Characterisation of Trichodorus similis (Nematoda: Trichodoridae) associated with potato from the Czech Republic

Specimens from the Kaliště field population were extracted by sieving on 1 mm, 150 μm and 75 μm and placing the residual on 99 μm sieves on a Baermann funnel for 24 – 48 hours (Brown & Boag, 1988). Nematodes were heat killed, fixed in TAF, processed in slow glycerin process and mounted in anhydrous glycerin on slides. Photographs were taken using an Axio lmager.M2—Carl Zeiss compound microscope with a digital camera (ProgRes C7) and specialised software (CapturePro Software 2.8). Measurements were made using an Olympus BX41 light microscope, a digitising tablet (CalComp Drawing Board III, GTCO CalCom Peripherals, Scottsdale, AZ, USA), and computer Digitrak 1.0f programme, (Philip Smith, Scottish Crop Research lnstitute, Dundee, UK).

Total genomic DNA was prepared by a rapid technique of Klimyuk et al., (1993) and subsequent modification of the technique for nematodes by Stanton et al., (1998). The DNA template was amplified by polymerase chain reaction (PCR) using the species-specific primer SIMIREV2 (5'-CAC TCG TCG GAC TCA AAC C-3') and an antisense primer UNIVERSAL (5'-CCC GTC GCT ACT ACC GAT T-3') (Boutsika et al., 2004b). Amplification was done using one cycle of 95 °C for 3 min, followed by 35 cycles of 94 °C of 30 s, 55 °C for 30 s and 72 °C for 30 s, and finally one cycle of 72 °C for 10 min for annealing, using a DNA Engine PTC-1148 thermal cycler (Bio-Rad, USA) with heated lid. An aliquot (4 μl) of each amplification product was mixed with 1 μl of 6x loading dye (Fermentas, MBI) and electrophoresed in high resolution 1.5 % agarose gel in TAE buffer. The bands were visualized and photographed under UV (312 nm) after syber safe (1 μg/ml) binding to the DNA fragments. A 100 base pair marker (Fermentas, MBI) was included on the gel.

Two individuals of the Kaliště population (out of 10 with positive species-specific PCR products) were used for sequencing D2–D3 expansion segments of the 28S and ITS1 regions of ribosomal DNA. D2–D3 expansion segment of the 28S gene was amplified and sequenced using the following D2A primers: 5'-ACA AGT ACC GTG AGG GAA AGT TG-3' and D3B: 5'-TCG GAA GGA ACC AGC TAC TA-3' (Nunn, 1992). Internal transcribed spacer 1 (ITS1) was amplified and sequenced using the primers BL18: 5'-CCC GTG GMT ACT ACC GAT T-3' + 5818: 5'-ACG ARC CGA GTG ATC CAC-3' (Boutsika et al., 2004a). PCR mix and cycling profile were same as described above for specific PCR. Aliquots of PCR products were analysed by gel electrophoresis and the remaining products were purified using High Pure Product Purification kit (Roche Diagnostics GmbH, Mannheim, Germany) and sequenced (Macrogen, Netherlands). SequencherTM 4.8 (Genes codes. Corp., Ann Arbor, MI, USA) was used to assemble and view each sequence and check for base-calling errors.

(Figs. 1, 2; Tables 1, 2)

Fig. 1

Fig. 2

Male. Habitus J-shaped when heat killed. Cuticle 2 μm in onchiostyle region and 2 – 2.5 μm along the body, slightly thickened on tail (3 – 6 μm). Anterior to sensillium pouch 15 μm. Three ventromedian cervical papillae, anterior to secretory-excretory pore (SE) pore (in one specimen 4, the fourth after S-E pore, Fig. 1C); the first one in the onchiostyle region; in one specimen CP2 opposite to posterior end of pharyngostom. S-E pore opposite the isthmus. Pharyngeal bulb offset. One dorsal and two pairs of ventrosublateral glands, nuclei of the first pair smaller. Spicule markedly and regularly curved; manubrium offset from the shaft, knob like, widened; shaft conical, smooth with few bristles. Gubernaculum slightly curved, hooked proximally and knobbed distally. Sperm large (5 – 6 × 8 – 10 μm) with sausage-shaped nucleus (2 – 3 × 5 – 6 μm). Ventromedian precloacal supplements three, the first just posterior the level of proximal end of retracted spicules.

Codes according to the polytomous key by Decraemer and Baujard (1998): F3 D3 P2 A210 B21 C11 E12 G0 H22 I11 J500 K22 L11 M16 N11 O1.

Female. Body slightly curved ventrally when heat killed. Cuticle 2 – 2.5 μm in onchiostyle region, 2 – 3 μm along the body, slightly thickened at tail (4 – 6 μm). Anterior to sensillium pouch 14 – 16 μm. Position of S-E pore as in males, pharyngeal bulb offset. Genital system didelphic, ovaries reflexed. Vulva a narrow transverse silt, post-equatorial (aver. V=55.8 %). Vagina length 48 – 59 % of corresponding body width. Vagina rhomboid, pars distalis 2 – 3 μm, pars proximalis 11 – 13 μm. Vaginal sclerotized pieces triangular, oblique (tips directed towards vulva), close to each other, up to 2 μm long. Proximal of vagina end surrounded by constrictor muscles. Sperm located in spermathecae and rarely in uterus.

Codes following Decraemer and Baujard (1998): D1 C1 L1 K300 A210 B210 E 200 F100 G2 H2 I32 J11 M1 N1 O12 P11 Q? R? S1. Remarks. Male and female morphometrics were compared with the type population from the Netherlands (Seinhorst, 1963), one from Germany (Wyss, 1974), two from Italy (Roca & Lamberti, 1984) and three populations from Bulgaria (Peneva, 1988). Male body is shorter L = 730 (658 – 783) μm vs L = 810 (740 – 910) μm, and L = 887 (803 – 976) μm and L = 973 (880 – 1059) μm in the German and Italian populations, respectively, and is longer compared to two Bulgarian populations (L = 678.3 (601 – 782) and L = 666.6 (576 – 771) μm). Onchiostyle is shorter 38.9 (37 – 40) μm compared to the Italian populations (43 (40 – 47) and 45.5 (43 – 49) μm). Average distance CP1-CP2 20.2 (17 – 22) μm is close to all three Bulgarian populations but lower range of two of them is lower 19.9 (8 – 33) and 18.7 (10 – 24) μm. Distance CP2-CP3 (14.8 (11 – 16)) is shorter than in Bulgarian populations (19.1 (11 – 23) and 18.1 (7 – 32) μm). Ratio ‘a’ 26 (23.6 – 29) is higher compared to the Bulgarian populations 22.3 (19.5 – 24.5) and 23.4 (20.3 – 25.8); ratio ‘b’ 5.3 (4.8 – 5.7) is smaller compared to the Italian specimens 10 (9.3 – 11.3) and 9.3 (8 – 10.3); ratio ‘c’ is smaller than all other seven populations.

Female body is shorter compared to German and Italian materials, (av 758 μm vs avs. 820 μm, and 855 and 952 μm) and longer compared to the Bulgarian populations (692 and 644 μm). Onchiostyle is shorter compared to one Italian and one Bulgarian population (39.3 (37.7 – 41.6) μm vs 45 (43 – 47) μm and 46.5 (42 – 52)) μm. Ratio ‘a’ 24.7 (21.3 – 28.1) is somewhat higher compared to the Bulgarian populations 20.9 (17.9 – 24.1) and 20.9 (19.2 – 23) and ‘b’ ratio 5.5 (4.7 – 6.3) is lower than in Italian specimens b=9.6 (8.4 – 11) and 9.5 (8.7 – 11).

Morphological identification of this population was reliably verified by PCR of ribosomal DNA from single specimens using species-specific primers (Boutsika et al., 2004b). A single fragment of approximately 452 bp was amplified for all studied individuals (Fig. 3). No PCR products were obtained in the negative controls lacking DNA template or containing DNA of T. pakistanensis, T. variopapillatus and T. viruliferus (data not shown).

Fig. 3

Identical sequences were obtained from two individuals of the Kaliště population processed, thus two sequences were deposited in the NCBI (National Center for Biotechnology Information) database with accession numbers KX522761 (D2–D3 segment of 28S gene) and KX522760 (ITS1). After a BLASTN search tool D2–D3 sequence showed 771/775 nucleotides identity to T. similis from United Kingdom (accession number AM180730, Holeva et al., unpublished) and 753/755 nucleotides identity to T. similis from deciduous forest in the Czech Republic (DQ832183, Kumari et al., 2007). Similarly, the ITS1 showed 1237/1255 identity to T. similis from the UK (AJ439523, Boutsika et al., 2004a) and 1359/1364 identity to T. similis from the Czech Republic (JN123317, Kumari and Subbotin, 2012). ITS1 showed polymorphisms at three sites.

Taking into account the agricultural importance of trichodorids and tobraviruses as pathogens of potato (Taylor & Brown, 1997), a study has been initiated in the Czech Republic. The damage-level threshold is in the case of virus vector species equivalent to a single nematode. Therefore, the information on T. similis would be useful for developing nematode management strategies in the country. In this study, both taxonomical and molecular analysis verified the presence of T. similis in association with potato in the Czech Republic and extends the knowledge on morphological variability and genetic diversity of the species.