Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

Abstract

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.

Sequence variability of the MspI satellite DNA family of the pinewood nematode Bursaphelenchus xylophilus at different geographic scales

“…Although this satDNA family has been recurrently used for species diagnosis (François et al, 2007;Cardoso et al, 2012), the available information regarding the structure and variation of this satDNA family is still scantily studied (Tarès et al, 1993;Castagnone-Sereno et al, 2008). Despite the recent release of the genome sequence of B. xylophilus (Kikuchi et al, 2011), no output was provided for such highly repetitive regions.…”

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 new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

“…Several gDNA extraction methods using CTAB buffer, Chelex-100 resin and proteinase K have been developed for extracting total gDNA from B. xylophilus in infected pinewood (Cardoso et al, 2012;Francois et al, 2007;Hu et al, 2011;Kikuchi et al, 2009;Takeuchi et al, 2005). These methods, however, take a long time and the processes includes freezing, boiling, precipitation and filtering to increase extraction efficiency of gDNA extraction (Gou et al, 2015;Kikuchi et al, 2009;Takeuchi & Futai, 2007).…”

“…Identification of Bursaphelenchus species based on the morphological characteristics is therefore problematic. Some of the methods were attractive since they are not using Baermann funnel method and are extracting gDNA of B. xylophilus directly from the wood samples using various gDNA extraction buffer such as CTAB, Chelex-100 and proteinase K (Cardoso et al, 2012;Hu et al, 2011;Kikuchi et al, 2009;Takeuchi et al, 2005). such as several PCR, quantitative real-time PCR and loop-mediated isothermal amplification (LAMP) based methods with species-specific primers by targeting either rDNA-ITS (Iwahori et al, 2000;Kikuchi, Aikawa, Oeda, Karim, & Kanzaki, 2009;Takeuchi, Kanzaki, & Futai, 2005), rDNA-IGS (Kang et al, 2004), hsp70 (Leal, Green, Allen, Humble, & Rott, 2007), satDNA (Cardoso, Fonseca, & Abrantes, 2012;Castagnone, Abad, & Castagnone-Sereno, 2005;Francois et al, 2007), DNA topoisomerase I (Huang et al, 2010), pectate lyase 3 (Kang, Kim, Han, Moon, & Koh, 2015) and expansin-like gene (Leal et al, 2015).…”