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
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).
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
D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and coxI mtDNA sequences of the unidentified
Soil samples from grapevine at Thessaloniki, North Greece yielded two
Morphometrics of
Thessaloniki, Greece | Idleb, Syria (Lamberti et al., 1999) | |||
---|---|---|---|---|
Charactera | Females | Males | Females | Males |
|
8 | 5 | 20 | 12 |
|
7.6 ± 0.66 (6.62-8.51) | 7.19 ± 0.27 (6.66-8.02) | 7.6 ± 0.51 (6.5-8.6) | 7.7 ± 0.64 (6.8-8.7) |
|
147.9 ± 15.3 (125.0-168.8) | 150.1 ± 8.9 (138.3-162.5) | 131.6 ± 8.5 (120.3-143.5) | 139.9 ± 7.9 (130.8-154.8) |
|
16.0 ± 1.7 (13.9-19.0) | 16.5 ± 1.3 (14.8-17.8) | 16.9 ± 1.6 (14.5-19.7) | 16.1 ± 1.8 (12.8-19.5) |
|
173.1 ± 29.0 (126.9-224.0) | 158.9 ± 12.3 (146.1-175.3) | 200.7 ± 17.9 (163.5-220.0) | 181.9 ± 11.7 (166.0-197.8) |
|
1.1 ± 0.1 (1.0-1.3) | 1.2 ± 0.1 (1.0-1.3) | 0.9 ± 0.1 (0.8-1.0) | 0.97 ± 0.05 (0.9-1.0) |
|
2.1 ± 0.2 (1.9-2.4) | 2.3 ± 0.2 (2.0-2.6) | – | – |
|
1.6 ± 0.1 (1.4-1.8) | 1.5 ± 0.1 (1.4-1.6) | – | – |
V/Spicules length | 52.1 ± 2.2 (49.4-56.0) | 57.6 ± 0.4 (54.0-60.0) | 51 ± 1.5 (49.0-54.0) | 64.6 ± 2.5 (61.0-69.0) |
Odontostyle length | 117.4 ± 5.5 (112.0-126.0) | 113.6 ± 7.4 (103.0-120.0) | 109.4 ± 3.6 (102.0-118.3) | 109.0 ± 4.7 (101.5-117.7) |
Odontophore length | 63.5 ± 2.2 (61.0-67.0) | 63.4 ± 3.3 (59.0.68.0) | 61.2 ± 2.5 (56.0-64.0) | 63.0 ± 1.8 (61.0-66.5) |
Total stylet length | 180.9 ± 6.8 (173.0-192.0) | 177.0 ± 6.7 (167.0-184.0) | – | – |
Anterior end to guide ring | 31.6 ± 1.9 (28.0-33.5) | 33.2 ± 1.3 (32.0-35.0) | 30.6 ± 2.1 (27.2-35.8) | 30.4 ± 1.6 (27.7-32.9) |
Tail length | 42.9 ± 2.1 (38.0-45.0) | 46.6 ± 1.3 (44.0-49.0) | 37.8 ± 3.2 (31.5-45.0) | 42.4 ± 3.3 (36.4-46.3) |
Hyaline part of tail length | 16.4 ± 1.5 (14.0-18.5) | 14.3 ± 2.0 (12.5-17.0) | 13.1 ± 1.1 (10.5-15.4) | 11.5 ± 1.6 (10.0-14.9) |
Body width at level of | ||||
lip region | 14.9 ± 0.7 (13.5-16.0) | 14.8 ± 1.0 (13.5-16.0) | 15.2 ± 0.9 (14.0-17.0) | 14.2 ± 0.5 (13.9-15.0) |
guide ring | 23.1 ± 1.5 (21-24.5) | 22.6 ± 1.5 (20.0-23.5) | 23.5 ± 1.1 (21.0-25.0) | 21.8 ± 1.0 (19.6-23.0) |
anus | 38.8 ± 1.3 (37.0-41.5) | 36.6 ± 1.5 (32.0-39.0) | 41.4 ± 2.4 (36.6-44.6) | 42.6 ± 2.3 (37.6-45.7) |
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.
The Greek population of
This species is morphological- and morphometrically close
Molecular characterization and phylogeny of
The ITS1 region also showed a low intraspecific variability by 0 to 2 nucleotides and 1 indel (99% similarity). ITS1 for
The four new coxI sequences for
Phylogenetic relationships among
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
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
In summary, the present study confirms the correct identity of this nematode and increase the great biodiversity of this genus in the Mediterranean Basin.