Detection of Bursaphelenchus xylophilus from old discs of dead Pinus armandii var. amamiana trees using a new detection kit

Abstract

We examined the effectiveness of a new Bursaphelenchus xylophilus detection kit, based on loop-mediated isothermal amplification (LAMP), in old discs taken from the stem base of B. xylophilus-infested dead trees of Pinus armandii var. amamiana (PAAm) occurring in their natural habitats. LAMP products, representing a past B. xylophilus infection, were detected in two consecutive trials from 16 of 20 discs collected from PAAm trees that died between 2003 and 2006. Bursaphelenchus xylophilus were more frequently detected using LAMP in wood samples taken from sapwood than from heartwood. No significant differences in the detection of B. xylophilus using LAMP were observed in relation to the disc collection time (from 3 to 6 years before the analysis). Bursaphelenchus xylophilus were not detected using LAMP in four discs, although a B. xylophilus infection had been confirmed for the original PAAm trees at the time they were found dead. This may have resulted from the small amount of wood chips needed for the LAMP test or the reduced number and uneven distribution of the nematode in the old dead trees. The results indicate that the new B. xylophilus detection kit will be a very efficient tool for conducting retrospective analysis of PAAm mortality factors.

Development of two alternative Loop‐mediated isothermal amplification tools for detecting pathogenic pine wood nematodes

“…ITS can be used to identify geographic races of organisms, because of variation within species. Because insect vectors and host trees for B. xylophilus differ between geographical regions and countries, and because the LAMP Detection Kit developed with the ITS of rRNA of B. xylophilus detects pathogens from PWD-confirmed trees only in Japan (Kanetani et al 2011), other LAMP Detection Kits using genes that are more likely to be conserved need to be developed. In this study, we report a new LAMP assay for B. xylophilus using pectate lyase 3 (PEL3) genes and demonstrate the improved detection efficiency and practicality of the LAMP assay compared with conventional or real-time PCR-based methods (Kang et al 2009a).…”

Detection of living Bursaphelenchus xylophilus in wood, using reverse transcriptase loop‐mediated isothermal amplification (RT‐LAMP)

“…In the case of PWN, neither the Baermann funnel extraction method followed by a DNA-based PCR diagnostic assay nor some diagnostic approaches that extract PWN DNA directly from wood (Takeuchi et al 2005;Franc ßois et al 2007;Kikuchi et al 2009a;Hu et al 2011;Kanetani et al 2011;and Cardoso et al 2012) are adequate to differentiate between dead and living PWN. Therefore, because of the prolonged persistence of PCR-detectable DNA in samples after death of target PWN cells, the RT-PCR method developed by Leal et al (2013), and the RT-LAMP assay reported here are more suitable methods to be used as indicators of viability of PWN.…”

“…As a result, numerous laboratories have developed molecular detection techniques using both endpoint and real-time PCR diagnostics to detect PWN by targeting DNA regions or genes such as ribosomal DNA, internal transcribed spacer (ITS) regions, satellite DNA or heat-shock protein 70 (Hsp70) and DNA topoisomerase 1 genes (Iwahori et al 2000;Kang et al 2004Kang et al , 2009Matsunaga and Togashi 2004;Cao et al 2005;Castagnone et al 2005;Leal et al 2005Leal et al , 2007Wang et al 2009;Huang et al 2010;and Zhuo et al 2011;Ye and Giblin-Davis 2013). In addition, some studies have carried out direct molecular detection of B. xylophilus by isolating DNA directly from either wood samples (Takeuchi et al 2005;Franc ßois et al 2007;Kikuchi et al 2009a;Hu et al 2011;Kanetani et al 2011;Cardoso et al 2012) or the Monochamus vector (Wang et al 2010(Wang et al , 2011. In all of the abovecited methods, the detection of PWN relies on the presence of genomic DNA, which means that it fails to differentiate between living and dead PWN, as intact DNA can be extracted from PWN that is no longer viable.…”

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

“…One previously described method, an adaptation of the cetylmethylammonium bromide (CTAB) method, was efficient, but laborious and time consuming (Takeuchi et al 2005;Futai 2007, 2009). Another method described by Kikuchi et al (2009) and Kanetani et al (2011), cannot be reproduced as there is no description of the buffers and the mentioned kit is not commercially available. The DNA extraction methods, directly from wood samples, described by Wang et al (2010) and Hu et al (2011) are time consuming and use only 5 mg of wood sample as starter material, which is a much smaller sample than the 100 mg used in this study.…”

“…Molecular detection methods are simpler, faster and reliable and thus several PCR based methods to detect B. xylophilus with species-specific primers have been developed by targeting either rDNA, ITS regions, satDNA or Hsp70 and DNA topoisomerase I genes (Iwahori et al 1998(Iwahori et al , 2000Kang et al 2004;Matsunaga and Togashi 2004;Cao et al 2005;Castagnone et al 2005;Leal et al 2005Leal et al , 2007Kang et al 2009;Huang et al 2010;Zhuo et al 2011). However, few studies on the use of these methodologies for direct detection of PWN in pine wood and insect vector, without the preliminary steps of nematode extraction, have been conducted (François et al 2007;Takeuchi and Futai 2007;Kikuchi et al 2009;Takeuchi and Futai 2009;Kanetani et al 2011;Wang et al 2010;Hu et al 2011;Wang et al 2011). In the present study, a new methodology was developed for the direct detection of PWN, performed by PCR amplification of the species specific MspI satDNA, leading to a pattern of monomer and multimers of the 160 bp monomer (Tarés et al 1994;Castagnone et al 2005), as recommended by the European and Mediterranean Plant Protection Organization (EPPO) (2009), using total DNA extracted from P. pinaster wood and bark samples and from the insect vector, M. galloprovincialis.…”