Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia

Abstract A new needle nematode, Longidorus carniolensis sp. n., recovered from the soil around the roots of grapevine Vitis vinifera L. from Slovenia, is described and illustrated. Longidorus carniolensisis an amphimictic species, characterised by females with a moderately long (L=5.6–8.2 mm) and plump (a=51–72.4, ave. 66.3) body, assuming a spiral to C-shape when heat relaxed. Head region continuous, anteriorly almost flat, lip region 23–25 µm wide; guiding ring situated posteriorly (42–47 μm, 43–50 μm in males), odontostyle long (ave. 146.6 (136–157) μm); pharyngeal glands with normal location, their nuclei of approximately equal size; tail bluntly conoidal to almost hemispherical. Males abundant, spicules slender and long (122–145 μm), ventromedian supplements 13–17, irregularly spaced, preceded by an adanal pair. Four juvenile stages present, the first stage juvenile with bluntly conoidal tail. Codes for identifying the new species when using the key by Chen et al. (1997) are: A 56, B 4, C 4, D 1, E 4, F 35, G 1, H 1, I 2. The new species is morphologically the most similar to Longidorus poessneckensis Altherr, 1974, Longidorus macrosoma Hooper, 1961, Longidorus caespiticola Hooper, 1961, Longidorus helveticus Lamberti et al., 2001, Longidorus macroteromucronatus Altherr, 1974, Longidorus pius Barsi & Lamberti, 2001, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram et al. 2008, Longidorus silvae Roca, 1993, Longidorus iuglandis Roca et al., 1985, Longidorus vinearum Bravo & Roca, 1995 and Longidorus major Roca & d’Erico, 1987, but differs from these species either by the body and odontostyle length, position of guide ring, head region and tail shape or the shape of the first stage juvenile tail. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other speciesof the genus Longidorus with known sequences. Relationships of Longidorus carniolensis sp. n. with other Longidorus species based on analysis of this DNA fragment and morphology are discussed.


introduction
The nematodes of the genus Longidorus Micoletzky, 1922 cause damage to many economically important crops by direct feeding on their roots. Additionally, they can cause indirect damage to the host plants by transmitting plant viruses. To date, six Longidorus species have been reported from Slovenia (Širca and Urek 2009): L. elongatus (de Man, 1876) Micoletzky, 1922, L. caespiticola Hooper, 1961, L. juvenilis Dalmasso, 1969 helveticus Lamberti, Kunz, Grunder, Molinari, De Luca, Agostinelli & Radicci, 2001, L. leptocephalus Hooper, 1961, L. moesicus Lamberti, Choleva & Agostinelli, 1983. The study of relationships between longidorids and Nepoviruses in Slovenia and Bulgaria in the frame of a bilateral project, revealed the presence of a new species described herein as L. carniolensis sp. n. The description of the new species is based both on morphological and molecular data, in particular the sequence of D2D3 expansion regions of the large subunit rDNA nuclear gene which proved to be useful in molecular phylogenetic analyses of Longidoridae (Rubtsova et al. 2001, Ye et al. 2004, He et al. 2005. Additionally, sequences of these domains allow species differentiation (Širca and Urek 2009).

Materials and methods
Soil samples were collected in July 2008 and October 2009 from the rhizosphere of Vitis vinifera L. in Drašiči and Krmačina localities in the southern part of Slovenia. The sampling was performed by digging holes beneath grapevine plants and carefully collecting soil around the roots at 40-50 cm depth. Approximately 500 cm 3 of a collected soil sample was gently mixed and two 200 cm 3 sub-samples were processed. Nematodes were extracted from the soil using a decanting method followed by the Baermann funnel technique. Longidorid nematodes for morphological study were hand-picked, fixed in TAF (7 ml 40% formalin, 2 ml tri-ethanolamine, and 91 ml distilled water), processed to glycerol (Seinhorst 1959) and mounted on glass microscope slides in anhydrous glycerol.
Drawings and photographs were taken using an Olympus BX51 compound microscope powered with differential interference contrast (DIC). Images were taken with a ColorView IIIu camera and cell^P software (Olympus Soft Imaging Solutions Gmbh). Measurements were made using an Olympus BX 41 light microscope, a digitising tablet (CalComp Drawing Board III, GTCO CalCom Peripherals, Scottsdale, AZ, USA), and Digitrak 1.0f programme (Philip Smith, Scottish Crop Research Institute, Dundee, UK).

Total DNA extraction and amplification
Extracted female nematodes for molecular study were transferred into 1.5 ml tube in a 1 μl drop of sterile water. DNA was extracted from a single female nematode from typelocality Drašiči and from Krmačina locality; 10 μl 1M EDTA pH 8 and 50 μl nucleic lysis solution (Promega Wizard DNA purification kit) mixture was added to each tube and homogenised with micropestle. Isolation of DNA was continued according to manufacturer's instructions. Isolated DNA was re-suspended in 10 µl of distilled water of which 2 μl was used in each PCR reaction. A fragment of the D2 and D3 expansion region of the 28S rDNA gene was amplified using the primers D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (Rubtsova et al. 2001) in a PCR cycler and conditions as described earlier (Širca et al. 2007).

Analyses of rDNA sequence
Obtained PCR products were purified using the JetQuick PCR purification spin kit (Genomed) and sequenced on an ABI PRISM 310 DNA Sequencer using BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), the sequences obtained were submitted to the GenBank. Cluster analyses were performed using sequences of several Longidorus species from the NCBI GenBank (http://www.ncbi.nlm. nih.gov/) obtained from different phylogenetic studies (Rubtsova et al. 2001, Handoo et al. 2005, He et al. 2005, Lišková 2007, Kumari et al. 2009, Širca and Urek 2009 (Table 1). Xiphinema index (AY601628) (He et al. 2005) was used as an out-group. For cluster analyses and tree construction a Neighbour-Joining method was applied using MEGA5 software (Tamura et al. 2011).
Juveniles. Four developmental stages clearly present (Fig. 11) as determined from the position of the replacement odontostyle and the principal morphometric characters of body, odontostyle and replacement odontostyle lengths, and developing gonad (genital primordium) size. The habitus of juveniles not changing considerably during successive stages, assuming J or C shape. In first stage juvenile, lip region somewhat different from the next stages, it is rounded with a very weak depression after the second circle of labial papillae, the latter slightly protruding and changing the lip region outline. Amphidial fovea in first two stages has no clearly visible receptors, only small refractive elements discernable. Both the tail and body width at anus is increasing in length and c' ratio is decreasing. Tail shape in J1 is conoidal, ventrally almost straight or slightly concave, dorsally convex, which gives asymmetrical appearance, in successive stages it gradually becomes rounded but always with the dorsal curvature more strongly expressed.
Etymology. The species epithet carniolensis was derived from Carniola which is the Latin name of the Kranjska province, a historical region that comprised parts of what is now Slovenia.
rDNA sequence analysis. Cluster analyses of the D2-D3 expansion regions of the 28S rDNA nuclear gene sequences of L. carniolensis sp. n. and closely related species (Table 1) were performed and a phylogenetic tree was constructed (Fig. 12). The sequences of both populations of L. carniolensis sp. n. from Drašiči and Krmačina were identical. They formed a distinct clade within a cluster of the closely related sequences of L. poessneckensis, L. helveticus, L. macrosoma, L. caespiticola and L. latocephalus. The closest sequence to L. carniolensis sp. n. was the sequence of L. poessneckensis (Acc. No EF538750) with 91.9% of similarity.

Discussion
There are some characteristic morphological features observed in L. carniolensis sp. n. such as the presence of vestigium, hemizonid and hemizonion, and the abberant ventral pore. The vestigium was present in all specimens (males and females), it was located in the slender pharynx, behind odontophore, in few specimens in the odontophore area. Such a vestigium has been reported also for Longidorus fursti Heyns, Coomans, Hutsebaut & Swart, 1987 from South Africa; two Chinese Longidorus species (Xu and Cheng 1992); it is more frequently observed in Xiphinema spp. (Kruger and Heyns 1987, Swart 1994, Swart and Quénéhervé 1998Mincheva et al. 2008), reported also for several species of Xiphidorus (Decraemer et al. 1996) and Paraxiphidorus brevistylus (Decraemer et al. 1998).

L. carpetanensis
The only available data on the excretory system in Longidorus refers to L. macrosoma in which a ventral excretory pore at the level of the nerve ring, leading to a noncanalicular tissue in its anterior part has been observed together with two nucleated glands embedded in the tissues of a ventrally located ampulla-like structure (Aboul-Eid 1969). In L. carniolensis sp. n. we observed an aberrant ventral pore in all adults, differing in structure from the other ventral pores and also having a longer duct (Figs 1A, 4F and 6H), it probably functions as more specialised part of the excretory system. It was also detected in juvenile stages (Fig. 8B, D).
The data on D2D3 rDNA regions of majority of longidorid species, particularly of those belonging to the genus Longidorus is far to be complete; this does not facilitate the reconstruction of the phylogenetic relationships among the members of this widely distributed group of ectoparasitic nematodes. Despite of this, based on the rDNA results as well as a combination of morphological features the new species is included in a clearly defined group of closely related species (L. poessneckensis (92% similarity), L. macrosoma and L. caespiticola (90%), L. helveticus (89%), sharing some common characters -amphids with pouch-like fovea, not bilobed, amphidial duct well discernable, tapering lip region, which is continuous with the rest of body, normal arrangement of pharyngeal glands, bluntly conoidal to hemispherical tail, much shorter or equal to the anal body width; and the development through 4 juvenile stages. All these species occur in Europe, more frequently in West and Central Europe. The correlation between the amphid structure and clustering of longidorid species has been underlined by Rubtsova et al. (2001) and He et al. (2005) and it is supported by our study.