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 in Pinus thunbergii, as well as other Pinus species that are also susceptible to PWD. Results A total F1 mapping population of 188 individuals derived from a cross between the PWD-resistant P. thunbergii varieties ‘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 of P. 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 designated PWD1 for PINE WILT DISEASE 1. Conclusions The PWD1 locus is a major resistance QTL located on the Pinus consensus LG03 that acts in a dominant manner to confer pine wood nematode resistance. Information from the present study will be useful for P. thunbergii breeding programs to improve resistance to PWD, and also to help identify susceptibility genes in Pinus species.

Knowledge of the genetic relationship between growth traits and wood properties is critical for their simultaneous genetic improvement. We measured the height and diameter at breast height (DBH) and wood quality traits, including stress wave velocity (SWV) as the selection criteria for wood stiffness, wood density, and Pilodyn penetration depth as selection criteria for wood density, at a progeny test site at stand age ca. 30, which comprised of full-sib families by a full diallel mating design with eight plus Larix kaempferi tree clones. We estimated the genetic parameters for each trait and phenotypic, genetic and residual correlation between traits. The contribution of specific combining ability and reciprocal effects were small for all traits. Growth traits showed high positive genetic correlation with average wood density of the outermost five rings (0.912 for height, 0.826 for DBH) and with SWV (0.738 for height, 0.762 for DBH), irrespective of small phenotypic correlations between them. Wood density and SWV also showed high genetic correlation. Pilodyn penetration depth showed high selection efficiency for average wood density of the outermost five rings (79.8 %) whereas SWV showed higher selection efficiency for wood density. Thus, simultaneous genetic improvement of growth traits and wood properties of L. kaempferi appears possible.

The among-tree differences in the inhibition of systemic dispersal of Bursaphelenchus xylophilus by Pinus densiflora were determined. Two-year-old branches were collected from four different aspects and at two different heights from six field grown P. densiflora. The virulent isolate of B. xylophilus, T-4, was inoculated on the upper end of 5-cm-long branch sections placed upright in a glass vial. The number of nematodes passing through the sections over 24 h was counted. Daily changes in the inhibition of systemic spread of the nematode were also determined. Two-year-old branches were collected at four hour intervals for 24 h from six additional trees. These branches were then inoculated with the nematode. The number of nematodes passing through the branch sections differed among the pine trees but did not differ between the branch heights or aspects. A diurnal change in the passing nematode number was observed only for a tree which grew on the top of a steep slope.

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.

Two species-specific PCR primer pairs were developed for identifying the two nematode species, Bursaphelenchus xylophilus and B. mucronatus. The primer pairs were developed from the sequence of ribosomal DNA (rDNA) repeats to produce DNA fragments of different lengths by PCR amplification. The DNA fragments for B. mucronatus and B. xylophilus were 210 bp and 557 bp, respectively. When mixed, neither primer pair inhibited the PCR amplification of the other. Five isolates of B. xylophilus and four isolates of B. mucronatus showed different band profiles of PCR products between the two species, but identical profiles among isolates of the same species.