAFFT v. 7.450 [31]. The sequence alignments were visualized using BioEdit [32] and edited by Gblocks ver. 0.91b [33] in the Castresana Laboratory server (http: //molevol.cmima.csic.es/castresana/Gblocks_server.html, accessed on 2 April 2021) 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 no conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half).
The phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MRBAYES 3.2.7a [34]. The best-fit model of DNA evolution was calculated with the Akaike information criterion (AIC) of JMODELTEST v. 2.1.7 [35]. The best-fit model, the base frequency, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates in the AIC were then used in the phylogenetic analyses.