Morphological and molecular characterisation of Longidorus pauli (Nematoda: Longidoridae), first report from Greece

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Morphological and molecular characterisation of Longidorus pauli (Nematoda: Longidoridae), first report from Greece

Emmanuel A. Tzortzakakis / Ilenia Clavero-Camacho / Carolina Cantalapiedra-Navarrete / Parthenopi Ralli / Juan E. Palomares-Rius / Pablo Castillo / Antonio Archidona-Yuste *

Keywords : Cytochrome oxidase c subunit 1, D2-D3 of 28S rRNA, Description, ITS1 rRNA, Longidorus, L. pauli, L. pisi, Needle nematodes, Taxonomy

Citation Information : Journal of Nematology. Volume 53, Pages 1-10, DOI: https://doi.org/10.21307/jofnem-2021-034

License : (CC-BY-4.0)

Received Date : 17-February-2021 / Published Online: 20-March-2021

ARTICLE

ABSTRACT

Sampling for needle nematodes was carried out in a grapevine area in Thessaloniki, North Greece and two nematode species of Longidorus (L. pauli and L. pisi) were collected. Nematodes were extracted from 500 cm3 of soil by modified sieving and decanting method, processed to glycerol and mounted on permanent slides, and subsequently identified morphologically and molecularly. Nematode DNA was extracted from single individuals and PCR assays were conducted to amplify D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial coxI regions. Morphology and morphometry data obtained from these populations were consistent with L. pauli and L. pisi identifications. To our knowledge, this is the first report of L. pauli for Greece, and the second world report after the original description from Idleb, Syria, extending the geographical distribution of this species in the Mediterranean Basin.

Graphical ABSTRACT

Needle nematodes are polyphagous root ectoparasites of a wide range of economically important plants by directly feeding on root cells. Some species of this genus are economically important pests of agricultural plants, and others are proved to transmit nepoviruses (Taylor and Brown, 1997). The genus Longidorus consists of more than 160 valid species (Archidona-Yuste et al., 2016; Cai et al., 2020), but currently only 12 have been reported from Greece. Seven of these have been molecularly identified Longidorus closelongatus (Stoyanov, 1964), Longidorus cretensis (Tzortzakakis et al., 2001), Longidorus euonymus (Mali and Hooper, 1974), Longidorus iranicus ( =  moesicus) (Sturhan and Barooti, 1983), Longidorus orientalis (Loof, 1982), Longidorus pisi ( =  latocephalus) (Edward et al., 1964), and Longidorus pseudoelongatus (Altherr, 1976; He et al., 2005, Tzortzakakis et al., 2014, 2017). The five remaining species L. africanus (Merny, 1966), L. elongatus (de Man, 1876; Micoletzky, 1922) L. fasciatus (Roca and Lamberti, 1981), L. intermedius (Kozlowska and Seinhorst, 1979), and L. proximus (Sturhan and Argo, 1983) lack of molecular characterization (Tzortzakakis et al., 2008). During a recent nematode sampling from a grapevine area in Northern Greece, two needle nematode populations were detected resembling L. pauli (Lamberti et al., 1999) and L. pisi. Longidorus pauli was previously reported only from original description on fig tree at Idleb, Syria (Lamberti et al., 1999). Therefore, the objective of the present study was to provide an accurate identification of Longidorus species detected in North Greece by an integrative approach of morphological and molecular characterization by using the D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial coxI regions.

Materials and methods

Nematode samples and morphological study

Soil samples were collected at a depth of 20 to 40 cm from the rhizosphere of a grapevine grafted on 1103-Paulsen of the Institute of Plant Breeding and Genetic Resources, Thermi, Thessaloniki, Greece. Nematodes were extracted from soil by modified sieving and decanting method (Brown and Boag, 1988). Extracted specimens were heat killed, fixed in TAF, processed to glycerol by a slow evaporation method, and mounted on permanent slides (Hooper, 1986). The light micrographs and measurements of nematode populations including the main diagnostic characteristics (i.e., de Man indices, body length, odontostyle length, lip region, tail shape, amphid shape, and oral aperture-guiding ring) were performed using a Leica DM6 compound microscope with a Leica DFC7000 T digital camera. All abbreviations were used as defined in Jairajpuri and Ahmad (1992).

Molecular characterization

For molecular analyses, and in order to avoid mistakes in case of mixed populations in the same sample, single specimens from the sample were temporarily mounted in a drop of 1 M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens conformed with the target population. All necessary morphological and morphometric data were recorded. This was followed by DNA extraction from single individuals as described by Archidona-Yuste et al. (2016). The D2-D3 segments were amplified using the D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) primers (De Ley et al., 1999). The Internal Transcribed Spacer region 1 (ITS1) separating the 18S rRNA gene from the 5.8S rRNA gene was amplified using forward primer 18S (5´-TTGATTACGTCCCTGCCCTTT-3´) (Vrain et al., 1992) and reverse primer rDNA1 5.8S (5´-ACGAGCCGAGTGATCCACCG-3´) (Cherry et al., 1997). Finally, the portion of the coxI gene was amplified as described by Lazarova et al. (2006) using the primers COIF (5´-GATTTTTTGGKCATCCWGARG-3´) and COIR (5´-CWACATAATAAGTATCATG-3´).

All PCR assays were done according to the conditions described by Archidona-Yuste et al. (2016). Then, the amplified PCR products were purified using ExoSAP-IT (Affimetrix, USB products. COUNTRY) and used for direct sequencing on a DNA multicapillary sequencer (Model 3130XL genetic analyzer; Applied Biosystems, Foster City, CA, USA), using the BigDye Terminator Sequencing Kit V.3.1 (Applied Biosystems, Foster City, CA, USA), at the Stab Vida sequencing facilities (Caparica, Portugal). The newly obtained sequences were submitted to the GenBank database under the accession numbers indicated on the phylogenetic trees. This population of Longidorus is proposed here as standard and reference population for L. pauli until topotype material becomes available and molecularly characterized. Voucher specimens of this described species have been deposited in the nematode collection of Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.

Phylogenetic analyses

D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and coxI mtDNA sequences of the unidentified Longidorus species population were obtained in this study. These sequences, and other sequences from species of Longidorus from GenBank, were used for phylogenetic analyses. Outgroup taxa for each dataset were chosen following previously published studies (Archidona-Yuste et al., 2019; Cai et al., 2020; He et al., 2005; Holterman et al., 2006). Multiple sequence alignments of the different genes were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al., 2019). Sequence alignments were manually visualized using BioEdit (Hall, 1999) and edited by Gblocks ver. 0.91b (Castresana, 2000) 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 non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al., 2012) with the Akaike information criterion (AIC). 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 MrBayes for the phylogenetic analyses. The general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2-D3 segments of 28S rRNA and the partial coxI gene, and the transitional model and a gamma-shaped distribution (TIM3+G) model for ITS1 rRNA were analyzed with four chains for 2 × 106 generations, respectively. A combined analysis of the three ribosomal genes was not undertaken due to some sequences not being available for all species. The Markov chains were sampled at intervals of 100 generations and two runs were conducted for each analysis. After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for in-depth analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. Trees from all analyses were visualized using FigTree software version v.1.42 (Rambaut, 2014).

Results and discussion

Soil samples from grapevine at Thessaloniki, North Greece yielded two Longidorus populations, including a moderately abundant population (5-10 needle nematodes/500 cm3 of soil) resembling L. pauli and two juvenile specimens of L. pisi that were confirmed by 28S rRNA, which were 100% coincident with a population from Bulgaria (LR032064) and 99% (AY601569, differing in 1 nucleotide) from a Greek population (He et al., 2005; Peneva et al., 2013). Since in recent studies, this species has been molecularly characterized by D2-D3 28S rRNA or the coxI gene for populations from Greece, South Africa, and Iran (He et al., 2005; Palomares-Rius et al., 2017; Pedram et al., 2008), no further studies were developed on this new Greek population.

Systematics

Longidorus pauli (Lamberti et al., 1999) (Fig. 1 and Table 1).

Table 1.

Morphometrics of Longidorus pauli (Lamberti et al., 1999) from Greece.

10.21307_jofnem-2021-034-t001.jpg
Figure 1:

Light micrographs of Longidorus pauli (Lamberti et al., 1999) from Greece (A-N). A-E: Female anterior regions. F: Detail of base of odontostyle and odontophore, G: Vulval region showing sperm cells in the uterus, H-K: Female tail regions, L: Detail of sperm cells in male genital tract, M: Male tail region, N: Detail of spicules. a = anus; af = amphidial fovea; as = adanal supplement; c = cloaca; gr = guiding ring; Odt = odontostyle; Odp = odontophore; sp = spicules; spc = sperm cells; V = vulva. (Scale bars: A-N = 20 µm).

10.21307_jofnem-2021-034-f001.jpg

Description

Female: body ventrally curved in a close C-shaped to single spiral when killed by gentle heat with greater curvature in the posterior half more pronounced in the case of male. Moderate long body length (6.6-8.5 mm in female; and 6.6-8.0 mm in male). Cuticle (3.0-4.0) µm thick at mid-body. Lip region rounded and set off by a slight depression from the rest of body, anteriorly slightly rounded to flattened (Fig. 1). Amphidial pouch slightly asymmetrically bilobed. Guiding ring single, located 1.9 to 2.4 times lip region diameter from anterior end. Odontostyle 1.8 to 2.0 times as long as odontophore; odontophore well developed, with slight basal swellings. Esophagus extending to a terminal esophageal bulb (107.0-127.0) μm long), with dorsal (DN) gland nucleus and ventrosublateral (SVN) gland nuclei separately located at (20.6-28.9)% and (51.4-57.0)% of distance from anterior end of esophageal bulb, respectively. Glandularium (97.0-118.0) μm long. Cardia conoid-rounded. Vulva located about mid-body or slightly posterior (49.4-56.0%). Vagina (9.0-12.0) µm wide, ovijector (23.0-34.0) µm wide. Genital tract amphidelphic, anterior and posterior genital branches equally developed, 414 to 772 and 407 to 647 µm long, respectively. Rectum 23 to 25 µm long. Sperm cells (5.0-6.0) μm long, frequently detected in both genital branches. Tail conoid-rounded with bluntly rounded terminus.

Male: morphologically similar to female and common. Testes paired, full of oblong sperm cells. Adanal supplements paired, at (10.5-14.5) µm from anus, preceded anteriorly by a row of 9 to 15 irregularly spaced ventromedians supplements. Spicules paired, robust and ventrally curved, approximately 1.1 to 1.3 times larger than tail length. Lateral guiding pieces with a curved proximal end. According to the polytomous key Chen et al. (1997), supplement by Loof and Chen (1999), and the addition of some characters by Peneva et al. (2013), the Greek population has the following codes (codes in parentheses are exceptions): A4 – B2(3) – C2(3) – D3 – E2(3) – F4 – G3 – H1 – I2 – J1 – K7.

Remarks

The Greek population of L. pauli was collected from the rhizosphere of grapevine at Thermi, Thessaloniki, Greece with a nematode density of 5 to 10 nematodes/500 cm3 soil. Up to our knowledge, this is the first report of this species from Greece and the second after original description from Syria by Lamberti et al. (1999). Morphology and morphometrics of Greek population agree with those of the type population of this species (Table 1). The main differences between Greek population of L. pauli and original population of this species are: a ratio ((125.0-168.8) vs (120.3-143.5)), ratio ((1.0-1.3) vs (0.8-1.0)), odontostyle length ((112.0-126.0) vs (102.0-118.3) µm), and spicules length ((54.0-60.0) vs (61.0-69.0) µm). These small morphometrics differences detected may be due to geographical intraspecific variability of them.

This species is morphological- and morphometrically close L. closelongatus, L. pseudoelongatus, L. apulus (Lamberti and Bleve Zacheo, 1977), and L. apuloides (Roca, 1996), from which can be separated by (i) L. closelongatus: longer body length ((6.6-8.5) vs (5.3-7.3) mm) and lower ratio ((1.0-1.3) vs (1.3-1.5)); (ii) L. pseudoelongatus: longer body length ((6.6-8.5) vs (5.1-5.6) mm), higher a ratio ((125.0-168.8) vs (73.0-110.0)), and slightly higher V ratio ((49.4-56.0) vs (45.0-48.0)); (iii) L. apulus: longer odontostyle length ((112.0-126.0) vs (91.0-112.0) µm); and (iv) L. apuloides slightly shorter body length ((6.6-8.5) vs (7.4-10.2) mm) and spicules length ((54.0-60.0) vs (52.5-86.0) µm). From all of them can be also separated by molecular markers 28S rRNA, ITS rRNA, and coxI regions, except for L. apuloides which has not been molecularly characterized yet.

Molecular characterization and phylogeny of Longidorus pauli Amplification and sequencing of the D2-D3 expansion domains of 28S rRNA, ITS1 rRNA, and partial coxI genes yielded sequences sizes of ca 750 bp, 1,000 bp, and 400 bp, respectively, based on gel electrophoresis. Five new D2-D3 of 28S rRNA gene sequences of L. pauli were obtained in the present study (MW598384-MW598388) and showed a low intraspecific variability with 0 to 1 different nucleotides and 0 indels (99% similarity). The D2-D3 for L. pauli. differed from the closest related species, L. proximus (MK894275) by 19 nucleotides and 0 indel (98% similarity), L. iranicus (MK894273) by 17 nucleotides and two indels (98% similarity), L. closelongatus (KJ802866) by 25 nucleotides and 0 indel (97% similarity), and from L. cretensis (KJ802868) by 27 nucleotides and 0 indel (96% similarity).

The ITS1 region also showed a low intraspecific variability by 0 to 2 nucleotides and 1 indel (99% similarity). ITS1 for L. pauli (MW598390-MW598392) showed low similarity with all the ITS1 sequences of Longidorus spp. deposited in NCBI, including the most similar species, L. cretensis (KJ802892), L. iranicus (KP222295), and L. closelongatus (KJ802891), by 159 to 179 different nucleotides and 58 indels (83-85% similarity).

The four new coxI sequences for L. pauli showed moderate intraspecific variability by 20 to 23 nucleotides and 0 indel (94% similarity). coxI for L. pauli (MW598436-MW598439) showed low similarity with all the coxI sequences of Longidorus spp. deposited in NCBI, including the most similar species by 70, 76, 81 and 77 nucleotides (78-80% similarity) and 0 to 2 indels from the closest related species, L. pseudoelongatus (KY816699), L. iranicus (KY816677), L. pini (MH454070) and L. cretensis (KY816670), respectively.

Phylogenetic relationships among Longidorus species, as inferred from analyses of D2-D3 expansion domains of 28S rRNA, ITS1, and the partial coxI mtDNA gene sequences using BI, are shown in Figures 2-4, respectively. The phylogenetic trees generated with the ribosomal and mitochondrial DNA markers included 109, 10 and 61 sequences with 749, 992 and 390 characters in length, respectively (Figs. 2-4). The D2-D3 region of the 28S rRNA tree of Longidorus spp. showed a well-supported subclade (PP = 1.00), including L. pauli (MW598384-MW598388), L. iranicus, L. pseudoelongatus, L. proximus, Longidorus sp. 4SAS2014 and L. cretensis (Fig. 2). Longidorus pisi from Thessaloniki clustered into a subclade with another L. pisi population from Iran (Fig. 2).

Figure 2:

Phylogenetic relationships of Longidorus pauli (Lamberti et al., 1999) within the genus Longidorus. Bayesian 50% majority rule consensus tree as inferred from D2 and D3 expansion domains of 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 more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

10.21307_jofnem-2021-034-f002.jpg
Figure 3:

Phylogenetic relationships of Longidorus pauli (Lamberti et al., 1999) from Greece within the genus Longidorus. Bayesian 50% majority rule consensus tree as inferred from ITS1 rRNA sequence alignment under the TIM3 + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

10.21307_jofnem-2021-034-f003.jpg
Figure 4:

Phylogenetic relationships of Longidorus pauli (Lamberti et al., 1999) within the genus Longidorus. Bayesian 50% majority rule consensus trees as inferred from cytochrome c oxidase subunit I (coxI) mtDNA gene sequence alignments under the GTR + I + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are in bold letters.

10.21307_jofnem-2021-034-f004.jpg

Due to scarce similarity with other sequences of the genus Longidorus, the phylogenetic reconstruction using the marker ITS1 sequences was difficult to obtain, therefore only related sequences were used for the phylogeny study. The 50% majority rule consensus ITS1 BI tree showed a low-supported clade (PP = 0.71) including L. pauli (MW598390-MW598392), L. iranicus, L. pseudoelongatus, and L. cretensis (Fig. 3). Finally, the phylogenetic relationships of Longidorus species inferred from analysis of the partial coxI gene sequences showed that L. pauli (MW598436-MW598439) clustered with the closed species in a well-supported clade (PP = 1.00) including L. cretensis, Longidorus sp. 4CCN2017, L. iranicus, L. pseudoelongatus, L. kuiperi, and L. closelongatus (Fig. 4).

Phylogenetic analyses based on three rDNA molecular markers (D2-D3 expansion domains of 28S rRNA gene and ITS1 region) and mitochondrial DNA coxI resulted in a general consensus of species phylogenetic positions for the majority, and were generally congruent with those given by previous phylogenetic analysis (Archidona-Yuste et al., 2019; Cai et al., 2020; Clavero-Camacho et al., 2021; Gutiérrez-Gutiérrez et al., 2013; Inserra et al., 2021). This research increased the number of Longidorus species in Greece, as well as the molecular diversity within Longidorus. In particular, phylogenetic results are congruent with morphological traits, since the new sequenced population of L. pauli clustered together with other species showing lip region rounded and set off by a slight depression from the rest of body, anteriorly slightly rounded to flattened, moderate long odontostyle, and tail conoid-rounded with bluntly rounded terminus.

In summary, the present study confirms the correct identity of this nematode and increase the great biodiversity of this genus in the Mediterranean Basin.

Acknowledgements

The authors thank Dr. Ricos Thanopoulos and Mr. Konstantinos Kotrotsis from Institute of Plant Genetic Resources and Breeding, Thermi, Thessaloniki, Greece for sample collection and shipping, and J. Martín Barbarroja and G. León Ropero (IAS-CSIC) for their excellent technical assistance. Spanish authors acknowledge grant 201740E042, ‘Análisis de diversidad molecular, barcoding, y relaciones filogenéticas de nematodos fitoparásitos en cultivos mediterráneos’ from Spanish National Research Council (CSIC).

References


  1. Altherr, E. 1976. La faune des eaux profondes interstitielles de la région de Wiesbaden. Bulletin de la Société Vaudoise des Sciences Naturelles 73:97–116.
  2. Archidona-Yuste, A. , Cantalapiedra-Navarrete, C. , Castillo, P. and Palomares-Rius, J. E. 2019. Molecular phylogenetic analysis and comparative morphology reveals the diversity and distribution of needle nematodes of the genus Longidorus (Dorylaimida: Longidoridae) from Spain. Contributions to Zoology 88:1–41.
  3. Archidona-Yuste, A. , Navas-Cortés, J. A. , Cantalapiedra-Navarrete, C. , Palomares-Rius, J. E. and Castillo, P. 2016. Unravelling the biodiversity and molecular phylogeny of needle nematodes of the genus Longidorus (Nematoda: Longidoridae) in olive and a description of six new species. PLoS ONE 11:e0147689.
  4. Brown, D. J. F. and Boag, B. 1988. An examination of methods used to extract virus-vector nematodes (Nematoda: Longidoridae and Trichodoridae) from soil samples. Nematologia Mediterranea 16:93–99.
  5. Brown, D. J. F. , Grunder, J. , Hooper, D. J. , Klinger, J. and Kunz, P. 1994. Longidorus arthensis sp. n. (Nematoda: Longidoridae) a vector of cherry rosette disease caused by a new nepovirus in cherry trees in Switzerland. Nematologica 40:133–149.
  6. Cai, R. , Archidona-Yuste, A. , Cantalapiedra-Navarrete, C. , Palomares-Rius, J. E. and Castillo, P. 2020. New evidence of cryptic speciation in needle and dagger nematodes of the genera Longidorus and Xiphinema (Nematoda: Longidoridae). Journal of Zoological Systematics and Evolutionary Research 58:869–899.
  7. Castresana, J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17:540–552.
  8. Chen, Q. W. , Hooper, D. J. , Loof, P. A. A. and Xu, J. 1997. A revised polytomous key for the identification of species of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Fundamental and Applied Nematology 20:15–28.
  9. Cherry, T. , Szalanski, A. L. , Todd, T. C. and Powers, T. O. 1997. The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology 29:23–29.
  10. Clavero-Camacho, I. , Liébanas, G. , Escuer, M. , Cantalapiedra-Navarrete, C. , Archidona-Yuste, A. , Castillo, P. and Palomares-Rius, J. E. 2021. Studies on Longidorus iberis (Escuer & Arias, 1997) n. comb. (Nematoda: Longidoridae) from Spain. Nematology 23 (in press).
  11. Darriba, D. , Taboada, G. L. , Doallo, R. and Posada, D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9:772.
  12. De Ley, P. , Félix, M. A. , Frisse, L. M. , Nadler, S. A. , Sternberg, P. W. and Thomas, K. W. 1999. Molecular and morphological characterization of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae). Nematology 1:591–612.
  13. de Man, J. G. 1876. Onderzoekingen over vrij in de aarde levende Nematoden. Tijdschrift der Nederlandsche Dierkundige Vereeniging 2:78–196.
  14. Edward, J. C., Misra, S. L. and Singh, G. R. 1964. Longidorus pisi n. sp. (Nematoda, Dorylaimoidea) associated with the rhizosphere of Pisum sativum, from Uttar Pradesh, India. Japanese Journal of Applied Entomology and Zoology 8:310–2.
  15. Gutiérrez-Gutiérrez, C. , Cantalapiedra-Navarrete, C. , Montes-Borrego, M. , Palomares-Rius, J. E. and Castillo, P. 2013. Molecular phylogeny of the nematode genus Longidorus (Nematoda: Longidoridae) with description of three new species. Zoological Journal of the Linnean Society 167:473–500.
  16. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series 41:95–98.
  17. He, Y. , Subbotin, S. , Rubtsova, T. V. , Lamberti, F. , Brown, D. J. F. and Moens, M. 2005. A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida). Nematology 7:111–124.
  18. Holterman, M. , Van Der Wurff, A. , van Den Elsen, S. , van Megen, H. , Bongers, T. , Holovachov, O. , Bakker, J. and Helder, J. 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Phylogenetics and Evolution 23:1792–1800.
  19. Hooper, D. G. 1986. “Handling, fixing, staining and mounting nematodes”, In Southey, J. F. (Ed.), Laboratory Methods for Work with Plant and Soil Nematodes Ministry of Agriculture, Fisheries and Food, England, pp. 39–54.
  20. Inserra, R. N. , Troccoli, A. , Vau, S. and Subbotin, S. A. 2021. Morphological and molecular characterisation of populations of Longidorus longicaudatus Siddiqi, 1962 and L. americanus Handoo, Carta, Skantar, Ye, Robbins, Subbotin, Fraedrich & Cram, 2005 from Florida, USA. Nematology 23 (in press).
  21. Jairajpuri, M. S. and Ahmad, W. 1992. Dorylaimida. Free-living, predaceous and plant-parasitic nematodes. New Delhi: Oxford and IBH Publishing Co., p. 458.
  22. Katoh, K. , Rozewicki, J. and Yamada, K. D. 2019. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinformatics 20:1160–1166.
  23. Kozlowska, J. and Seinhorst, J. W. 1979. Longidorus elongatus and closely related species in the Netherlands and lower Saxony (Germany), with the description of two new species, L. cylindricaudatus and L. intermedius (Nematoda: Dorylaimida). Nematologica 25:42–53.
  24. Lamberti, F. and Bleve Zacheo, T. 1977. Two new species of Longidorus (Nematoda: Longidoridae) from Italy. Nematologia Mediterranea 5:73–78.
  25. Lamberti, F. , Molinari, S. , De Luca, F. , Agostinelli, A. and Di Vito, M. 1999. Longidorids (Nematoda, Dorylaimida) from Syria with description of Longidorus pauli sp. n. and Paralongidorus halepensis sp. n. with SOD isozymes and PCR-RFLP profiles. Nematologia Mediterranea 27:63–78.
  26. Lazarova, S. S. , Malloch, G. , Oliveira, C. M. G. , Hübschen, J. and Neilson, R. 2006. Ribosomal and mitochondrial DNA analyses of Xiphinema americanum-group populations. Journal of Nematology 38:404–410.
  27. Loof, P. A. A. 1982. Two new species of Longidoridae (Dorylaimida) from Saudi Arabia. Nematologica 28:307–317
  28. Loof, P. A. A. and Chen, Q. 1999. A revised polytomous key for the identification of the species of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Supplement 1. Nematology 1:55–59.
  29. Mali, V. R. and Hooper, D. J. 1974. Observations on Longidorus euonymus n. sp. and Xiphinema vuittenezi Luc et al., 1964 (Nematoda: Dorylaimida) associated with spindle trees infected with Euonymus Mosaic Virus in Czechoslovakia. Nematologica 19:459–467.
  30. Merny, G. 1966. Nematodes from tropical Africa: a new Paratylenchus (Criconematidae) two new Longidorus and observations on Longidorus laevicapitatus Williams, 1959 (Dorylaimidae). Nematologica 12:385–395.
  31. Micoletzky, H. 1922. Die freilebenden Erd-Nematoden. Archiv fur Naturgesrchichte 87:1–650.
  32. Palomares-Rius, J. E. , Cantalapiedra-Navarrete, C. , Archidona-Yuste, A. , Subbotin, S. A. and Castillo, P. 2017. The utility of mtDNA and rDNA for barcoding and phylogeny of plant-parasitic nematodes from Longidoridae (Nematoda, Enoplea). Scientific Reports 7:10905.
  33. Pedram, M. , Niknam, G. , Robbins, R. T. , Ye, W. and Karegar, A. 2008. Longidorus kheirii n. sp. (Nematoda: Longidoridae) from Iran. Systematic Parasitology 71:199–211.
  34. Peneva, V. K. , Lazarova, S. S. , De Luca, F. and Brown, D. J. F. 2013. Description of Longidorus cholevae sp. n. (Nematoda, Dorylaimida) from a riparian habitat in the Rila Mountains, Bulgaria. ZooKeys 330:1–26.
  35. Rambaut, A. 2014. FigTree v1.4.2, A Graphical Viewer of Phylogenetic Trees, available at: http://tree.bio.ed.ac.uk/software/figtree/.
  36. Roca, F. 1996. Longidorus apuloides n. sp. (Nematoda: Longidoridae) from Italy with a hierarchical cluster analysis of the closely related species. Fundamental and Applied Nematology 19:315–320.
  37. Roca, F. and Lamberti, F. 1981. Longidorus fasciatus sp. n. from Greece and Italy. Nematologia Mediterranea 9:175–179.
  38. Ronquist, F. and Huelsenbeck, J. P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574.
  39. Stoyanov, D. 1964. A contribution to the nematofauna of the grapevine. Rastitelna Zashtita Sofia 12:16–24
  40. Sturhan, D. and Argo, D. 1983. Studies on Longidorus closelongatus Stoyanov and L. cohni Heyns, with description of L. proximus sp. nov. (Nematoda, Dorylaimida). Revue Nematologie 6:57–64.
  41. Sturhan, D. and Barooti, S. 1983. Longidorus iranicus n sp. (Nematoda: Dorylaimida). Systematic Parasitology 5:21–4.
  42. Taylor, C. A. and Brown, D. J. F. 1997. Nematode vectors of plant viruses CAB International. Wallingford: CAB International.
  43. Tzortzakakis, E. A. , Peneva, V. , Brown, D. J. F. and Avgelis, A. D. 2008. A literature review on the occurrence of nematodes of the family Longidoridae in Greece. Nematologia Mediterranea 36:153–156.
  44. Tzortzakakis, E. A., Peneva, V., Terzakis, M., Neilson, R. and Brown, D. J. F. 2001. Longidorus cretensis n. sp. (Nematoda: Longidoridae) from a vineyard infected with a foliar ‘yellow mosaic’ on Crete, Greece. Systematic Parasitology 48:131–9.
  45. Tzortzakakis, E. A. , Cantalapiedra-Navarrete, C. , Castillo, P. , Palomares-Rius, J. E. and Archidona-Yuste, A. 2017. Morphological & molecular identification of Longidorus euonymus and Helicotylenchus multicinctus from the rhizosphere of grapevine & banana in Greece. Journal of Nematology 49:233–235.
  46. Tzortzakakis, E. A. , Archidona-Yuste, A. , Cantalapiedra-Navarrete, C. , Nasiou, E. , Lazanaki, M. S. , Kabourakis, E. A. , Palomares-Rius, J. E. and Castillo, P. 2014. Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete, Greece, with description of Xiphinema cretense n. sp. (Nematoda, Longidoridae). European Journal of Plant Pathology 140:563–590.
  47. Vrain, T. C. , Wakarchuk, D. A. , Levesque, A. C. and Hamilton, R. I. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15:563–573.
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FIGURES & TABLES

Figure 1:

Light micrographs of Longidorus pauli (Lamberti et al., 1999) from Greece (A-N). A-E: Female anterior regions. F: Detail of base of odontostyle and odontophore, G: Vulval region showing sperm cells in the uterus, H-K: Female tail regions, L: Detail of sperm cells in male genital tract, M: Male tail region, N: Detail of spicules. a = anus; af = amphidial fovea; as = adanal supplement; c = cloaca; gr = guiding ring; Odt = odontostyle; Odp = odontophore; sp = spicules; spc = sperm cells; V = vulva. (Scale bars: A-N = 20 µm).

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Figure 2:

Phylogenetic relationships of Longidorus pauli (Lamberti et al., 1999) within the genus Longidorus. Bayesian 50% majority rule consensus tree as inferred from D2 and D3 expansion domains of 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 more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

Full Size   |   Slide (.pptx)

Figure 3:

Phylogenetic relationships of Longidorus pauli (Lamberti et al., 1999) from Greece within the genus Longidorus. Bayesian 50% majority rule consensus tree as inferred from ITS1 rRNA sequence alignment under the TIM3 + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

Full Size   |   Slide (.pptx)

Figure 4:

Phylogenetic relationships of Longidorus pauli (Lamberti et al., 1999) within the genus Longidorus. Bayesian 50% majority rule consensus trees as inferred from cytochrome c oxidase subunit I (coxI) mtDNA gene sequence alignments under the GTR + I + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are in bold letters.

Full Size   |   Slide (.pptx)

REFERENCES

  1. Altherr, E. 1976. La faune des eaux profondes interstitielles de la région de Wiesbaden. Bulletin de la Société Vaudoise des Sciences Naturelles 73:97–116.
  2. Archidona-Yuste, A. , Cantalapiedra-Navarrete, C. , Castillo, P. and Palomares-Rius, J. E. 2019. Molecular phylogenetic analysis and comparative morphology reveals the diversity and distribution of needle nematodes of the genus Longidorus (Dorylaimida: Longidoridae) from Spain. Contributions to Zoology 88:1–41.
  3. Archidona-Yuste, A. , Navas-Cortés, J. A. , Cantalapiedra-Navarrete, C. , Palomares-Rius, J. E. and Castillo, P. 2016. Unravelling the biodiversity and molecular phylogeny of needle nematodes of the genus Longidorus (Nematoda: Longidoridae) in olive and a description of six new species. PLoS ONE 11:e0147689.
  4. Brown, D. J. F. and Boag, B. 1988. An examination of methods used to extract virus-vector nematodes (Nematoda: Longidoridae and Trichodoridae) from soil samples. Nematologia Mediterranea 16:93–99.
  5. Brown, D. J. F. , Grunder, J. , Hooper, D. J. , Klinger, J. and Kunz, P. 1994. Longidorus arthensis sp. n. (Nematoda: Longidoridae) a vector of cherry rosette disease caused by a new nepovirus in cherry trees in Switzerland. Nematologica 40:133–149.
  6. Cai, R. , Archidona-Yuste, A. , Cantalapiedra-Navarrete, C. , Palomares-Rius, J. E. and Castillo, P. 2020. New evidence of cryptic speciation in needle and dagger nematodes of the genera Longidorus and Xiphinema (Nematoda: Longidoridae). Journal of Zoological Systematics and Evolutionary Research 58:869–899.
  7. Castresana, J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17:540–552.
  8. Chen, Q. W. , Hooper, D. J. , Loof, P. A. A. and Xu, J. 1997. A revised polytomous key for the identification of species of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Fundamental and Applied Nematology 20:15–28.
  9. Cherry, T. , Szalanski, A. L. , Todd, T. C. and Powers, T. O. 1997. The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology 29:23–29.
  10. Clavero-Camacho, I. , Liébanas, G. , Escuer, M. , Cantalapiedra-Navarrete, C. , Archidona-Yuste, A. , Castillo, P. and Palomares-Rius, J. E. 2021. Studies on Longidorus iberis (Escuer & Arias, 1997) n. comb. (Nematoda: Longidoridae) from Spain. Nematology 23 (in press).
  11. Darriba, D. , Taboada, G. L. , Doallo, R. and Posada, D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9:772.
  12. De Ley, P. , Félix, M. A. , Frisse, L. M. , Nadler, S. A. , Sternberg, P. W. and Thomas, K. W. 1999. Molecular and morphological characterization of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae). Nematology 1:591–612.
  13. de Man, J. G. 1876. Onderzoekingen over vrij in de aarde levende Nematoden. Tijdschrift der Nederlandsche Dierkundige Vereeniging 2:78–196.
  14. Edward, J. C., Misra, S. L. and Singh, G. R. 1964. Longidorus pisi n. sp. (Nematoda, Dorylaimoidea) associated with the rhizosphere of Pisum sativum, from Uttar Pradesh, India. Japanese Journal of Applied Entomology and Zoology 8:310–2.
  15. Gutiérrez-Gutiérrez, C. , Cantalapiedra-Navarrete, C. , Montes-Borrego, M. , Palomares-Rius, J. E. and Castillo, P. 2013. Molecular phylogeny of the nematode genus Longidorus (Nematoda: Longidoridae) with description of three new species. Zoological Journal of the Linnean Society 167:473–500.
  16. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series 41:95–98.
  17. He, Y. , Subbotin, S. , Rubtsova, T. V. , Lamberti, F. , Brown, D. J. F. and Moens, M. 2005. A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida). Nematology 7:111–124.
  18. Holterman, M. , Van Der Wurff, A. , van Den Elsen, S. , van Megen, H. , Bongers, T. , Holovachov, O. , Bakker, J. and Helder, J. 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Phylogenetics and Evolution 23:1792–1800.
  19. Hooper, D. G. 1986. “Handling, fixing, staining and mounting nematodes”, In Southey, J. F. (Ed.), Laboratory Methods for Work with Plant and Soil Nematodes Ministry of Agriculture, Fisheries and Food, England, pp. 39–54.
  20. Inserra, R. N. , Troccoli, A. , Vau, S. and Subbotin, S. A. 2021. Morphological and molecular characterisation of populations of Longidorus longicaudatus Siddiqi, 1962 and L. americanus Handoo, Carta, Skantar, Ye, Robbins, Subbotin, Fraedrich & Cram, 2005 from Florida, USA. Nematology 23 (in press).
  21. Jairajpuri, M. S. and Ahmad, W. 1992. Dorylaimida. Free-living, predaceous and plant-parasitic nematodes. New Delhi: Oxford and IBH Publishing Co., p. 458.
  22. Katoh, K. , Rozewicki, J. and Yamada, K. D. 2019. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinformatics 20:1160–1166.
  23. Kozlowska, J. and Seinhorst, J. W. 1979. Longidorus elongatus and closely related species in the Netherlands and lower Saxony (Germany), with the description of two new species, L. cylindricaudatus and L. intermedius (Nematoda: Dorylaimida). Nematologica 25:42–53.
  24. Lamberti, F. and Bleve Zacheo, T. 1977. Two new species of Longidorus (Nematoda: Longidoridae) from Italy. Nematologia Mediterranea 5:73–78.
  25. Lamberti, F. , Molinari, S. , De Luca, F. , Agostinelli, A. and Di Vito, M. 1999. Longidorids (Nematoda, Dorylaimida) from Syria with description of Longidorus pauli sp. n. and Paralongidorus halepensis sp. n. with SOD isozymes and PCR-RFLP profiles. Nematologia Mediterranea 27:63–78.
  26. Lazarova, S. S. , Malloch, G. , Oliveira, C. M. G. , Hübschen, J. and Neilson, R. 2006. Ribosomal and mitochondrial DNA analyses of Xiphinema americanum-group populations. Journal of Nematology 38:404–410.
  27. Loof, P. A. A. 1982. Two new species of Longidoridae (Dorylaimida) from Saudi Arabia. Nematologica 28:307–317
  28. Loof, P. A. A. and Chen, Q. 1999. A revised polytomous key for the identification of the species of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Supplement 1. Nematology 1:55–59.
  29. Mali, V. R. and Hooper, D. J. 1974. Observations on Longidorus euonymus n. sp. and Xiphinema vuittenezi Luc et al., 1964 (Nematoda: Dorylaimida) associated with spindle trees infected with Euonymus Mosaic Virus in Czechoslovakia. Nematologica 19:459–467.
  30. Merny, G. 1966. Nematodes from tropical Africa: a new Paratylenchus (Criconematidae) two new Longidorus and observations on Longidorus laevicapitatus Williams, 1959 (Dorylaimidae). Nematologica 12:385–395.
  31. Micoletzky, H. 1922. Die freilebenden Erd-Nematoden. Archiv fur Naturgesrchichte 87:1–650.
  32. Palomares-Rius, J. E. , Cantalapiedra-Navarrete, C. , Archidona-Yuste, A. , Subbotin, S. A. and Castillo, P. 2017. The utility of mtDNA and rDNA for barcoding and phylogeny of plant-parasitic nematodes from Longidoridae (Nematoda, Enoplea). Scientific Reports 7:10905.
  33. Pedram, M. , Niknam, G. , Robbins, R. T. , Ye, W. and Karegar, A. 2008. Longidorus kheirii n. sp. (Nematoda: Longidoridae) from Iran. Systematic Parasitology 71:199–211.
  34. Peneva, V. K. , Lazarova, S. S. , De Luca, F. and Brown, D. J. F. 2013. Description of Longidorus cholevae sp. n. (Nematoda, Dorylaimida) from a riparian habitat in the Rila Mountains, Bulgaria. ZooKeys 330:1–26.
  35. Rambaut, A. 2014. FigTree v1.4.2, A Graphical Viewer of Phylogenetic Trees, available at: http://tree.bio.ed.ac.uk/software/figtree/.
  36. Roca, F. 1996. Longidorus apuloides n. sp. (Nematoda: Longidoridae) from Italy with a hierarchical cluster analysis of the closely related species. Fundamental and Applied Nematology 19:315–320.
  37. Roca, F. and Lamberti, F. 1981. Longidorus fasciatus sp. n. from Greece and Italy. Nematologia Mediterranea 9:175–179.
  38. Ronquist, F. and Huelsenbeck, J. P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574.
  39. Stoyanov, D. 1964. A contribution to the nematofauna of the grapevine. Rastitelna Zashtita Sofia 12:16–24
  40. Sturhan, D. and Argo, D. 1983. Studies on Longidorus closelongatus Stoyanov and L. cohni Heyns, with description of L. proximus sp. nov. (Nematoda, Dorylaimida). Revue Nematologie 6:57–64.
  41. Sturhan, D. and Barooti, S. 1983. Longidorus iranicus n sp. (Nematoda: Dorylaimida). Systematic Parasitology 5:21–4.
  42. Taylor, C. A. and Brown, D. J. F. 1997. Nematode vectors of plant viruses CAB International. Wallingford: CAB International.
  43. Tzortzakakis, E. A. , Peneva, V. , Brown, D. J. F. and Avgelis, A. D. 2008. A literature review on the occurrence of nematodes of the family Longidoridae in Greece. Nematologia Mediterranea 36:153–156.
  44. Tzortzakakis, E. A., Peneva, V., Terzakis, M., Neilson, R. and Brown, D. J. F. 2001. Longidorus cretensis n. sp. (Nematoda: Longidoridae) from a vineyard infected with a foliar ‘yellow mosaic’ on Crete, Greece. Systematic Parasitology 48:131–9.
  45. Tzortzakakis, E. A. , Cantalapiedra-Navarrete, C. , Castillo, P. , Palomares-Rius, J. E. and Archidona-Yuste, A. 2017. Morphological & molecular identification of Longidorus euonymus and Helicotylenchus multicinctus from the rhizosphere of grapevine & banana in Greece. Journal of Nematology 49:233–235.
  46. Tzortzakakis, E. A. , Archidona-Yuste, A. , Cantalapiedra-Navarrete, C. , Nasiou, E. , Lazanaki, M. S. , Kabourakis, E. A. , Palomares-Rius, J. E. and Castillo, P. 2014. Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete, Greece, with description of Xiphinema cretense n. sp. (Nematoda, Longidoridae). European Journal of Plant Pathology 140:563–590.
  47. Vrain, T. C. , Wakarchuk, D. A. , Levesque, A. C. and Hamilton, R. I. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15:563–573.

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