Assessment of the Geographic Origins of Pinewood Nematode Isolates via Single Nucleotide Polymorphism in Effector Genes

Abstract

The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification.

Bursaphelenchus xylophilus and B. mucronatus secretomes: a comparative proteomic analysis

“…15,000 nematodes as previously described39. A fraction of 2.0 μg was used as starting material for cDNA synthesis using the MINT cDNA synthesis kit (Evrogen), where a strategy based on SMART double stranded cDNA synthesis was applied40.…”

Worldwide invasion routes of the pinewood nematode: What can we infer from population genetics analyses?

“…However, these classical and Bayesian methods have proved both useful and conclusive in other cases of invasion (Ciosi et al 2008;Facon et al 2003;Kolbe et al 2004;Papura et al 2012;Perdereau et al 2013;Wan et al 2012). We can therefore put forward several hypotheses to account for the conflicting results obtained here: (1) the low level of diversity may have resulted in a lack of power to discriminate between the two possible alternatives, (2) the strong spatial genetic structure observed in the USA, requiring a very large sampling scheme to embrace most genetic variation of the native and (3) the lack of samples from some of the existing invaded areas, such as China or South Korea, potentially serving as sources for the invasion of Europe, as suggested by Figueiredo et al (2013) on the basis of analyses of collection samples for PWN.…”

“…Many studies have attempted to establish the origin of invasive outbreaks of PWN in different geographic areas (Cheng et al 2008;Figueiredo et al 2013;Fonseca et al 2012;Metge and Burgermeister 2008;Pereira et al 2013;Tares et al 1992;Valadas et al 2012a, b;Vieira et al 2007;Zhang et al 2008;Zhou et al 2007). An Asian origin for European invasive populations of PWN was thus proposed (Figueiredo et al 2013;Fonseca et al 2012;Metge and Burgermeister 2008;Valadas et al 2012b).…”

“…An Asian origin for European invasive populations of PWN was thus proposed (Figueiredo et al 2013;Fonseca et al 2012;Metge and Burgermeister 2008;Valadas et al 2012b). However, these studies were subject to experimental limitations, such as the use of low-resolution markers (e.g.…”

EU Legislation on Forest Plant Health: an Overview with a Focus on <em>Fusarium Circinatum</em>

“…also transmitted to the wood by ovipositing beetles. They rapidly multiple in the resin canals leading to tree death within a fewmonths [49,50].…”