Background: Pine wilt disease (PWD), which is caused by the pine wood nematode (PWN)Bursaphelenchus xylophilus, is currently the greatest threat to pine forests in Europe and East Asian countries including Japan. Constructing a detailed linkage map of DNA markers and identifying PWD resistance genes/loci lead to improved resistance inPinus thunbergii, as well as otherPinusspecies that are also susceptible to PWD.Results: A total F₁mapping population of 188 individuals derived from a cross between the PWD-resistantP. thunbergiivarieties ‘Tanabe 54’ (resistant rank 2 to PWD) and ‘Tosashimizu 63’ (resistant rank 4 to PWD) was inoculated with PWN, and was evaluated for disease symptoms. To perform linkage analysis for PWN resistance, a set of three maps was constructed; two parental maps generated using the integrated two-way pseudo-testcross method, and a consensus map with population-type cross-pollination. The linkage map of ‘Tanabe 54’ consisted of 167 loci, and covered 14 linkage groups (LGs), with a total genetic distance of 1214.6 cM. The linkage map of ‘Tosashimizu 63’ consisted of 252 loci, and covered 14 LGs, with a total genetic distance of 1422.1 cM. The integrated consensus map comprised 12 LGs with the basic chromosome number ofP. thunbergii, and a total genetic distance of 1403.6 cM. Results from quantitative trait loci (QTL) analysis using phenotype data and linkage maps indicated that PWN resistance is controlled by a single dominant allele, which was derived from the ‘Tanabe 54’ female parent. This major QTL was located on linkage group 3 and was designatedPWD1forPINE WILT DISEASE 1.Conclusions: ThePWD1locus is a major resistance QTL located on thePinusconsensus LG03 that acts in a dominant manner to confer pine wood nematode resistance. Information from the present study will be useful forP. thunbergiibreeding programs to improve resistance to PWD, and also to help identify susceptibility genes inPinusspecies.

Differences among four isolates of Bursaphelenchus xylophilus and one of B. mucronatus in vulnerability to the inhibitory effect of Pinus densiflora on nematode dispersal were investigated by inoculating boiled and living branch sections 5 cm long. The intrinsic dispersal ability and vulnerability to the inhibitory effect of living branches differed among isolates. Additionally, the degree of branch inhibition of nematode dispersal was examined by inoculating the five isolates on 2.5 and 5.0 cm long, P. densiflora living branch sections. An increase in section length induced an 80% decrease in the number of nematodes of all isolates passing through the branch. The extent to which passing nematode number decreased as branch section length increased can be used to express susceptibility of pine species to pine wilt disease. Virulence had no relation to intrinsic and realised dispersal rates in B. xylophilus.

Summary: The pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer), is the causative agent of pine wilt disease. It is native to North America and has been devastating pine forests in East Asia and West Europe since its introduction. The pathogenic nematode is transmitted by cerambycid beetle adults of Monochamus alternatus Hope in East Asia and M. galloprovincialis (Olivier) in West Europe. Bursaphelenchus mucronatus Mamiya et Enda is widely distributed in Eurasia and has no or little pathogenicity to pine species. If hybrids between the two species are backcrossed with B. xylophilus nematodes repeatedly, a small proportion of B. mucronatus genes will be infused into B. xylophilus genome (introgression) and may lead to adaptation to locally harsh environments. Inoculation tests in this study revealed that two of eight hybrid-derived populations from crossing B. xylophilus and B. mucronatus caused pine seedling mortalities of 36-70%. The pathogenic hybrid-derived populations carried nuclear ribosomal DNA (rDNA) of two species and B. xylophilus extra-nuclear genes, whereas non-pathogenic populations carried B. mucronatus rDNA and extranuclear genes of B. xylophilus or B. mucronatus. Thus, it is inferred that a gene or genes associated with pathogenicity are present in the nuclear genome of B. xylophilus. A laboratory experiment revealed that the pathogenic hybrid-derived population had as great an ability to board M. alternatus adults as the parental-lineage B. xylophilus isolate. Thus, the introgressive hybridisation is considered to occur in pine forests if two species cohabit in host trees.

In the twentieth century, a substantial decline in Pinus thunbergii populations in Japan occurred due to the outbreak of pine wood nematode (PWN), Burshaphelencus xylophilus. A PWN-P. thunbergii resistant trees-breeding project was developed in the 1980s to provide reforestation materials to minimalize the pest damage within the population. Since climate change can also contribute to PWN outbreaks, an intensive reforestation plan instated without much consideration can impact on the genetic diversity of P. thunbergii populations. The usage and deployment of PWN-P. thunbergii resistant trees to a given site without genetic management can lead to a genetic disturbance. The Iki-no-Matsubara population was used as a model to design an approach for the deployment management. This research aimed to preserve local genetic diversity, genetic structure, and relatedness by developing a method for deploying Kyushu PWN-P. thunbergii resistant trees as reforestation-material plants into Iki-no-Matsubara. The local genotypes of the Iki-no-Matsubara population and the Kyushu PWN-P. thunbergii resistant trees were analyzed using six microsatellite markers. Genotype origins, relatedness, diversity, and structure of both were investigated and compared with the genetic results previously obtained for old populations of P. thunbergii throughout Japan. A sufficient number of Kyushu PWN-P. thunbergii resistant trees, as mother trees, within seed orchards and sufficient status number of the seedlings to deploy are needed when deploying the Kyushu PWN-P. thunbergii resistant trees as reforestation material planting into Iki-no-Matsubara population. This approach not only be used to preserve Iki-no-Matsubara population (genetic diversity, genetic structure, relatedness, and resilience of the forests) but can also be applied to minimize PWN damage. These results provide a baseline for further seed sourcing as well as develop genetic management strategies within P. thunbergii populations, including Kyushu PWN-P. thunbergii resistant trees.

Background and Objectives: To determine whether the progeny of pinewood nematode-resistant Pinus thunbergii Parl. clones selected in the southwestern region of Japan could be successful in reforestation in the northern region, we investigated the magnitude of the genotype–environment interaction effect on the resistance against Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle in P. thunbergii. Materials and Methods: We inoculated P. thunbergii seedlings of six full-sib families, with various resistance levels, with B. xylophilus in nurseries at three experimental sites in the northern and southern regions of Japan. All parental clones of the tested families originated from southwestern Japan, and selection of parental clones for resistance was performed in the same region. Sound rates after nematode inoculation were calculated, and survival analysis, correlation analysis and variance component analysis were performed. Results and Conclusions: Families with high sound rate in the southern region also showed a high sound rate in the northern region. In almost all cases, Spearman’s correlation coefficients for sound rates were more than 0.698 among sites. The variance component of the interaction between site and family was small compared to that of site and family separately. Thus, we conclude that the resistant clones selected in the southern region would retain their genetic resistance in the northern regions.

Pine wilt disease (PWD) caused by the pinewood nematode (PWN) (Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle) is a worldwide issue. Infection is considered to be promoted mainly by the increased air temperature, but it is important to investigate whether the effect of high temperature similarly influences the different ranks of resistant clone. In the present study, we conducted PWN inoculation tests using six common open-pollinated families of resistant Pinus thunbergii Parl. The tests were conducted at nurseries of five test sites across Japanese archipelago between 2015 and 2017. Our analysis focused specifically on temperature. Firstly, we examined the effects of test sites, inoculation year, and their interaction on unaffected seedling rate and found that the unaffected seedling rate of all tested pine families decreased as the cumulative temperature increased. We found that the unaffected seedling rate decreased as the cumulative temperature increased for all tested pine families. In general, higher cumulative temperatures were required for having an effect on the unaffected seedling rates of higher PWN-resistant families. Typically, early cumulative temperatures, i.e., 19 days after inoculation, had the greatest effect on the unaffected seedling rates of PWN-resistant pines. However, the relationship between cumulative temperature and predicted unaffected seedling rate follow similar rate for all families. Thus, the order of resistance level is maintained in terms of the cumulative temperature required for having an effect.