ZooKeys 141: 1–27, doi: 10.3897/zookeys.141.1906
Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia
Saša Širca1,†, Gregor Urek1,‡, Stela Lazarova2,§, Milka Elshishka2,|, Vlada Peneva2,¶
1 Agricultural Institute of Slovenia, Hacquetova ulica 17, 1001 Ljubljana, Slovenia
2 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2, Yu. Gagarin Street, 1113 Sofia, Bulgaria

Corresponding author: Vlada Peneva (vpeneva@ecolab.bas.bg)

Academic editor: S. Subbotin

received 12 July 2011 | accepted 24 October 2011 | Published 28 October 2011


(C) 2011 Saša Širca. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


For reference, use of the paginated PDF or printed version of this article is recommended.

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.

Keywords

grapevine, morphology, taxonomy, 28S rDNA

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): Longidorus elongatus (de Man, 1876) Micoletzky, 1922, Longidorus caespiticola Hooper, 1961, Longidorus juvenilis Dalmasso, 1969, Longidorus helveticus Lamberti, Kunz, Grunder, Molinari, De Luca, Agostinelli & Radicci, 2001, Longidorus leptocephalus Hooper, 1961, Longidorus 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 Longidorus 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 cm3 of a collected soil sample was gently mixed and two 200 cm3 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 ml drop of sterile water. DNA was extracted from a single female nematode from type-locality Drašiči and from Krmačina locality; 10 ml 1M EDTA pH 8 and 50 ml 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).

Table 1.

Species of fam. Longidoridae used in phylogenetic reconstructions.

GenBank accession number Nematode species Origin Reference
AY601583 Longidorus africanus Merny, 1966 California, USA He et al. 2005
AY494715 Longidorus americanum Handoo, Carta & Skantar, 2005 Georgia, USA Handoo et al. 2005
AY601571 Longidorus apulus Lamberti & Bleve-Zacheo, 1977 Mola di Bari, Italy He et al. 2005
AY601570 Longidorus arthensis Brown, Grunder, Hooper, Klingler & Kunz, 1974 Suter, Switzerland He et al. 2005
AY601574 Longidorus athesinus Lamberti, Coiro & Agostinelli, 1991 Italy He et al. 2005
AY601572 Longidorus attenuatus Hooper, 1961 Germany He et al. 2005
AY601576 Longidorus breviannulatus Norton & Hoffmann, 1975 Nebraska, USA He et al. 2005
HM447030 Longidorus caespiticola Brdo, Slovenia Širca and Urek 2009
AY601585 Longidorus camelliae Zheng, Peneva & Brown, 2000 Hangzhou, China He et al. 2005
JN631811 Longidorus carniolensis sp. n. Krmačina, Slovenia This study
JN631812 Longidorus carniolensis sp. n. Drašiči, Slovenia This study
AF480072 Longidorus carpathicus Lišková, Robbins & Brown, 1997 Germany Rubtsova 2001
EF654539 Longidorus distinctus Lamberti, Choleva & Agostinelli, 1983 Kráľovský Chlmec, Slovakia Lišková 2007
AY593057 Longidorus dunensis Brinkman, Loof & Barbez, 1987 Holterman et al.
Unpublished
AY601575 Longidorus edmundsi Hunt & Siddiqi Caribbean sea beach, Cuba He et al. 2005
HM447032 Longidorus elongatus Maribor, Slovenia Širca and Urek 2009
AY601573 Longidorus euonymus Mali & Hooper, 1973 Zabagr, Hungary He et al. 2005
AY601581 Longidorus goodeyi Hooper, 1961 Peebles, Scotland, UK He et al. 2005
HM447031 Longidorus helveticus Trška gora, Slovenia Širca and Urek 2009
AF480074 Longidorus intermedius Kozlowska & Seinhorst, 1979 Germany Rubtsova et al. 2001
DQ364599 Longidorus juvenilis Svetinje, Slovenia Širca et al. 2007
AY601568 Longidorus latocephalus Lamberti, Choleva & Agostinelli, 1983 Greece He et al. 2005
DQ364600 Longidorus leptocephalus Juršinci, Slovenia Širca et al. 2007
AY601565 Longidorus macrosoma Hooper, 1961 Switzerland He et al. 2005
HM447029 Longidorus moesicus Vrhpolje, Slovenia Širca and Urek 2009
AY601577 Longidorus piceicola Lišková, Robbins & Brown, 1997 Branisko, Slovakia He et al. 2005
EF538750 Longidorus poessneckensis Altherr, 1974 Cerne Voderady, Czech Republic Kumari et al. 2009
AF480073 Longidorus profundorum Hooper, 1965 Germany Rubtsova et al. 2001
AF480071 Longidorus sturhani Rubtsova, Subbotin, Brown & Moens, 2001 Belgium Rubtsova et al. 2001
EF538754 Longidorus uroshis Krnjaic, Lamberti, Krnjaic, Agostinelli & Radicci, 2002 Velke Pole, Slovakia Kumari et al. 2009
AY601628 Xiphinema index Thorne & Allen Argentina He et al. 2005
Taxonomy
Measurements.

See Table 2.

Table 2.

Measurements of females, males and juvenile stages of Longidorus carniolensis sp. n., from Slovenia (mean ± standard deviation, with range). All measurements in micrometers.

Character Holotype Females Males J1 J2 J3 J4
n n=1 n=13 n=14 n=15 n=6 n=11 n=9
L 7089 7447.5±679.0
5653-8226
7917.7±753.9
6702-9525
1349.9±53.8
1283-1449
2584.2±228.0
2329-2872
3692.2±238.0
3305-4149
5441.2±700.7
4677-6647
a 65.2 66.3±6.1
51.0-72.4
72.5±6.4
59.6-81.2
44.0±2.3
39.3-46.2
46.5±4.3
39.8-50.6
50.5±4.7
42.8-57.4
60.6±3.7
53.2-65.6
b 14.2 12.7±0.9
11.6-14.3
12.8±0.8
11.8-14.9
4.9±0.4
4.3-5.6
6.3±0.3
5.8-6.6
7.9±0.7
7.0-9.0
9.7±1.7
8.4-13.9
c 165.7 177.9±35.5
108.1-224.5
173.7±27.5
127.6-241.8
41.2±2.7
36.8-45.9
71.9±4.8
66.4-79.4
94.2±9.2
82.5-113.6
132.6±17.6
98.2-155.1
c’ 0.6 0.6±0.1
0.5-1.0
0.8±0.1
0.6-1.1
1.4±0.1
1.2-1.5
0.8±0.04
0.7-0.9
0.7±0.1
0.6-0.8
0.7±0.04
0.6-0.8
V (%) 49.7 49.4±1.4
47.1-51.5
G1 (%) 11.9 13.4±2.6
10.6-17.4
G2 (%) 11.8 13.3±2.6
9.9-17.3
d 2.0 1.8±0.1
1.7-2.0
2.1±1.1
1.0-5.8
2.0±0.1
1.8-2.2
1.9±0.1
1.8-2.1
1.9±0.1
1.7-2.1
1.7±0.6
0.3-2.0
d’ 2.0 2.0±0.2
1.8-2.3
1.9±0.1
1.8-2.1
1.7±0.1
1.6-1.9
1.8±0.2
1.7-2.1
1.9±0.1
1.7-2.2
1.9±0.4
0.8-2.2
Anterior end to guiding ring 45.4 44.6±1.6
42-47
46.5±2.3
43-50
21±0.7
20–22
28.7±1.6
26.5-30.5
33.9±1.0
32-35
39.8±1.2
38-42
Anterior end to nerve ring 267.5 258.6±15.4
220-275
270.7±9.5
249-289
129.6±8.9
114-145
182.1±31.0
153-228
207.5±24.1
176-273
231.1±15.9
213-252
Hemizonid 237.5 253.7±18.9
204-270
10.4±0.5
10-11
205.9±7.4
195-216, n=7
234.6±19.2
210-216, n=4
Odontostyle 144 147.5±4.7
136-157
149.1±6.6
132-159
81.9±3.5
76-88
88.2±5.0
79-94
107.7±4.4
98-114
125.9±3.6
120-131
Replacement odontostyle 87.6±2.5
85-93
104.9±2.4
101-108
123.7±3.2
119-130
146.0±3.9
142-152
Odontophore 92 91.4±4.1
85-97
90.2±6.4
75-99
49.6±3.9
43-56
62.4±2.3
59-66
72.9±4.2
64-78
85.1±3.4
81-91
Neck length 500 590.5±59.9
462-674
618.6±56.6
510-703
274.1±20.8
237-317
408.3±33.4
370-448
472.5±48.7
409-524
553.6±47.5
478-628
Pharyngeal bulb length 139 142.7±6.0
133-152
141.1±6.9
129-157
72.6±2.3
67-75
89.6±3.1
84-93
106.8±6.1
96-114
123.6±5.6
118-135
Pharyngeal bulb width 39 40.2±2.8
36-44
37.9±2.1
32-41
15.6±0.9
14-17
22.7±1.4
21-24
28.1±1.6
26-31
33.3±1.9
31-37
DO* 11.4 11.6±0.3
11.2-11.9
11.0±0.8
9.8-11.8
14.0±2.4
11.3-18.1
10.9±1.3
9.1-12.0
11.3±0.7
10.3-11.9
11.7±1.6
9.9-14.8
DN 37.1 38.1±2.6
32.8-40.2
37.9±3.0
33.2-42.9
38.6±1.9
36.7-42.7
37.2±1.7
35.5-40.3
37.0±1.0
35.6-38.4
36.8±3.7
30.0-42.6
LS1N 53.7 56.1±2.6
52.3-61.0
55.0±3.0
49.6-62.3
52.0±2.6
47.9-57.3
50.6±1.7
48.6-52.7
52.9±2.1
48.1-55.3
54.3±2.2
52.2-57.7
RS1N 53.7 54.6±2.5
51.5-58.6
55.5±2.6
52.4-61.2
51.5±2.7
46.3-55.3
50.9±1.4
48.9-52.7
53.3±2.3
48.1-56.3
53.5±2.5
50.7-57.4
S2O 83.4 84.9±1.6
82.3-87.1
86.3±5.7
81.9-102.5
84.2±1.0
82.7-85.8
84.8±2.7
83.1-90.2
85.0±1.6
81.6-87.1
82.8±0.9
81.6-84.1
Prerectum 347.5
419.0±85.9
280-550
576.8±182.2
248-832
141.7±60.7
81-290
183.8±30.1
150-224
289.1±50.0
175-363
363.6±91.7
275-538
Rectum 51.5
50.3±3.0
47-57
- 14.3±1.9
12-19
22.8±0.8
22-24
32.6±2.6
30-38
45.3±2.4
42- 49
Tail 42.8 43.3±9.2
34-69
46.4 ± 6.8
32-61
32.9±1.4
31-35
35.9±1.6
34-38
39.4±2.9
35.5-45
41.2±3.2
38-48
Length of hyaline part 20.2 17.8±0.7
17-19
16.9±1.2
14-19
7.9±0.9
6-9
10.4±0.7
9-11
13.1±0.9
11-15
14.9±0.8
13-16
Body diameter at:
- lip region
22.9 24.2±0.8
23-25
24.8±1.3
22-26.5
10.6±0.4
10-11
15.0±0.5
15-16
18.4±1.1
17-20
20.9±0.5
20-21
- guiding ring 46.6 48.5±3.3
44-55
48.0±2.3
45-52
18.3±0.5
18-19
26.8±2.2
25-31
35.7±2.7
31-41
43.0± 3.9
38.7-51
- base of pharynx 95.4 93.4±4.5
89-101
93.8±4.8
84-103
30.2±1.2
29-33
50.5±3.4
46-55
63.9±3.8
59-73
76.4±5.6
69.5-84
- mid-body/at vulva 108.7 112.9±9.5
97-127
109.3±5.4
98-117
30.7±1.2
29-33
55.8±4.0
49-60
73.5±5.6
66-85
89.8±10.0
79-105
- anus 68.5 68.2±4.3
60-75
61.2±3.2
55-66
24.3±1.0
22-26
44.4±1.7
42-46
55.2±3.6
51.3-60.5
63.1±3.6
59-70
- hyaline part 49.5 49.0±1.7
47-52
39.5±2.2
37-43
16.0±0.9
15-17
26.8±2.7
24-32
37.6±2.5
32-41
42.9±2.7
39-47
Spicules 126.9±5.8
122-145

* Following Loof and Coomans 1972


Description.

Female. Body moderately long (L=5.6–8.2 mm) and plump (a=51–72.4), assuming a spiral to C shape when heat relaxed. Cuticle consisting of several layers under light microscope: 11–14 µm thick at guiding ring level; 7–8 µm along the body; 13–15 µm on tail posterior to anus. Lateral pores number 10–14 in pharyngeal region: a single pore in front of guide ring, rarely two or none; 3–5 in odontostyle and 1–3 in odontophore regions; 3–4 dorsal pores and 7–10 ventral pores; numerous lateral body pores. Usually the fifth ventral pore (sometimes the fourth) differs in size (Figs 1A, 4F and 6H) compared to the other ventral pores. Lip region continuous, anteriorly almost flat, 7–9 μm high. Labial papillae prominent. Amphid aperture assumed to be a minute pore, difficult to be observed under light microscope. Pouch-like amphidial fovea with convoluted fine dendritic branches (receptors), extending to 1/2 - 2/3 the distance between anterior end and guiding ring, fovea slightly longer (15–18 μm, n=5) than wide (14–16 μm, n=4) with no distinct margins. Fusus (sensillium pouch) at 57±1.9 (55–60) μm from anterior end. Guiding ring 7–9 μm wide. Odontostyle long and very slender, 2 μm wide at the base. Odontophore with weakly developed flanges. In all females a small (2–3 μm long) rudimentary odontostyle tip (vestigium) present, directed forward, and observed in the slender pharynx at 300.5±40.3 (224–350) μm from anterior end; in two specimens the vestigium located in odontophore area. Slender pharynx often coiled in its posterior part. In this region 5–7 glandular bodies are observed in all females. Nerve ring surrounding odontophore base, rarely surrounding mid-odontophore, or just behind it, second nerve ring at a distance of 85.2±6.6 (78–98) μm behind the first one. Hemizonid flat, 10–11 μm long. Pharyngeal bulb about 1/4 of the neck length. Normal arrangement of pharyngeal glands, the nuclei of dorsal and ventrosublateral glands approximately the same size, their diameters 3.4±0.4 (3–4) μm, n=7 and 3.9±0.2 (3.5–4) μm, n=11, respectively. Cardia small, broadly rounded, wider than long, variable in size: 20.1±1.8 (10–23) × 10.1±1.8 (7–12) μm . Reproductive system amphidelphic, varying in dimensions due to the stage of maturity of female. Vagina extending about half body width. Pars distalis vaginae with characteristic shape (Fig. 2F, G), 26–28 µm and pars proximalis vaginae 32–38 µm long, respectively; muscular walls of the latter almost parallel. Uteri very long, anterior uterus 494.6±52 (430–563) µm long, posterior uterus 510.0±88.7 (357–643) µm long, differentiated, filled with sperm cells in all females examined; well developed sphincter between uterus and pars dilatata oviductus also containing numerous sperm cells. Anterior and posterior oviduct of similar size, measured in four specimens: 275–348 μm, and 283–330 μm. Anterior ovarium 263.4±51.8 (210–347) μm long, n=7, posterior ovarium 234.3±35.8 (183–309) μm long, n=5; in older mature specimens the length is about 3 times greater (1055–1060 μm for anterior and 1020 μm for posterior ovary). One egg in anterior pars dilatata oviductus measuring 227 × 87.5 μm and one uterine egg measuring 225 × 77.5 μm. A weakly developed ovijector present, 112.0±12 (95–125) μm long. In one female a rudimentary adanal pair of supplementary papillae was observed (Fig. 9E). Prerectum variable in length; rectum 0.7±0.1 (0.6–0.8) body width at anus. A short post-intestinal sac present. Tail bluntly conoidal, rounded to almost hemispherical; ventral side straight or slightly convex, the dorsal curvature greater. Two pairs of lateral pores.

Male. Body C shaped when heat relaxed, posterior part more strongly coiled ventrally. Similar to females in general morphology except for genital system. Lateral pores number 10–15 in pharyngeal region: a single pore in front of guide ring, 3–5 in odontostyle and 1–2 in odontophore regions; 2–5 dorsal pores, mostly 3–4, and 7–10 ventral pores. Cuticle in post-labial region at the guiding ring level 10.5–13.5 µm thick, 6.5–9 µm along body, 9–10 µm in post-cloacal area. Second nerve ring at 80.7±14 (50–100) μm behind the first one (n=14). In all males a small vestigium (2–3 μm, in one specimen 6 μm long), directed forward (in two specimens directed rearward), is observed in the slender pharynx at 300.5±40.3 (224–350) μm from anterior end; in two specimens the vestigium detected in odontophore area. Two to eleven glandular bodies observed in all males in posterior part of the slender pharynx and pharyngeal bulb. In two specimens lens-like hemizonion at a distance of 242 and 271 μm from anterior end observed. Pharyngeal bulb slightly less than 1/4 of neck length (22.9±1.6 (20.9–26.7%). Ventromedian supplements composed of one adanal pair and a row of 13–17 irregularly spaced single ones, the first three appear as double in some specimens. Spicules comparatively slender, of almost equal width along the length, curved to almost at right angle. Lateral guiding piece not bifid, with uneven internal walls. Post-cloacal papilla well developed. Tail short, bluntly conoidal, ventral side almost straight, dorsal side convex. Two or three pairs of lateral caudal pores.

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.

Figure 1.

Longidorus carniolensis sp. n. Female: A Neck region F Habitus H Pharyngeal bulb Male: G Habitus I Pharyngeal bulb; Juveniles: B–E Habitus of first, second, third and forth juvenile stages J–M Pharyngeal bulb of first, second, third and forth juvenile stages. Scale bars: B–G 1 mm; A, H–M 100 μm.

Figure 2.

Longidorus carniolensis sp. n. Female: A–D Anterior ends E Vestigium in the walls of the slender part of pharynx F, G Vulval region G Anterior genital branch. Scale bars: A–I 50 μm, J 100 μm.

Figure 3.

Longidorus carniolensis sp. n. Female: A Anterior region B–D Amphidial fovea E Vestigium F–H Vulval region I Vulval region, uterus and egg J Pharyngeal bulb, dorsal and subventral glands K, L Tail – different optical sections M Sphincter N Prerectum O–Q Variation in tail shape. Scale bars: I, N 200 μm; A–G, H–M, O–Q 50 μm.

Figure 4.

Longidorus carniolensis sp. n. Male: A–E Anterior end B, D, E in sublateral view F Excretory pore and ventral pores G Partly protracted spicules H–I Tail end. Scale bar: 50 μm.

Figure 5.

Longidorus carniolensis sp. n. Male: A, B Variation in tail shape. Scale bar: 50 μm.

Figure 6.

Longidorus carniolensis sp. n. Male: A Anterior region B, C Head region D–G Amphidial fovea H Vestigium (white arrow), excretory pore (thick arrow) and ventral pores (slender arrows) I Ejaculatory glands (marked by arrows) J Lateral field K, L Pharyngeal bulb with glandular bodies (marked by arrows) M, N Sperm cells at different stage of development. Scale bars: A 200 μm; B–N 50 μm.

Figure 7.

Longidorus carniolensis sp. n. Male: A Posterior genital branch B, C, E, F Tail and copulatory apparatus – different optical sections D, G Posterior end H Rectum (marked by arrow), spicules and lateral piece I Partly protracted spicules. Scale bars: A, D, G – 200 μm; B, C, E–F, H, I – 50 μm.

Figure 8.

Longidorus carniolensis sp. n. Juveniles: A Neck region of first stage B–D Spear region of second, third and fourth stage. Scale bar: 50 μm.

Figure 9.

Longidorus carniolensis sp. n. Evolution of the tail. A–D Tail of first–fourth juvenile stage E Tail of female. Scale bar: 100 μm.

Figure 10.

Longidorus carniolensis sp. n. Juvenile: A–D Anterior region of first, second, third and forth stages H–K Pharyngeal bulb of first, second, third and forth juvenile stages M, F, G, R genital primordium of first, second, third and forth stages N, S Tail shape of first stage O, T Tail shape of second stage P, U Tail shape of third stage Q, V Tail shape of forth stage Female: E Anterior region L Pharyngeal bulb W Tail shape. Scale bar: 50 μm.

Figure 11.

Longidorus carniolensis sp. n. Scatter plot of the functional (, dark blue) and replacement odontostyle (, light blue) in relation to the body length of the juvenile stages and adults: females (, dark blue) and males (, red), female with very short odontostyle (, green).

Differential diagnosis and relationships.

Longidorus carniolensisis an amphimictic species, characterized by females with a moderately long (L=5.6–8.2 mm) and plump (a=51–72) 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), long odontostyle (146.6 (136–157) μm), distribution of pharyngeal glands normal, nuclei of approximately equal size, tail bluntly conoid to hemispherical. Males abundant, spicules slender and long (122–145 μm), ventromedian supplements 13–17, irregularly spaced and preceded by an adanal pair. Postembrional development through four juvenile stages.

The codes for identifying the new species when using the polytomous 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 species belongs to the group of species with long odontostyle – over 100 μm and bluntly conoid to hemispherical tail: Longidorus poessneckensis, Longidorus macrosoma, Longidorus caespiticola, Longidorus helveticus, Longidorus macroteromucronatus Altherr, 1974, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram, Niknam, Robbins, Ye & Karegar, 2008, Longidorus pius Barsi & Lamberti, 2001, L. nevesi Macara, 1986, Longidorus major Roca & d’Erico, 1987, Longidorus carpathicus, Longidorus piceicola, Longidorus vinearum Bravo & Roca, 1995, Longidorus pauli Lamberti, Molinari, De Luca, Agostinelli & Di Vito, 1999, Longidorus arthensis, Longidorus iuglandis Roca, Lamberti & Agostinelli, 1985, Longidorus picenus Roca, Lamberti & Agostinelli, 1985, Longidorus silvae Roca, 1993, Longidorus uroshis, Longidorus saginus Khan, Seshardi, Weischer & Mathen, 1971, Longidorus orongorongensis Yeates & Van Etteger, 1992, Longidorus cretensis Tzortzakakis, Peneva, Terzakis, Neilson & Brown, 2001, Longidorus cylindricaudatus Krnjaić, Roca, Krnjaic & Agostinelli, 2005, Longidorus fasciatus Roca & Lamberti, 1981 and Longidorus litchii. Longidorus carniolensis sp. n. can be differentiated from all these species either by morphometrics or/and quantitative characters. It differs from:

Longidorus poessneckensis – by its somewhat longer odontostyle (ave. 147.5 (136–157) vs ave. 133 (122–142), ave. 126 (122–130) and ave. 140.2 (132–148) μm); more posteriorly sitated guiding ring (ave. 44.6 (42–47) vs ave. 40 (36–43) and 39 ave. (37–40) μm); tail short conoidal vs elongate conoid in J1 (c’=1.2–1.5 vs 1.8–2.2 and 1.8–2.5); males abundant vs males very rare (Sturhan and Loof 2001, Lišková and Kumari 2010, Kornobis and Peneva 2011);

Longidorus macrosoma – by its shorter (5.6–8.2 vs 8.4–11.9 mm) and more plump (a=51–72.4 vs 77–113 and 105–126) body; differently shaped lip region (laterally rounded vs truncated, flattened); somewhat longer odontostyle (136–157 vs 113–148 μm); different tail shape in J1, bluntly conoidal vs mucronate; longer spicules in males 122–145 vs 105 μm and ave. 116.2 (112–121) μm (Hooper 1961, Lamberti et al. 2001);

Longidorus caespiticola – by its longer odontostyle (136–157 vs 100–120, 96–109 μm); different numbers of dorsal (2–5 vs a single pore) and ventral (7–10 vs 5–6) pores; more posteriorly sitated guiding ring (42–47 vs 30–41, 37, 42.5 μm); longer spicules in males (122–145 vs 90, 80–95 μm), smaller c’ value in J1 (1.2–1.5 vs almost 2) (Hooper 1961, Širca and Urek 2009);

Longidorus helveticus – by different tail shape of J1 being bluntly conoid vs mucronated; longer (122–145 vs 104–118 μm ) and differently shaped spicules (Lamberti et al. 2001);

Longidorus macroteromucronatus – by having more posteriorly situated guide ring (42–47 vs 38 μm); thicker cuticle along the body (6–7 vs 4 μm) and on tail region (9–10.5 vs 13–15 μm); longer odontostyle (136–157 vs 133 μm) (Altherr 1974);

Longidorus raskii – by its wider lip region (23–25 vs 15–19 and 14–16 μm); more posteriorly situated guiding ring (42–47 vs 33–38 and 33–43 μm); longer odontostyle (136–157 vs 90–103 and 98–100 μm);longer spicules (122–145 vs 82–103 and 79–90 μm) (Lamberti and Agostinelli 1993, Lamberti et al. 2001, Krnjaić et al. 2002);

Longidorus kheirii – by itslonger odontostyle (136–157 vs 111–130 μm); different tail shape in J1 (bluntly conoidal vs mucronated); males abundant vs males rare; longer spicules (122–145 vs 80 μm) (Pedram et al. 2008);

Longidorus pius – by its more posterior position of the guiding ring (42–47 vs 35–42 and 37–42.5 μm); different tail shape in J1 (bluntly conoidal vs mucronated); males abundant vs males absent (Barsi and Lamberti 2001a, Barsi and De Luca 2008)

Longidorus nevesi – by having wider lip region (23–25 vs 16–22 μm), different amphidial fovea shape (pouch like, not bilobed vs bilobed); differently shaped and longer spicules in males (122–145 vs 87–100 μm) (Macara 1986);

Longidorus major – by having shorter body (L=5.6–8.2 vs 8.5–12 mm); somewhat narrower lip region (23–25 vs 22–27 μm); different tail shape in J1 (bluntly conoidal vs mucronate) and amphidial fovea (pouch like, not bilobed vs bilobed), males abundant vs males absent (Roca and d’Erico 1987);

Longidorus carpathicus – by its longer body (L=5.6–8.2 mm vs 6.2–6.5 mm); wider (23–25 vs 16–18 μm) and differently shaped lip region; lower c’ value (c’= ave. 0.6 (0.5–1.0) vs c’= 0.8); different shape in J1 (bluntly conoidal vs mucronated with a rather long mucro); males abundant vs males absent (Lišková et al. 1997);

Longidorus piceicola – by having longer body (L=5.6–8.2 vs 4.2–6.5, 4.4–8.0 and 5.2–7.9 mm); wider (23–25 vs 14–17 μm) and differently shaped lip region (continious, almost flat vs broadly rounded); lower c’ value (c’=ave. 0.6 (0.5–1.0) vs c’=0.9–1.3); differently shaped tail in J1 (bluntly conoidal vs elongate conoid) (Lišková et al. 1997, Barsi and Lamberti 2001b);

Longidorus vinearum – by having different lip region shape (abruptly vs gradually tapering), different shape of amphidial fovea (pouch like not bilobed vs irregularly bilobed); longer odontostyle (136–157 vs 105.5–132.5 μm); different tail shape in J1 (bluntly conoidal vs conical, c’=1.2–1.5 vs 1.9–2.8) (Bravo and Roca 1995);

Longidorus pauli – by having different (continious vs slightly offset) and wider (23–25 vs 14–17 μm) lip region, amphidial fovea pouch like, not bilobed vs bilobed; longer odontostyle (136–157 vs 102–118 μm); lower a and c’ values (a=51.0–72.4 vs a=120.3–143.5; c’= ave. 0.6 (0.5–1.0) vs c’=0.9 (0.8–1.0), respectively); more posteriorly situated guiding ring (42–47 vs 27–36 μm); longer spicules (122–145 vs 61–69 μm); different tail shape in J1 (bluntly conoidal vs subdigitate) (Lamberti et al. 1999);

Longidorus arthensis – by itswider (23–25 vs 14–17 μm) lip region, amphidial fovea pouch like not bilobed vs bilobed; longer odontostyle (136–157 vs 102–111 μm); lower c’ values (c’=av 0.6 (0.5–0.1) vs c’=av 0.9 (0.8–1.1); more posteriorly sitated guiding ring (42–47 vs 30–38 μm); longer spicules (122–145 vs 60–66 μm); different tail shape in J1 (bluntly conoidal vs mucronated) (Brown et al. 1994);

Longidorus iuglandis – by its wider lip region (23–25 vs 14–16 μm); amphidial pouches not bilobed vs bilobed; longer odonostyle (136–157 vs 112–128 μm); more posterior position of the guiding ring (42–47 vs 31–41 μm); longer tail (34–69 vs 33–41 μm); longer spicules (122–145 vs 93–99 μm); different tail shape in J1 (bluntly conoidal vs mucronated) (Roca et al. 1985);

Longidorus picenus - by its wider lip region (23–25 vs 19–22 μm); amphidial fovea not bilobed vs bilobed; more posterior position of the guiding ring (42–47 vs 31–41 μm); longer spicules (122–145 vs 103–112 μm); different tail shape in J1 (bluntly conoidal vs mucronated) (Roca et al. 1985);

Longidorus silvae - by its more plump body (a=51.0–72.4 vs a=87.5–137.5 in Italian population and a=87.4–116 in Serbian populations), wider lip region (23–25 vs 14–17 μm); amphidial fovea not bilobed vs bilobed; longer odontostyle (136–157 vs 113.5–133 μm (Italian population) and 108–136 μm (Serbian populations)); different tail shape in J1 (bluntly conoidal vs mucronated); males abundant vs males rare; longer spicules (122–145 vs 77–78 μm) (Roca 1993, Barsi and Lamberti 2004, Barsi et al. 2007);

Longidorus uroshis – by having wider (23–25 vs 15–20.5 μm) lip region; lower a values (a=51.0–72.4 vs a=96.9–108.9); different tail shape in J1 (bluntly conoidal vs mucronated); longer spicules (122–145 vs 59–72 μm) (Krnjaić et al. 2000);

Longidorus saginus – by having longer body (L=5.6–9.5 vs 4.8–6.4 mm); amphidial fovea pouch shaped not bilobed vs asymetrically bilobed; longer tail (34–69 vs 21–33 μm) (Khan et al. 1971);

Longidorus orongorongensis – by its more anterior position of the guiding ring (42 -47 vs 63–73 μm); shorter odontostyle (136–157 vs 152–166 μm); longer spicules (122–145 vs 84–87 μm) (Yeates et al. 1992);

Longidorus cretensis – by having normal vs abnormal location of pharyngeal glands; wider lip region (23–25 vs 17–21 μm); longer spicules (122–145 vs 71–91 μm); different tail shape in J1 (bluntly conoidal vs conoid pointed) (Tzortzakakis et al. 2001);

Longidorus cylindricaudatus – by having lip region abruptly vs gradually tapering; amphidial fovea not bilobed vs bilobed; shorter odontostyle (136–157 vs 164–178 μm); lower a values (a=51–72.4 vs a=94.4–113.4); males abundant vs males absent (Krnjaić et al. 2005);

Longidorus fasciatus – by its wider lip region (23–25 vs 12–14 μm); different amphidial poches (not bilobed vs asymmetrically bilobed); longer odontostyle (136–157 vs 102–119 μm; male abundant vs males absent (Roca and Lamberti 1981);

Longidorus litchii – by its somewhat shorter odontostyle (136–157 vs 138–171 mm); different amphidial poches (not bilobed vs bilobed); more anterior postion of the guiding ring (42–47 vs 82.5–96.5 μm); different tail shape in J1 (bluntly conoidal vs elongate conoid with long digitate tip, c’=1.2–1.5 vs c’=2.7–3.4); longer spicules (122–145 vs 68.5–71 μm) (Xu and Cheng 1992).

Type-locality and plant association.

An old vineyard with roots of several Vitis vinifera varieties close to Drašiči village in southern part of Slovenia (45°39'N; 15°23'E), 229 m above sea-level.

Other-locality: a vineyard close to Krmačina village in southern part of Slovenia.

Distribution notes.

Longidorus carniolensis n. sp were detected in 6 out of 10 soil samples from locations of Drašiči and Krmačina. Population density was 4–15 specimens of all developmental stages per 200 cm3 of soil sample.

Type-material.

Holotype female and 2 female, 5 male and 8 juvenile (3 J1, 1 J2, 2 J3, 2 J4) paratypes deposited in the Nematode Collection of Agricultural Institute of Slovenia, Ljubljana, Slovenia; two female, two male and 10 juvenile (3 J1, 2 J2, 2 J3, 2 J4) paratypes - in the Wageningen Nematode Collection (WaNeCo), Wageningen, the Netherlands; one female, one male and 6 juvenile (3 J1, 1 J2, 1 J3, 1 J4) paratypes - in the Nematode Collection of The Food and Environment Research Agency, Sand Hutton, UK (former Rothamsted Nematode Collection); one female, three male and 6 juvenile (3 J1, 2 J3, 1 J4) paratypes - in the Nematode Collection of the Zoology Museum, Ghent University, Belgium; one female, three male and two juvenile (1 J3, 1 J4) paratypes - in the Nematode Collection of the Institute of Plant Protection, Bari, Italy; two female, one male and 6 juvenile (3 J1, 1 J2, 1 J3, 1 J4) paratypes - in the Nematode Collection of the University of California at Riverside, USA; one female, one male and two juvenile (1 J3, 1 J4) paratypes - in the USDA Nematode Collection, Beltsville, Maryland, USA; 4 female, 5 male and 8 juvenile (3 J1, 1 J2, 2 J3, 2 J4) paratypes - in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, BAS, Sofia.

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 Longidorus 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 Longidorus carniolensis sp. n.from Drašiči and Krmačina were identical. They formed a distinct clade within a cluster of the closely related sequences of Longidorus poessneckensis, Longidorus helveticus, Longidorus macrosoma, Longidorus caespiticola and Longidorus latocephalus. The closest sequence to Longidorus carniolensis sp. n.was the sequence of Longidorus poessneckensis (Acc. No EF538750) with 91.9% of similarity.

Figure 12.

Phylogenetic tree of rDNA D2/D3 expansion region sequences of Longidorus carniolensis sp. n. from Slovenia (square mark) and sequences of closely related Longidorus species (NCBI GenBank). Sequences were analysed using Neighbour Joining Method. Bootstrap support values higher than 50% are presented.

Discussion

There are some characteristic morphological features observed in Longidorus 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 et 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 1998; Mincheva et al. 2008), reported also for several species of Xiphidorus (Decraemer et al. 1996) and Paraxiphidorus brevistylus (Decraemer et al. 1998).

Hemizonid and hemizonion are not commonly observed structures in dorylaimids (Jairajpuri and Ahmad 1992), the hemizonid was seen both in adults and in the last two juvenile stages of the new species and hemizonion in only two male specimens. Both structures were reported also for Longidorus fursti (Heyns et al. 1987), Longidorus iranicus (Sturhan and Barooti 1983); only hemizonid – for Longidorus litchii, Longidorus henanus (Xu and Cheng 1992). Longidorus carpetanensis Arias, Fé Andres & Navas, 1986, Longidorus pawneensis Luc & Coomans, 1988, Longidorus brevis Swart et al., 1996, Longidorus africanus (Bravo and Roca 1995), Longidorus kheirii (Pedram et al. 2008), Longidorus laevicapitatus Williams, 1959 (Heyns and Luc 1987), in a few specimens of Longidorus fagi Peneva, Choleva & Nedelchev, 1997, for one Xiphidorus and some Paralongidorus species (Siddiqi et al. 1963, Fisher 1964, Luc and Doucet 1984).

The only available data on the excretory system in Longidorus refers to Longidorus macrosoma in which a ventral excretory pore at the level of the nerve ring, leading to a non-canalicular 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 Longidorus 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 (Longidorus poessneckensis (92% similarity), Longidorus macrosoma and Longidorus caespiticola (90%), Longidorus 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.

Acknowledgements

The study was supported by the National Science Fund, project N DO 02-101/2008 and Slovenian Research Agency, project BI-BG/09-10-009. We are grateful to Dr Nathalie Yonow, Swansea University, Wales, UK for linguistic editing as well as to Dr Plamen Pankov, IBER, BAS, Sofia for his technical assistance.

References
Aboul-Eid HZ (1969) Histological anatomy of the excretory and reproductive systems of Longidorus macrosoma. Nematologica 15: 437-450. doi: 10.1163/187529269X00777
Altherr E (1974) Nématodes de la nappe phréatique du réseau fluvial de Saale (Turinge) II. Limnologica 9: 81-132.
Arias M, Fé Andres M, Navas A (1986) Longidorus carpetanensis sp. n. and Longidorus unedoi sp. n. (Nematoda: Longidoridae) from Spain. Revue Nématologie 9: 101-106.
Barsi LB, Lamberti F (2004) Longidorus silvae Roca, 1993 (Nematoda: Dorylaimida): a first record from the former territory of Yugoslavia and the description of a male specimen. Russian Journal of Nematology 12 (2): 97-105.
Barsi L, De Luca F (2008) Morphological and molecular characterisation of Longidorus pius (Neamatoda: Dorylaimida) from the Republic of Macedonia. Nematology 10: 63-68. doi: 10.1163/156854108783360069
Barsi L, Lamberti F (2001a) Longidorus pius sp. n. (Nematoda: Longidoridae) from Macedonia. Nematologia mediterranea 29: 207-213.
Barsi L, Lamberti F (2001b) Morphometric variation and juvenile stages of Longidorus piceicola Liskova et al., 1997 (Nematoda: Longidoridae) from the former territory of Yugoslavia. Russian Journal of Nematology 9: 77-83.
Barsi L, Lamberti F, De Luca F (2007) Morphological and molecular characterisation of Longidorus danuvii sp. n. and Longidorus silvae Roca, 1993 (Nematoda: Dorylaimida) from Serbia. Nematology 9 (4): 585-59. doi: 10.1163/156854107781487279
Bravo MA, Roca F (1995) Observations on Longidorus africanus Merny from Portugal with description of Longidorus vinearum sp. n. (Nematoda: Longidoridae). Fundamental and Applied Nematology 18: 97-84
Brown DJF, Grunder J, Hooper DJ, Klingler J, 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. doi: 10.1163/003525994X00094
Chen Q, Hooper DJ, Loof PAA, Xu J (1997) A revised polytomous key for the identification of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Fundamental and Applied Nematology 20: 15-28.
Decraemer W, Doucet M, Coomans A (1998)Longidoridae from Argentina with the description of Paraxiphidorus brevistylus sp. n. (Nematoda: Longidoridae). Fundamental and Applied Nematology 21: 371-388.
Decraemer W, Luc M, Doucet M, Coomans A (1996) Study of the genus Xiphidorus Monteiro, 1976 (Nematoda: Longidoridae). Fundamental and Applied Nematology 19: 207-225.
Fisher JM (1964) Dolichodorus adelaidensis sp. n. and Paralongidorus eucalypti sp. n. from S. Australia. Nematologica 10: 464-470 doi: 10.1163/187529264X00484
Handoo ZA, Carta LK, Skantar AM (2005) Morphological and molecular characterization of Longidorus americanum sp. n. (Nematoda: Longidoridae), a needle nematode parasitizing pine in Georgia. Journal of Nematology 37: 91-104.
He Y, Subbotin SA, Rubtsova T, Lamberti F, Brown DJF, Moens M (2005) A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida). Nematology 7: 111-124. doi: 10.1163/1568541054192108
Heyns J, Coomans A, Hutsebaut M, Swart A (1987) Longidorus fursti sp. n. from South Africa with a discussion of its relationships (Nematoda: Longidoridae). Fundamental and Applied Nematology 10: 381-385
Heyns J, Luc M (1987) A first report on Longidoridae from Swaziland. Phytophylactìca 19: 41-44.
Hooper D (1961) A redescription of Longidorus elongatus (de Man, 1876) Thorne & Swanger, 1936 (Nematoda, Dorylaimidae), and description of five new species of Longidorus from Great Britain. Nematologica 6: 237-257. doi: 10.1163/187529261X00072
Jairajpuri MS, Ahmad W (1992) Dorylaimida: Free-Living, Predaceous and Plant-Parasitic Nematodes, 458 pp, Brill.
Khan E, Seshardi AR, Weischer B, Mathen K (1971) Five new species associated with coconut in Kerala, India. Indian Journal of Nematology 1: 116-127.
Kornobis FW, Peneva V (2011) Longidorus poessneckensis Altherr, 1974 and Longidorus piceicola Lišková, Robbins & Brown, 1997 (Nematoda: Longidoridae): new records from Poland and the first description of the Longidorus poessneckensis male and a bivulval female. Systematic Parasitology 80 (3): 205-216. doi: 10.1007/s11230-011-9325-8
Krnjaić D, Lamberti F, Krnjaić S, Agostinelli A, Raddicci V (2002) Longidoridae (Nematoda) occurring in the Topčider Park of Belgrade, with the description of Paralongidorus serbicus sp. n. Nematologia mediterranea 30: 185-200.
Krnjaić D, Lamberti F, Krnjaić S, Agostinelli A, Radicci V (2000) Three new longidorids (Nematoda: Dorylaimida) from Montenegro. Nematologia Mediterranea 28: 235-248.
Krnjaić D, Roca F, Krnjaić S, Agostinelli A (2005) Longidorus cylindricapitatus sp. n. (Nematoda: Longidoridae) from Serbia. Nematology 7: 803-808. doi: 10.1163/156854105776186280
Kruger JC de W, Heyns J (1987) The genus Xiphinema in southern Africa. XIV. Description of two new species from Bourke’s Luck, eastern Transvaal and a redescription of X. rarum Heyns, 1979 (Nematoda: Dorylaimida). Revue de Nématologie 10: 269-287.
Kumari S, Decraemer W, Liskova M (2009) Molecular and morphological delineation of Longidorus poessneckensis Altherr, 1974 (Nematoda: Dorylaimida). European Journal of Plant Pathology 123: 125-137. doi: 10.1007/s10658-008-9348-4
Lamberti F, Agostinelli A (1993) Longidorus raskii sp. n. (Nematoda: Dorylaimida) from Switzerland. Nematologia mediterranea 21: 243-246.
Lamberti F, Choleva B, Agostinelli A (1983) Nematologia mediterranea 11: 49-72.
Lamberti F, Kunz P, Grunder J, Molinari S, De Luca F, Agostinelli A, Radicci V (2001) Molecular characterization of six Longidorus species from Switzerland with the description of Longidorus helveticus sp. n. (Nematoda, Dorylamida). Nematologia mediterranea 29: 181-205.
Lamberti F, Molinari S, De Luca F, Agostinelli A, Di Vito M (1999) Longidorids (Nematoda: Dorylaimida) from Syria with a description of Longidorus pauli sp. n. and Paralongidorus halepensis sp. n. with sod isozymes and PCR RLFP profiles. Nematologia mediterranea 29: 181-205
Lišková M, Kumari S (2010) The notes on the occurrence of Longidorus poessneckensis in the Slovak Republic. Helmintologia 47: 264-268. doi: 10.2478/s11687-010-0041-8
Lišková M, Robbins RT, Brown DJF (1997) Description of three new Longidorus species from Slovakia ( Nemata: Longidoridae). Journal of Nematology 3: 336-348.
Lišková M (2007) Morphometrics of females, juveniles and a hermaphrodite of Longidorus distinctus Lamberti et al., 1983 (Nematoda: Longidoridae) from Slovakia. Helminthologia 44: 210-213. doi: 10.2478/s11687-007-0033-5
Loof PAA, Coomans A (1972) The oesophageal gland nuclei of Longidoridae (Dorylaimida). Nematologica 18: 213-233. doi: 10.1163/187529272X00458
Luc M, Coomans A (1988) Xiphinema smoliki sp. n. and Longidorus pawneensis sp. n. (Nemata: Longidoridae) from Colorado, USA. Revue de Nématologie 11: 137-142.
Luc M, Doucet M (1984) Description of Xiphidorus achalae sp. n. and proposa1 for a classification of longidorids (Nematoda: Dorylairnoidea). Revue de Nématologie 7: 103-112.
Macara M (1986) Two new species of Longidorus (Nematoda: Longidoridae) associated with forest plants in Portugal. Nematologica 31: 410-423. doi: 10.1163/187529285X00508
Mincheva Y, Lazarova S, Peneva V (2008) Xiphinema pirinense sp. n. (Nematoda: Dorylaimida: Longidoridae), a new species from Bulgaria with a digitate tail. Systematic Parasitolgy 70: 215-222. doi: 10.1007/s11230-008-9139-5
Pedram M, Niknam G, Robert RT, Ye W, Karegar A (2008) Longidorus kheirii sp. n. Nematoda: Longidoridae) from Iran. Systematic Parasitology 71: 199-211. doi: 10.1007/s11230-008-9148-4
Peneva V, Choleva B, Nedelchev S (1997) Description of Longidorus fagi sp. n. (Nematoda, Dorylaimida) with an identification key to the species of the genus occurring in Bulgaria. Systematic Parasitology 36: 115-122. doi: 10.1023/A:1005731707842
Roca F (1993) Langidorus silvae sp. n. (Nematoda Longidoridae) from Italy. Fundamental and Appied Nematology 16: 211-214.
Roca F, d’Errico FP (1987) Longidorus major, a new Longidoridae species from Italy. Nematologia mediterranea 15: 59-63.
Roca F, Lamberti F (1981) Longidorus fasciatus sp. n. from Greece and Italy. Nematologia mediterranea 9: 175-179.
Roca F, Lamberti F, Agostinelli A (1985) Three new species of Longidorus (Nematoda, Dorylaimida) from Italy. Nematologia mediterranea 12: 187-200.
Rubtsova TV, Subbotin SA, Brown DJF, Moens M (2001) Description of Longidorus sturhani sp. n. (Nematoda: Longidoridae) and molecular characterisation of several longidorid species from western Europe. Russian Journal of Nematology 9: 127-136.
Seinhorst JW (1959) A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4: 67-69. doi: 10.1163/187529259X00381
Siddiqi MR, Hooper D, Khan E (1963) A new nematode genus Paralongidorus (Nematoda: Dorylaimoidea) with descriptions of two new species and observations on Paralongidorus citri (Siddiqi, 1959) n. comb. Nematologica 9: 7-14. doi: 10.1163/187529263X00025
Širca S, Stare BG, Pleško IM, Marn MV, Urek G (2007) First record of Longidorus juvenilis and Longidorus leptocephalus (Nematoda: Dorylaimida) in Slovenia and their morphometrical and ribosomal DNA sequence analysis. Russian Journal of Nematology 15 (1): 1-8.
Širca S, Urek G (2009) Morphological and molecular characterization of six Longidorus species (Nematoda: Longidoridae) from Slovenia. Russian Journal of Nematology 17 (2): 95-105.
Sturhan D, Barooti Sh (1983) Longidorus iranicus sp. n. (Nematoda: Dorylaimida). Systematic Parasitology 5: 21-24.
Sturhan D, Loof PAA (2001) Redescription of Longidorus poessneckensis Altherr, 1974 (Nematoda: Dorylaimida). Russian Journal of Nematology 9: 43-49. doi: 10.1007/BF00010982
Swart A (1994) Description of three new Xiphinema species from South Africa (Nematoda: Dorylaimida). Fundamental and Applied Nematology 17: 455-467.
Swart A, Cadet P, N’Diaye SB (1996) Longidorus brevis sp. n. from Senegal, West Africa, Russian Journal of Nematology 4: 139-143.
Swart A, Quénéhervé P (1998) The genus Xiphinema (Nematoda: Longidoridae) in Guyane and Martinique. Fundamental and Applied Nematology 21: 581-604.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution. doi: 10.1093/molbev/msr121
Tzortzakakis E, Peneva V, Terzakis, M, Neilson R, Brown DJF (2001) Longidorus cretensis sp. n. (Nematoda: Longidoridae) from a vineyard infected with a foliar ‘yellow mosaic’ on Crete, Greece. Systematic Prasitology 48: 131-139. doi: 10.1023/A:1006436718672
Xu J, Cheng H (1992) Longidorus litchii sp. n. and Longidorus henanus sp. n. (Nemata: Longidoridae) from China. Fundamental and Applied Nematology 15: 517-523.
Ye WM, Szalanski AL, Robbins RT (2004) Phylogenetic relationships and genetic variation in Longidorus and Xiphinema species (Nematoda: Longidoridae) using ITS1 sequences of nuclear ribosomal DNA. Journal of Nematology 36: 14-19.
Yeates G, Van Etteger M, Hooper DJ (1992) Longidorus orongorongensis sp. n. (Nematoda: Dorylaimida) from subsoil of conifer/broadleaved forest. New Zealand Journal of Zoology 19(1/2): 25–31.