Eric Allen

Application of a real-time PCR method for the detection of pine wood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine

A real-time PCR polymerase chain reaction (real-time PCR) method was developed to detect and differentiate Bursaphelenchus xylophilus (pine wood nematode, PWN) from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus heat shock protein 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 0.005 ng of B. xylophilus genomic DNA, as well as DNA extracted from single nematodes. The practical application of this real-time PCR diagnostic method for the detection of B. xylophilus from actual wood samples of lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees containing a heterogeneous population of nematodes, rather than pure cultures or individual nematodes, is demonstrated. This method works well in the presence of potential inhibitors associated with wood after Baermann extraction and thus eliminates the need to produce pure nematode samples through further culturing and/or isolation of nematodes with a high-power microscope.

An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine

A molecular diagnostic method has been designed for the detection and identification of Bursaphelenchus xylophilus. Heat shock protein 70 gene sequences from B. xylophilus and the closely related B. mucronatus were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by PCR. As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimisation, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than one nematode. Species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta var. latifolia) trees from British Columbia, Canada, containing an unknown nematode population, thus bypassing the need for culturing or recovering the nematode before analysis.

Phytosanitary measures to reduce the movement of forest pests with the international trade of wood products

International trade in wood products brings the risk of the movement of tree pests, which can cause devastating ecosystem and economic damage. International phytosanitary guidelines have been created to help countries that import wood products develop import requirements to minimize pest movement. Requirements may include specific phytosanitary measures, including treatments such as heat, fumigation, chemical, or systems approaches that combine phytosanitary measures. This paper provides an overview of phytosanitary measures for the international trade of wood commodities and the regulatory framework in which they are applied.

Application of Conventional PCR and Real-Time PCR Diagnostic Methods for Detection of the PineWood Nematode, Bursaphelenchus xylophilus, in Wood Samples from Lodgepole Pine

Molecular diagnostic methods have been designed for the detection and identification of the pinewood nematode, Bursaphelenchus xylophilus. Heat shock protein 70 (Hsp 70) gene sequences from B. xylophilus and the closely related B. mucronatus, were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by polymerase chain reaction (PCR). As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimization, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than 1 nematode. In addition, a real-time PCR method was developed to detect and differentiate B. xylophilus from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus Hsp 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 5 pg of B. xylophilus genomic DNA, as well as DNA extracted from individual nematodes. The species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees.