TaqMan REAL-Time PCR-based approach for differentiation between Globodera rostochiensis (golden nematode) and Globodera artemisiae species

Abstract

Cyst nematodes from the genus Globodera are common, and widely distributed parasites of Solaraceae. Intact cysts persist in soil even up to 10 years without detriment. Out of two Globodera species occurring in Poland, Globodera rostochiensis is considered by European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest, while Globodera artemisiae is not. Therefore, the distinction between these two species is crucial. Classic methods of detection and differentiation are laborious and time-consuming. Instead, application of molecular biology techniques allows obtaining of rapid and reliable results. The aim of this study was to establish detection and differentiation method of two species, G. rostochiensis and G. artemisiae, based upon real-time polymerase chain reaction with the use of TaqMan probes. In reaction with primers and probes specific for the nematodes' ribosomal DNA (rDNA), the samples used were DNAs isolated from the two species, alone or in mixture, as well as crushed single cysts. Applied probes enable not only to identify the species in DNA mixtures but also in a single cyst. The use of a crushed cyst eliminates long-lasting procedure of DNA isolation and reduces costs of analysis.

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi and X. italiae, and for the quantification of X. index numbers

“…These molecular methods have been used for the detection of both mammal‐parasitic (Fink et al ., ; Höglund et al ., ) and plant‐parasitic nematodes. In plants, these assays allow the identification of some of the major nematode threats to crops such as soybean cyst, potato cyst, root‐knot or lesion nematodes (Berry et al ., ; Nowaczyk et al ., ; Yan et al ., ; Ye, ). With a few exceptions, such as an assay based on the Msp I satellite DNA in Bursaphelenchus xylophilus (François et al ., ), the ITS1 (Yan et al ., ) and ITS2 (Höglund et al ., ) regions and the 18S ribosomal RNA genes (Fink et al ., ) are commonly used as targets to detect those pests.…”

“…These molecular methods have been used for the detection of both mammal-parasitic (Fink et al, 2011;H€ oglund et al, 2013) and plant-parasitic nematodes. In plants, these assays allow the identification of some of the major nematode threats to crops such as soybean cyst, potato cyst, rootknot or lesion nematodes (Berry et al, 2008;Nowaczyk et al, 2008;Yan et al, 2012;Ye, 2012 Table 1.…”

Hsp90 gene, an additional target for discrimination between the potato cyst nematodes, Globodera rostochiensis and G. pallida, and the related species, G. tabacum tabacum

“…The presence of weed-associated cysts of other Globodera sp. such as G. artemisiae and G. achilleae, in European and Asian agricultural crop land has been noted recently but has not yet been studied extensively (Dobosz et al 2006;Hockland 2005;Nowaczyk et al 2008). In any case, it would seem prudent to investigate additional genomic regions for useful discriminatory sequences to supplement ITSbased and other identification methods.…”

Molecular Diagnostic Tools for Nematodes

“…qPCR is used for the quantitative detection, species identiication and discrimination in plant and in veterinary parasitic nematodes [8,23,24,[26][27][28][29][30].…”