The first country suffering from pine wilt disease (PWD) is Japan, and the disease has recently spread to some other countries including Korea and China. Although devastated areas by PWD are now gradually decreasing through continual applications of various control methods, a great amount of pine trees still are lost. For effective control of this disease, we need to know the mechanism of symptom development more precisely.

After invasion of a pathogenic pine wood nematode (PWN), Bursaphelenchus xylophilus, into a pine tree, PWNs are thought to migrate to every branch through pine tissues, proliferate, kill pine cells, and thereby lead the pine tree to death. Since PWN migration is a prerequisite to development of all symptoms, it is important to know how PWNs migrate within pine tissues. PWN migration has been investigated using the Bearmann funnel technique by which the number of PWNs in a branch segment could be counted, and migration patterns within pine trees after PWN inoculation have been outlined. Such kind of investigation, however, could not directly reveal migration routes of invading PWNs on the scale of each tissue because segments for the Bearmann funnel technique can be prepared only as assemblages of various tissues. Although some investigators infer migration routes on the scale of each tissue from the distribution of discolored and damaged regions on the assumption that pine tissue damage after PWN inoculation coincides with PWN presence, none of them provide direct evidence for PWN migration routes. Migration of individual PWNs in each tissue needs to be traced more precisely to clarify the causal relationship between PWN presence and pine symptom. Recently, a new staining technique in which fluorescein isothiocyanate-conjugated wheat germ agglutinin (F-WGA) intensely stains PWNs in pine tissues without staining background has been developed and enabled us to know PWNs distribution within each pine tissue much easily.

In this thesis, I investigated time-course changes in distribution of pathogenic and nonpathogenic PWNs in PWD-susceptible and resistant pines after PWNs inoculation using the F-WGA staining technique and inferred PWN migration routes.

Distribution and migration routes of Bursaphelenchus xylophilus in Pinus thunbergii

Distribution of a pathogenic PWN (B. xylophilus) in the tissues of susceptible Pinus thunbergii seedlings was investigated. After PWNs were inoculated into current-year stems of pine seedlings, 1-cm blocks excised from the stems at about 5 cm below the inoculation site were thin sectioned and stained with F-WGA. Distribution of PWNs in each tissue of cross thin sections was observed under epifluorescence microscope. PWN distribution was confined only to cortical resin canals at 1 day after inoculation (dai), and then spread to xylem axial resin canals, pith and resin canals of short branches at 3 dai. PWNs were newly detected in cortical tissues and tracheids at 7 dai. Lots of PWN were additively distributed in cortical tissues and cambial region at and after 14 dai. A new finding that PWNs invade cortical resin canals of short branches at as early as 3 dai may suggest that the leaf wilting appearance specific to PWD results from damaging of the leaf bases by the PWNs.

To estimate vertical or horizontal migration speed of PWNs, PWNs were inoculated onto the cross or tangential cut surfaces of P thunbergii stem segments and time-course changes in PWN distribution were chased using F-WGA staining technique. Maximal speed of PWN migration was estimated to be much faster through cortical and xylem axial resin canals (more than 6.7 and 2.2-2.3 mm per hour, respectively) than through cortical tissues both vertically and horizontally (1.0-1.2 and 0.2 mm per hour) at 6 hours after inoculation (hai).

To examine whether PWNs in resin canals could invade surrounding tissues, pulse-chase experiments were performed. Segments in which PWNs resided only in cortical resin canals were prepared by removing 2 cm portions including the inoculation site (top cross-cut surface) from 5 cm stem segments at 6 hai. Additional incubation of residual segments caused extended PWN distribution to xylem axial resin canals and then to other tissues. Segments in which PWNs resided only in xylem axial resin canals and pith were also prepared by removing 2 cm portion including the inoculation site at 12 hai from 5 cm stem segments girdled just before PWN inoculation at 1-2 cm from the top cross-cut surface. Additional incubation of those segments also caused extended PWN distribution to cortical resin canals and then to other tissues. These results indicate that PWNs have an ability to migrate from cortical resin canals and xylem axial resin canals to other tissues. The present pulse-chase experiments provided direct evidence for the routes of PWN migration in susceptible P thunbergii: PWNs migrate quickly through cortical and xylem resin canals from an infected site to remote site in a pine tree and thereafter PWNs within resin canals invade surrounding tissues.

Migration of Bursaphelenchus xylophilus in resistant pine species

It is known that some pine species and some families of susceptible pine species have resistance to PWD. Such host resistance may reflect the difference in PWN behavior in each pine tissue. Therefore, I tried to obtain information on resistance mechanisms against PWD from PWN migration patterns in each tissue of resistant pines.

PWNs (B. xylophilus) were inoculated onto top cross-cut surfaces of 20 cm stem cuttings of resistant pines, i.e. P strobus, P rigida and a P thunbergii resistant family Namikata, as well as susceptible P thunbergii as a control. Time-course changes in PWN distribution within these resistant pines were traced using the F-WGA staining technique up to 8 dai. PWNs were absent from entire tissues in a remote region (11 to 19 cm from the top surfaces) of P strobus cuttings at 6 and 24 hai, and still absent from almost all tissues in the region except for cortical resin canals containing a few PWNs at 3 and 8 dai, when many PWNs already spread to the remote region in susceptible P thunbergii. In P rigida, PWNs were absent from both cortex and xylem in the remote region during entire experiment time. In a resistant family of P thunbergii, PWNs were absent from entire tissues in the remote region at 6 hai, and thereafter remained absent form xylem with a few PWN in pith at 8 dai. These results indicate that PWN migration were completely inhibited in xylem and highly restrained in cortex of resistant pine species. Since the complete inhibition of PWN migration in xylem prevents PWNs widely dispersing in a pine tree, it may be at least one reason for resistance of resistant pines to PWD.

Migration in pine species and ability to kill pine cells of Bursaphelenchus mucronatus

A nonpathogenic PWN, B. mucronatus, has been isolated from pine trees. Differences in pathogenicity between B. xylophilus and B. mucronatus may reflect difference in behavior in pine tissues. Information on the differences would provide a hint on the mechanism of PWD symptom development.

B. mucronatus was inoculated onto top cross-cut surfaces of 20 cm stem cuttings of susceptible P thunbergii, P strobus, P rigida and a P thunbergii resistant family Namikata. Time-course changes in distribution of B. mucronatus in tissues of those pines were investigated using the F-WGA staining technique up to 8 dai. Distribution patterns of B. mucronatus in tissues of susceptible and resistant pines were essentially the same as those of B. xylophilus; B. mucronatus spread to the remote region in susceptible P thunbergii at 8dai, whereas migration of B. mucronatus was inhibited in xylem of P strobus, P rigida and a P thunbergii resistant family Namikata. This result indicates that nonpathogenicity of B. mucronatus may not be derived from weakened migration ability.

To estimate the ability of PWN to kill pine cells, I developed the following method. A pine cutting is inoculated with PWNs onto top cross-cut surface. Several days after inoculation, the cutting is divided into small segments (about 2.5 cm long) and tangentially cut with a razor blade to expose epithelial cells inside cortical resin canals longitudinally. Distribution of dead epithelial cells inside cortical resin canals is observed after staining with Evans' blue which stains dead cells but not living ones. While almost no dead cell in non-inoculated P thunbergii cuttings throughout the experiment for 7 days, dead cells were distributed sparsely and in an isolated manner among epithelial cells in B. xylophilus-inoculated cuttings even at 1 dai. The sparse distribution of isolated dead cells may interestingly mean that a PWN attacks one epithelial cell at once and randomly. Although density of dead cells increased at 3 dai, the dead cells were still distributed in a sparse and isolated manner. At 7 dai, dead epithelial cells increased, and in some resin canals, all cells were dead. The pattern of cell death caused by B. mucronatus inoculation was essentially the same as that by B. xylophilus inoculation. The results suggest that B. mucronatus has the same ability to kill epithelial cells and probably other pine cells as B. xylophilus.

Discoloration of pine bark peelings caused by PWN inoculation on its cambium side was also compared between B. xylophilus and B. mucronatus. Discoloration rates of non-inoculated and B. mucronatus- and B. xylophilus-inoculated bark peelings were 13%, 27% and 100% at 7 dai, and 20%, 60% and 100% at 11 dai, respectively. This result also indicates that B. mucronatus has ability to discolor pine cells although it is weaker than that of B. xylophilus.

From the above results, I concluded that lack of pathogenicity in B. mucronatus is not derived from lack of migration ability or ability to kill pine cells, but deficiency of some other ability.

Conclusion

The present time-course chases of individual PWN distribution within each pine tissue provided new findings on PWN migration routes, PWD-resistance, and PWN-pathogenicity. Pulse-chase experiments first provided direct evidence for detailed migration routes of B. xylophilus in P thunbergii; from cortical resin canals to xylem axial resin canals and vice versa, and thereafter from both cortical and xylem resin canals to surrounding tissues. Inoculation experiments of resistant pine species, P strobus, P rigida and a resistant family of P thunbergii, showed that migration of B. xylophilus in these pine species was inhibited especially in xylem at the early days after inoculation. The inoculation experiment with nonpathogenic B. mucronatus showed that the PWN has ability to migrate in resin canals and spread to surrounding tissues. Moreover, I developed a new technique to estimate PWN attack to epithelial cells of cortical resin canals, and using it, found that B. mucronatus have ability to kill pine epithelial cells in the same manner as B. xylophilus.

マツ材線虫病は、マツノマダラカミキリMonochamus alternatusの後食によってマツ樹体内に持ち込まれる病原線虫マツノザイセンチュウBursaphellenchus xylophilusが引き起こす萎凋病である。空中散布等、様々な防除対策がとられてきたが、依然として我が国で最大の樹木病害であることに変わりはない。本病のより効果的な防除方法を確立するには、今後も病原線虫の樹体内における行動をさらに詳しく理解する必要がある。本研究は、このマツ材線虫病における線虫の樹体内行動のうち移動に着目して、その特性を明らかにしたものである。また、線虫のマツ細胞に対する加害力についても、新たなアッセイ系を考案し検定している。

第一章では、マツ材線虫病の歴史とこれまでの研究を総括している。

第二章では、病原線虫一頭一頭の分布を調べることによって、線虫の移動の特徴を推定している。病原線虫を切り枝の上端に接種した後、経時的にサンプリングしてパラフィン切片を作製し、F-WGA染色により線虫のみを染色して落射型蛍光顕微鏡で線虫の分布を組織ごとに調べている。その結果、樹体内に侵入した線虫は、まず皮層樹脂道内に分布すること、時間とともに分布は他の組織へも広がることが分かった。また、接種後上端を切除して、皮層樹脂道あるいは木部樹脂道にのみ線虫が存在する状態を作ると、それらの線虫が時間とともに他の組織に広がって分布することも示している。これは、樹脂道内の線虫が周辺の組織へ移動することを示す新たな直接的証拠である。

第三章では、抵抗性マツ内での病原線虫分布の経時的変化を、同様の方法で組織ごとに調べている。その結果、抵抗性マツでは共通して木部への分布が阻害されること、また皮層樹脂道では樹種によっては阻害されないことを新たに明らかにしている。従来の研究では、べールマン法により枝断片といった組織複合体内の線虫数を調べたり、切り枝全体を通過する線虫数を数えたりして移動を推定していたため、線虫移動を組織ごとに把握することはできなかった。例えば、本研究では抵抗性マツの木部での移動阻害を明らかにしているが、皮層樹脂道での移動が阻害されない場合には、枝全体での移動を見ている限り、このような移動阻害は検出されない。本研究は、個々の線虫の移動を組織ごとに調べることの重要性、必要性を示した点でも意義深い。

第四章では、非病原性線虫ニセマツノザイセンチュウB.mucronatusの分布の経時的変化を組織ごとに調べている。その結果非病原性線虫が病原性線虫と同様の分布変化を示し、同様の移動をし得ることを明らかにしている。一方、線虫による細胞加害力を評価する方法を新たに確立し、非病原性線虫が細胞加害力を持つことも明らかにしている。この方法では、線虫接種後の枝断片をそぎ切りして皮層樹脂道のエピセリウムを露出させる等、独創的な工夫をしており、加害され死んだ個々のエピセリウム細胞を明確に検出することに成功している。今後この新たな実験系は、マツ材線虫病の病理学的研究に様々に応用されることが期待される。

第五章では、以上の結果をもとに、病原線虫移動について総合考察を行っている。

以上のように本研究では、線虫一頭一頭の樹体内分布を調べることにより、マツ材線虫病病原線虫の樹体内移動に関する新知見を得ている。とりわけ、線虫が樹脂道内から周辺の組織へ移動すること、抵抗性マツでは木部の線虫移動が共通して阻害されていること、非病原性線虫の移動様式、細胞加害力が病原性線虫と差がないこと等の新知見は、発病過程を理解し防除法を考える上での重要な手がかりを与えるものである。また、病原線虫によるマツ柔細胞への加害を細胞単位で直接検出できる実験系を確立したことも、今後本病の病理を研究する上で大きな貢献が期待できる重要な研究成果である。以上のように、得られた知見は独創的、先駆的でありかつ応用的意義も大きい。従って、本研究は応用上、学術上の貢献が極めて大きく、審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。