| Inoculated one-year old pine seedlings with Bursaphelenchus. xylophilus, the experiment studied the course and manner of host parenchymatous cells death in pine wilt disease. The stem was cut into several segments away from the pine seeding, arranged and collected in the order of chronology time and distance from where they were inoculated. To separate B.xylophilus coarsely out from the stems and made them into paraffin slices. And be observed under the potics microscope by dyed with safranin-fast green contrast-stain method and PI fluorescence stain method. In order to find out how about the nematode rigour distributing , but also ebb and flow of their amounts after invading the host pine seeding. Seek the pathological changing orderliness of host cells in different position along with the remotion of nematodes. By deeply analysing the variety of host cell nucleolus to estimate whether the host cell took place the programmed cell death after inoculated B.xylophilus.The nematodes can immediately intrude the host pine xylem from resin duct beside exposed incision. They overspread along radial xylem in landscape orientationin while spread vertically along resin duct, distributing just like a network. In initial inoculated stages, the nematodes diffused downwards more rapidly than upwards. While the pine resin ducts were widely destroyed, the nematodes expedited their diffusing speed both upwards and downwards. And at that time nematodes can be found in resin duct, radial xylem, cambium and cortex. As soon as the host pine appeared symptom, most of the pine tissues had inanition. Then nematodes were getting along with vigorous multiplying stage and be separated from the pin root.For the quantity and speed of host pine cell, it has a compactness correlation with nematodes'motion and diffusion. The injured parenchymatous and resinosis cell had been dead before the nematodes arrived. In the same time, the pathological changes degree of parenchymatous cells which far away inoculated dot were more gently than those were near. Parenchyma axial died earlier than parenchyma radial. When the pine resin ducts were widely destroyed, there were no obvious differences between the cells in different positions.The stem segment cells upwards the inoculated dot had difference with those downwards, and the changing trend were same with the nematodes. In the early stage, the pathological change degree of parenchymatous cells upwards inoculated dot were more gently than those downwards. Then they went to be consistent later.It is the first time that using PI fluorescence single stain method to check whether B.xylophilus can arouse PCD (programmed cell death) in plant. We can find part ofrepresentative configuration character of host cells PCD. The most apoptosis behaved were shown below. After stained by PI, the nucleolus can fluoresce red fluorescence, condensation and distortion. It is obvious that chromatin has condensation and margination like the shape of crescent. Then the nucleolus ruptures into several fragment, distributing separately with no regulation, till disintegrated finally. All of the characters were not only different far from the cell of control specimen, but also from the necrosis cell died of nature drought. It can offer sub-microcosmic level cytology evidence for the supposing that B.xylophilus could induce the host pine parenchymatous cells to take place PCD.This kind of PCD phenomenon weren't happen by accident inside the pine seedling, neither inside some stem segment. It took place not only beside the inoculated dot, but also in the position where the position nematodes never arrived. Configuration characte of PCD appeared evidently in those cells. Especially for the quantity and speed of the PCD of cortex parenchymatous cells, they increased gradually together with the motion and diffusion of nematodes. Even more we discovered though some cells border upon at the same position, the degree they apoptosis weren't assimilation estate. |
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