Cell Biology, Reactive Oxygen Metabolism And Cell Hypersensitive Response Of Poplar-Canker Pathogen Interaction

During the interaction of poplar and canker pathogen, the infected cells will go through aseries of responsive events related to plant resistance, including recognition of plant cell topathogen, intracellular signal transduction, initiation of defense responses, reactive oxygenburst, generation of SAR resistance, etc., which in the time and space have a close correlationwith structural change, necrosis and pathogen depression of plant tissue and cells. The relatedresearches have obtained much tremendous accomplishment on the interactions of crops andpathogens, but far from sufficient on that of trees and pathogens.In this paper, callus, excised branches and potted cuttings of two poplar cultivars, Populustomentosa and P. beijingensis, were used as materials to study the generation or accumulationof cell membrane meability, soluble sugar, malonadehyde (MDA), reactive oxygen species(ROS), defensive enzymes. The difference between susceptible and resistant poplar cultivarswas analyzed by the means of electron microscopy, fluorescence microscopy, molecular cellbiology and cell chemical, etc. The ultrastructural changes of cells during the infection processof B. dothidea on host were observed and the relationship between water stress and diseasedevelopment was also analyzed. At the meantime, the function of POD in the resistant materialwas discussed by the cytochemical location. The cell hypersensitive response (HR) in theprocess of the interaction of poplar and pathogen was preliminarily studied by the means ofelectron and fluorescence microscopy and DNA Ladder in order to understand the interactionmechanisms of host and pathogen from different angles.The main results were as following:1. Relative electrical conductivity, soluble sugar and MDA content of cells weredetermined in two poplar callus, P. tomentosa and P. beijingensis, after inoculation with B.dothidea. The results showed that the cell membrane and relative electrical conductivitysignificantly increased, and the increased range of susceptible showed greater than that ofresistant one; there was a significant change of soluble sugar content between susceptible andresistant species. The soluble sugar content of susceptible species increased and reached peaksat 24 h and 120 h after inoculation, while that in resistant one was a little higher than that ofcontrol at 48 h, which was 24.96μg·g^-1FW with an 11.43% increase rate compared with that of the control. Soluble sugar content in the resistant cultivar had no significant difference thanthat in control. The MDA content in susceptible cultivar increased and did not change evidentlyin resistant one, which was verified that an evident membrane lipid peroxidation occurred inthe cells of susceptible cultivar.2. Under simulated drought stress in the greenhouse, the characteristics of canker diseasedevelopment and plant cell ultrastructure under the coaction of drought and pathogen werestudied on one-year-old potted cuttings of P. tomentosa and P. beijingensis under three droughttreatments, normal, moderate and severe stresses. The results showed: (1) the canker disease ofboth cultivars was serious gradually with increased drought; (2) the bark tissue cells sufferedplasmolysis, more evident with the severity of drought stress; (3) under the drought andinoculation with B. dothidea, cells of two cultivars damaged at different degree, mailyrepresented in the changes of organelles, such as chloroplast swollen and distorted, number ofmitochondria increased and membrane system indistinct; then organelles suffered furtherdamagement with inoculation time, thinned mitochondrias stroma, decreased cristae, crumpledand partly broken membrane of chloroplasts with stroma exosmosis. At last, the chloroplastspartly disorganized; (4) the hyphae growed mainly intercellular in resistant cultivar and notonly intercellular but also intracellular in susceptible cultivar, which directly caused thenecrosis of cells; (5) under the severe drought, the damage of cells enhanced the infection ofpathogen and drought and pathogen stressed together and promoted the disease development;the damage from pathogen on cells was more serious than that from drought. The double stressof drought and canker pathogen enhanced and increased the disease development and severity.3. The cytochemical localization of POD showed that the peroxidase was found in cells ofboth cultivars after inoculation. The activity of POD in resistant cultivar was higher than that innot only the uninoculated controls but also the inoculated susceptible ones and localized morewidely, such as localized in mesophyll cell wall, intercellular spaces, membranes of chloroplastand mitochondrion and vacuolar membrane. Whereas the POD in susceptible cultivar waslocalized mainly in cell walls but little in plasma membrane with a lower range and intensitythan that in resistant cultivar. The POD in resistant plant cells generated earlier andaccumulated greater than that in susceptible ones. The accumulation of POD in susceptibleplant cells was obvious fewer than that in resistant ones. 4. The dynamic changes of O₂^·-, H₂O₂, SOD, POD, CAT, APX and MDA in poplar specieswith different resistance were measured after inoculation by B. dothidea and the relation ofROS generation, defensive enzymes and membrane lipid peroxidation and diseasedevelopment were analyzed. The results showed that the generation rate of O₂^·- and H₂O₂ oftwo poplar cultivars increased with the inoculation time and there was an obvious oxygen burstin resistant cultivar and unobvious in susceptible one. The activity of POD increased with agreater increased rate in resistant plant cells. The activities of SOD and CAT went up at thebeginning and down later, which in susceptible cells were depressed evidently at the late stage.There was a great change of APX activity in two poplar cultivars, greater range in resistantcultivars than that in susceptible one. The increased MDA content in two poplar cultivarsdemonstrated that oxygen burst initiated the membrane lipid peroxidation. In the susceptiblecultivar, the MDA content greatly raised and there was a significant positive correlationbetween MDA and O₂^·- generation rate.5. Nucleus in different time in poplar callus after inoculation with B. dothidea were dyedby DAPI and observed under fluorescence microscopy. The results showed that some nucleusof P. tomentosa was uneven and shrunken in chromatin 24 hours after inoculation, which wasstick or half-moon in shape, whereas most nucleus were approximately round. Most nucleusesof host cells become abnormal 48 hs after inoculation, and there was obvious chromatinuneven and DNA broken partly. Hyphae could be seen cellular with fewer branches, while at144 hs there were multi-branched hyphae cellular at 72 hs, which infected neighborhood cells,whose nucleus showed no fluorescence and become vacuolar. In the cells of P. beijingensis 48hs after inoculation, intercellular hyphae was found and more branched at 72 hs with vacuolarneighbor cells without evident chromatin condensation. DNA extracted from bark tissue by1.6% agarose-gel electrophoresis indicated there were evident DNA Ladder at 5 to 6 days afterinoculation by B. dothidea in resistant cultivar, but no the same phenomenece in susceptiblecultivar and no systematic HR in both vultivars. DNA extracted form poplar callus showed thatthere was no DNA Ladder in two poplar cultivars. There was tailed DNA in both cultivars,which was thought as a necrosis by cytology results.