Corresponding author: Vlada Peneva (
Academic editor: S. Subbotin
A new needle nematode,
The nematodes of the genus
Soil samples were collected in July 2008 and October 2009 from the rhizosphere of
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).
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 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‘) (
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
Species of fam.
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AY601583 | California, USA |
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AY494715 | Georgia, USA |
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AY601571 | Mola di Bari, Italy |
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AY601570 | Suter, Switzerland |
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AY601574 | Italy |
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AY601572 | Germany |
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AY601576 | Nebraska, USA |
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HM447030 |
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Brdo, Slovenia |
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AY601585 | Hangzhou, China |
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JN631811 | Krmačina, Slovenia | This study | |
JN631812 | Drašiči, Slovenia | This study | |
AF480072 | Germany | Rubtsova 2001 | |
EF654539 | Kráľovský Chlmec, Slovakia |
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AY593057 | Holterman et al. Unpublished | ||
AY601575 | Caribbean sea beach, Cuba |
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HM447032 |
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Maribor, Slovenia |
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AY601573 | Zabagr, Hungary |
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AY601581 | Peebles, Scotland, UK |
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HM447031 |
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Trška gora, Slovenia |
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AF480074 | Germany |
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DQ364599 |
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Svetinje, Slovenia |
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AY601568 | Greece |
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DQ364600 |
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Juršinci, Slovenia |
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AY601565 | Switzerland |
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HM447029 |
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Vrhpolje, Slovenia |
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AY601577 | Branisko, Slovakia |
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EF538750 | Cerne Voderady, Czech Republic |
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AF480073 | Germany |
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AF480071 | Belgium |
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EF538754 | Velke Pole, Slovakia |
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AY601628 | Argentina |
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See
Measurements of females, males and juvenile stages of
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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
The codes for identifying the new species when using the polytomous key by
An old vineyard with roots of several
Other-locality: a vineyard close to Krmačina village in southern part of Slovenia.
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.
The species epithet
Cluster analyses of the D2-D3 expansion regions of the 28S rDNA nuclear gene sequences of
Phylogenetic tree of rDNA D2/D3 expansion region sequences of
There are some characteristic morphological features observed in
Hemizonid and hemizonion are not commonly observed structures in dorylaimids (
The only available data on the excretory system in
The data on D2D3 rDNA regions of majority of longidorid species, particularly of those belonging to the genus
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.