To study the relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes (PWN) (Bursaphelenchus xylophilus), we conducted experiments using clonallypropagated Japanese black pines (Pinus thunbergii) with pre-evaluated individual resistance levels. Bark including the cortical resin canals-one of the main migration pathways of PWN-was removed by girdling, but neither the migration of PWN nor symptom development of pine wilt disease were inhibited by this treatment in non-resistant clones. Histological observations showed no significant differences in the lumen area or the number of cortical-and xylem-axial resin canals between resistant and susceptible clone groups from a half-sib family. A bioassay using methanol extracts from resistant and susceptible clones showed that extracts from both clones showed similar attractant effects to PWN, but neither had repellent effects. The resistant clones were multi-inoculated with PWN into three split points to mimic migration in the stem. The proportion of damaged plants was not significantly different from that in single-inoculated plants (control). In this experiment, the number of PWN detected from partially-damaged plants was much higher than that from non-damaged plants. An inoculation test using stem cuttings showed that the population of PWN increased in susceptible cuttings at 1-20 days after inoculation (dai), while it remained unchanged or gradually decreased in resistant cuttings. These findings suggested that the factors contributing to resistance were associated with inhibiting the proliferation of PWN, rather than inhibiting their migration.

Adventitious rooting is essential for cutting propagation of pine wilt-resistant Pinus thunbergii. To examine a variety of adventitious rooting potentials among donor plants, cuttings were taken from 31 seedlings within a half-sib family. Rooting abilities of cuttings from each seedling ranged from 0 to 100%. When 11 ortets and 11 ramets (clonally propagated from each ortet) were used as donor plants, there was a positive correlation between rooting abilities of cuttings from ortets and ramets, suggesting that adventitious rooting is dependent on genetic factors in the donor plants. To promote adventitious rooting of cuttings by hormonal treatment, we examined the effect of soaking time in Oxyberon (19.7 mM indole-3-butyric acid (IBA) solution) on rooting. Ten minutes was the best soaking time for rooted cuttings to produce more adventitious roots without impairing normal growth. When cuttings were soaked in Ethrel diluent (69.2 lM ethephon) for 24 h before soaking in Oxyberon for 10 min, a significantly higher rooting ability was observed than those soaked in Oxyberon alone. Ethrel on its own barely affected rooting ability. The positive effect of the combinational treatment was confirmed in a similar experiment using authentic ethephon and IBA instead of Ethrel and Oxyberon. When cuttings were soaked in a mixture of ethephon and silver thiosulfate (STS), an ethylene action inhibitor, before IBA-soaking, the effect was partially diminished compared with combinational treatment without STS. These findings suggest that ethylene action caused by ethephon treatment promotes IBA-mediated adventitious rooting of P. thunbergii cuttings.

Random amplified polymorphic DNA (RAPD) markers were used to identify the male parents of the half-sib progeny from a single clone within a Japanese black pine (Pinus thunbergii Parl.) clonal seed orchard consisting of 16 pinewood-nematode [Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle] resistant clones. Twenty-one primers, with 28 polymorphic and reproducible fragments, were selected from 320 primers examined. The genotypes of orchard clones at each fragment were fingerprinted by RAPD analyses of both diploid needles and haploid tissues of megagametophytes from each clone. We used all 28 RAPD markers to identify the male parents of the half-sib progeny of a single orchard clone, Tanabe-(t)54. By comparing the genotype of orchard clones with the phenotype in the seedling at each RAPD locus, we were able to identify the male parents of 82 out of 85 seedlings. Pollen contamination and natural selfing occurred at rates of only 2.4 % and 1.2 %, respectively. Mating with the clone deviated significantly from panmixia. More than half of the progeny of the clone were produced through fertilization by only two clones. Seven clones did not contribute at all to fertilization as the male parent. Deviations from panmixia have been caused by a difference in the distance between clones, variation in the amounts of pollen, and phenological synchrony between clones. The possibility and limitations of RAPD markers for paternity analysis in conifer seed orchards are discussed.

Overall male reproductive success was investigated in a Japanese black pine (Pinus thunbergii Parl.) clonal seed orchard consisting of 16 nematode-resistant clones, using random amplified polymorphic DNA (RAPD) markers. We genotyped all 16 of the orchard clones at selected RAPD loci by analyzing diploid needles and 10 haploid megagametophytes from each of them. In addition, the male parent of open-pollinated seedlings was identified by comparing their phenotypes with the genotypes of the orchard clones. In this way the male parents of 559 seedlings of 648 seedlings of progenies from 14 parental (orchard) clones were determined. The level of external pollen contamination in the seed orchard was estimated to be 2.2% for the total population. The effective mean selfing rate observed was 1.5%. Male reproductive success among clones varied widely, from 0.8 to 24.4%, for the total population. Our data suggest that the significant deviation in male reproductive success of individual clones from panmictic ratios may be a problem for the genetic quality in seed orchard crops. To improve the parental balance of seed orchard crops, the use of artificial management techniques such as gibberellin A4/7 treatment, supplemental mass pollination, and rearrangement of clonal ramets is recommended in seed orchards.