First Report of the Dagger Nematode Xiphinema dentatum (Nematoda: Longidoridae) in a Deciduous Forest in the Czech Republic

Xiphinema species are migratory ectoparasitic nematodes that feed on an extensive range of hosts and several species are vectors of nepoviruses. These long nematodes are readily distinguished from most other plant parasitic nematodes by a long stylet with forked odontostyle and flanged odontophore. In May of 2005, a sample from the rhizosphere of Carpinus betulus and Acer platanoides in a forest near Silnicna, South Moravia yielded a population of Xiphinema dentatum Sturhan, 1978. X. dentatum previously has been reported to be associated with several forest and grassland species in Germany, the former Yugoslavia, and Slovakia. Specimens were extracted from soil by decanting-sieving. A few female specimens were stored at –20°C in 1 M NaCl, and the rest of the specimens were heat killed, fixed in triethanolamine formalin, and mounted in anhydrous glycerin. In 2007, nematodes from permanent slides were identified by morphological and morphometrical characters (3): female body C shaped in fixed specimens, lip region offset by a depression, reproductive system amphidelphic with the presence of well developed pseudo Z-organ, and tail broadly convex-conoid to regularly hemispherical; main average morphometric of females were body length 3.6 mm, total stylet length 220 μm, vulva position 46%, and tail ratio 0.66. Identification of these nematodes was further verified by sequencing cytochrome oxidase subunit 1 (cox1) of mtDNA and D2/D3 expansion segments of large subunit rDNA. Two individual female specimens from NaCl storage were transferred to 0.5-ml Eppendorf tubes containing 0.25 M NaOH. Total genomic DNA was prepared by a rapid technique (4). The cox1 gene was amplified using forward primer COIF (5′-GAT TTT TTG GKC ATC CWG ARG-3′) and reverse primer COIR (5′-CWA CAT AAT AAG TAT CAT G-3′) (2). D2/D3 expansion segments of large subunit of rDNA were amplified using forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG -3′) and reverse primer D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) (1). The regions were sequenced in both directions after purification of PCR products from gel slices with a Qiagen gel extraction kit (Qiagen, Hilden, Germany). The sequences of two individual females were identical. The sequences were deposited in GenBank (Accession Nos. EU781537 [cox1] and EU781538 [D2/D3]). The length of cox1 was 393 bp and D2/D3 was 786 bp. The obtained sequences were compared by BLAST in NCBI. The cox1 gene sequence is not available in GenBank for X. dentatum, but the best BLAST hits were logically obtained with Xiphinema species. BLAST results of D2/D3 sequence showed strong similarities (99.6%) with X. dentatum Accession No. AY601627 and only a three nucleotide difference was observed in the beginning of the 5′ end. To our knowledge, this is the first report of X. dentatum associated with deciduous forest trees in the Czech Republic. Forests are the main terrestrial ecosystems and rich in species diversity and are of great importance as natural resources. Therefore, information on these plant parasitic nematodes from forests would be useful because they are a component of the continental forest diversity. References: (1) P. De Ley et al. Nematology 2:591, 1999. (2) Y. He et al. J. Mol. Evol. 61:819, 2005. (3) P. A. A. Loof and M. Luc. Syst. Parasitol. 16:35, 1990. (4) J. M. Stanton. Australas. Plant Pathol. 27:112, 1998.