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

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.


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 mis takes 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´-ACAAGTACCGTGAGGGAAAGT TG-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´-AC GAGCCGAGTGATCCACCG-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 × 10 6 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 cm 3 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;Pedram et al., 2008), no further studies were developed on this new Greek population.

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 ( 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)

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 cm 3 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) 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    from Greece.    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. (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 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.
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   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.
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.