New strategies for protecting pine trees from pine wilt disease

在显微镜下，通过视频监视器记录了显微松（松）切片的栓塞，并对该切片的声发射进行了计数。栓塞发生时，事件数增加。这一观察得出结论，栓子在线虫接种的松树中没有恢复，而在正常的松树中则没有恢复。因此，这种不可逆的栓塞使水流断裂，导致树木死亡。采用线虫接种试验对松苗进行了系统化学物质筛选。我们发现一种新的化学物质可以诱导幼苗抵抗线虫诱导的萎蔫。在3年生苗木中，一种商业系统化学品对萎蔫有效，其症状有所减轻，例如，接种的枝条只有枯死，萎蔫延迟。该药剂在枝条基部诱导出杀线虫的芪类，但在幼芽中没有。因此，树枝的基部对阻止线虫的运动起着重要的作用。另一点是松树种群中芪类化合物含量的变化。这种变异表明，线虫抗性品系可能是通过苯乙烯的含量来选择的。研究发现，苯乙烯类化合物的形成是预防松材萎蔫病的关键策略之一，因此从密松中克隆了苯乙烯合成酶基因，并对其进行了鉴定。在大肠杆菌中成功表达了三个具有完整编码序列的新基因。其中1个被证实具有二苯乙烯合成酶活性。因此，获得的克隆将可用于构建具有抗松树枯萎病的转基因松树。

Embolism in a microtomed pine (Pinus densiflora) section was recorded by a video-monitor under a microscope during counting the acoustic emission from the section. The event numbers were increased when embolism occurred. This observation led a conclusion that the embolism was not recovered in nematode inoculated pine trees while it does in the sound tree. Thus, this irreversible embolism splits water conducts and it results in the tree death.Systemic chemicals were screened by nematode inoculation test in pine (Pinus densiflora ) seedlings. We found that a new chemical was induced resistance against the nematode induced wilting in the seedlings. In 3-year-old nursery stocks, a commercial systemic chemical was effective against the wilting, the symptom of which was reduced, e.g., only dieback of the inoculated branches, delay of wilting. The chemical had induced nematicidal stilbenoids in the base of branches but not in young shoots. Thus, the base of branches plays important role for stopping the nematode movement. Another point is variation of the stilbenoid levels in the pine population. The variation suggest that nematode resistant lines may be selected by stilbenoid level.Because we found that stilbenoid formation is one of the key strategies for preventing pine wilt diseases, stilbene synthase genes, a step of which is a rate limiting for stilbenoid formation, were cloned from Pinus densiflora and characterized. Three new genes with full coding sequence was successfully expressed in E.coli. One of them was confirmed stilbene synthase activity. Thus the clones obtained will be available to construct a transgenic pine tree that carries resistance against pine wilt diseases.