Research Of Programmed Cell Death In Interaction Between Pinus Thunbergii And Bursaphelenchus Xylophilus

Studies on development of pathogenesis within 2-year-old seedlings of Pinus thunbergii infected with Bursaphelenchus xylophihts were conducted systematically in this paper. Methods of histology, cytobiology immunology, molecular biology and biochemistry were used. Results were described as follows.1. Results showed that behaviors of nematode dispersal in seedlings caused by different virulent isolates varied considerably, which resulted in the development of histopathology within host differed significantly, the velocity of disease occurrence was related to the survival rate of nematodes inside the seedlings and its development was directly influenced by dispersal rate and multiplication capability of nematodes.The virulent isolate of B. xylophihts dispersed throughout the host via axial and radial resin duct. And nematodes had great dispersal ability as the resin duct is a main reticular system of pine. Population of avirulent isolate of B. xylophilus mainly dispersed into cortical layer which led to a lower dispersal rate and killing power.Results also indicated that no nematode dispersal was detected in cambium of pine regardless of the inoculated isolate was virulent or avirulent. Which suggested the cell death of cambium was a bionecrosis and the phenomenon was similar to that of parenchyma cells such as epithelial cell in resin passage, resulted indirectly from nematode dispersal.2. Results showed that the pine wilt disease was caused by programmed cell death of the seedlings induced by B. xylophilus.Transmission electronic microscopy (TEM) showed the features of programmed cell death of cambium in stem as well as parenchyma around it. Variations of ultrastructure of mesophyllous cells in needles further confirmed that phenomena of programmed cell death existed in seedlings infected with B. xylophilus.The above phenomena suggested that the pine wilt disease was caused by systematically destruction, and reasons of which was indirect to the cell death of stems and leaves induced by the pathogens but direct to intrinsic gene program within the host.3. TUNEL technicque test showed that the majority of parenchyma cells of 2-year-old seedlings of Pinus thunbergii which infected with Bursaphelenchus xylophilus showed positive reaction, explaining that the DNA of parenchyma nucleolus degradate ordered. Systematic and persistent PCD was found in the host through the electronic microscopy (TEM) which was an evidence of the TEM observation. The result of garose gel electrophoresis didn't show tipical DNA ladder.4. Studies on the development of water potentials, activities of root system and polyamine in seedlings inoculated with virulent and avirulent B. xylophihts and B. mucronatus, respectively, further identified that the pine wilt disease was caused by programmed cell death but not because of the abnormality of water metabolism.5.Studies on variation of polyamine inside seedlings showed that increasing of polyamine had positive function on the resistance to B. xylophilus for Pinus thunbergii, in which 'put' played the most important role.6. Studies also indicated that occurrence of programmed cell death inside infectious seedlings might be correlated with concentration of Ca²⁺ inside cells.Distribution of subcellular free Ca²⁺ in parenchyma cells at different time periods after Pinus thunbergii being inoculated with B. xylophilus was tested, using the method of Nymphoides precipitation. Results showed that, with the development of disease, changes in the concentration of free Ca²⁺ in parenchyma cells were characterized as "low-high-low-high".