A simple, polymerase chain reaction‐restriction fragment length polymorphism‐aided diagnosis method for pine wilt disease

Abstract

For diagnosis of pine wilt disease, a simple PCR-RFLP method was developed to identify and to differentiate two similar nematode species, based on a living or preserved single specimen. Pinewood nematodes, Bursaphelenchus xylophilus, and Bursaphelenchus mucronatus were examined. A single nematode in 1 ml of distilled water was put on a glass slide. When the water had almost dried the nematode was crushed with a filter paper chip, 1.5 mm × 1.5 mm, with the aid of forceps. The filter paper chip containing nematode remains was immediately placed into PCR buffer as the DNA template. The primer set used was to amplify ribosomal DNA containing the inter-transcribed spacer (ITS) 1, 5.8S and ITS2 regions. The PCR product was consistently obtained from a single nematode, and digesting the product with restriction endonuclease, Hinf I, enabled discrimination between B. xylophilus and B. mucronatus. This method was simple, convenient and definitive, and could successfully determine the pathogen in the diagnosis of pine wilt disease. This method was applicable also to nematode specimens preserved under various conditions except in the case of those preserved in aldehyde-containing fixatives.

Genetic variation in the invasive process of Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae) and its possible spread routes in China

“…We used the restriction endonuclease HinfI (New England BioLabs Inc., Ipswich, MA, USA) to digest the product of PCR. The RFLP patterns in each species were distinctly different (Iwahori et al, 2000). The pattern of B. xylophilus was characterized by band sizes of 150, 155, 250 and 255 bp, whereas the pattern of B. mucronatus had band sizes of 110, 160, 242 and 404 bp.…”

Genetic diversity of ITS sequences of Bursaphelenchus xylophilus

“…Additionally, PWN morphological identification can sometimes be difficult or impossible when only juvenile stages are detected or owing to the variation in the female tail of some isolates (Fonseca et al, 2008). Therefore, several molecular methods have been developed that frequently use species-specific primers by targeting several genomic regions such as satellite DNA (Castagnone et al, 2005;Cardoso et al, 2012), the heat shock protein 70 gene (Leal et al, 2005(Leal et al, , 2007, the topoisomerase I gene (Huang et al, 2010), and internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) (Cao et al, 2005;Takeuchi et al, 2005) or by using nonspecific primers for polymerase chain reaction (PCR) amplification of the ITS regions and subsequent analysis with restriction fragment length polymorphism (RFLP) (Iwahori et al, 2000;Burgermeister et al, 2009). The ITS regions are located between the repeating array of nuclear 18S, 5.8S, and 28S ribosomal RNA genes and have 100-200 copies/genome.…”

A DNA extraction method with SDS from single nematodes for direct application to PCR amplification

“…A single nematode was placed into a drop of sterile distilled water on a glass slide. After the water dried, the nematode was crushed with a small sterile filter paper chip under the stereo microscope using forceps (Iwahori et al, 2000). The paper chip was dropped into a 0.5 ml plastic tube containing 4 µl of 0.1% SDS lysis buffer (10 mM Tris-HCl (pH8.0), 5 mM EDTA (pH8.0), 500 µg/ml Proteinase K, and 0.1% SDS [the contents except for SDS were prepared as a 2 stock premixed solution and frozen at 20°C until used; SDS was added just before use at room temperature]), and then the tube was incubated under the following conditions: 50°C for 2 hr, followed by 95°C for 10 min.…”