First Report of Trichodorus variopapillatus (Nematoda: Trichodoridae) from the Czech Republic

Members of the Trichodoridae can cause substantial crop losses directly by feeding on plant roots and indirectly as vectors of tobraviruses; both vector and virus are polyphagous. In April of 2008, soil samples from the rhizosphere of Ulmus minor Mill in a deciduous broadleaf forest at Krivoklat yielded a population of Trichodorus variopapillatus Hooper, 1972. Nematodes were identified by morphological and morphometric characters as well as by molecular analysis. For classical identification, specimens were extracted from soil by a decanting-sieving method, heat killed and fixed in triethanolamine formalin, and processed and mounted in anhydrous glycerin. For molecular analysis, specimens were stored at -20°C in 1 M NaCl. Specimens largely agreed with T. variopapillatus (1,3). Average morphometric data of five male specimens are: body length 766 μm; body width 33 μm; onchiostyle length 55 μm; and spicule length 43 μm. Number of anterior ventromedian cervical papillae and number of precloacal supplements was three each. Spicules are regularly curved and the manubrium is knob-like. Morphometric data of two female specimens are: body length 663 and 858 μm; body width 29 and 38 μm; onchiostyle length 52 and 53 μm; V 54 and 57%. Refractive thickenings at the vulva are very large and quandrangular in shape in the lateral optical section. Identification of these nematodes was further verified by sequencing two regions of rDNA (18S gene and D2/D3 expansion segments of the 28S gene). Single female and male specimens from NaCl storage were transferred to 0.5-ml Eppendorf tubes containing 0.25M NaOH. Total genomic DNA was prepared by a rapid technique (4). The 18S gene was amplified in three fragments using the primer SSU_F_04 + SSU_R_09 (first fragment), SSU_F_22 + SSU_R_13 (second fragment), and SSU_F_23 + SSU_R_81 (third fragment). D2/D3 expansion segments of the large subunit of rDNA were amplified using the forward primer D2A and the reverse primer D3B (2). The regions were sequenced in both directions after purification of PCR products. The sequences of female and male specimens were identical. The sequences were deposited in GenBank with Accession Nos. GQ148719 (28S) and GQ148719 (18S). The length of 18S was 1,760 bp and D2/D3 was 786 bp. The obtained sequences were compared by BLAST in NCBI. The D2/D3 sequence is not available in GenBank for T. variopapillatus. The best BLAST hits were obtained with Trichodorus species. BLAST results of 18S sequence showed 5%