Identification and Characterization of Pale Potato Cyst Nematode (Globodera pallida) in Teplá, the Czech Republic.

Abstract

Potato cyst nematode poses a significant threat to potato producers in the Czech Republic. Both species of potato cyst nematodes (Globodera rostochiensis and G. pallida) are listed as quarantine pests in the Czech Republic and also by the European Union, European and Mediterranean Plant Protection Organization, and North American Plant Protection Organization. To date, G. rostochiensis was responsible for all damage to potatoes caused by cyst nematodes in the Czech Republic, while G. pallida was recorded only once in the Czech Republic (2) 8 years ago. It is important to note that this occurrence of G. pallida was not located in the free-cultivation area. In July 2011, soil samples from a potato field located in the area of Teplá (Karlovy Vary Region) were collected, and the cysts extracted were identified as G. pallida according to microscopic observation of cyst fenestra and morphology of juveniles (1). Cyst morphometrics (means from 10 cysts) included: fenestra diameter 21.2 μm, distance fenestra to anus 56.8 μm, Granek's ratio 2.7, number of cuticular ridges between fenestra and anus 14; while second stage juvenile morphometrics (means of 13 specimens) were: L 466.7 μm, stylet 24.2 μm, tail 53.2 μm, body width at anus 13.2, h 28.6, c 8.8, c' 4.0. Terminus of juvenile tails was rounded, and stylet knobs possessed distinct forward projections. Total DNA was extracted from single cysts using the TriPure reagent (Roche), and the DNA samples were used to amplify cistron rDNA with the following primers: 18S, 5'-TTGATTAGGTCCCTGCCCTTT-3', and 21S, 5'-TTTCACTCGCCGTTACTAAGG-3'. The amplified region contains the 3' end of the 18S gene, ITS1, 5.8S, ITS2, and the 5' end of the 28S gene. Pfu DNA polymerase (Fermentas) was used for accurate amplification. A PCR product of approximately 1.0 kb was amplified from three individual cysts. The PCR amplicons were cloned into pJET1.2 using the CloneJET PCR Cloning Kit (Fermentas) and sequenced in both directions. The sequences of representative isolates were deposited in GenBank (Accession Nos. JQ692592, JQ692593, and JQ692594). The resultant 1.0 kb sequences showed 99% nucleotide identity to sequences of G. pallida from Canada (GenBank Accession Nos. GQ294522.1, GQ355975.1, and GQ294523.1), thus confirming the results of the morphological analyses. To our knowledge, this is the first detection of G. pallida in the free-cultivation area of Karlovy Vary Region, and only the second in the Czech Republic since the first report in 2003 (2).