Study On The Physiological Response Mechanism Of Pear Callus To Pathogenic Fungi Infection

Plants have developed different adaptive mechanisms and defense responses to protectthemselves against disease, which are same with suffering other stress. The infected cells willgo through a series of responsive events related to plant resistance, including cells structuralchange，physiological and biochemical responses, necrosis and pathogen depression of planttissue and cells. It is very important to studying on the defense resistance mechanisms of thehost to pathogen infect for improve plant disease. The related studies have obtained muchtremendous accomplishments on the interactions of crops and pathogens，but far fromsufficient on that of fruits and pathogens.In this study, callus, elicitation from Jinmi pear fruits, was used as materials to study thegeneration or accumulation of cell membrane meability, soluble sugar, malonadehyde (MDA),the defensive enzymes. The difference between susceptible and resistant of callus to fungiwas analyzed by the means of fluorescence microscopy，molecular cell biology and cellchemical，ete. The ultrastructural changes of cells during the infection process of two differentfungi on callus were observed. At the meantime，the function of defensive enzymes andisozyme in the defense response were discussed. The cell hypersensitive response (HR) in theprocess of the interaction of pear callus and pathogen was preliminarily studied by the meansof electron and fluorescence microscopy and DNA ladder in order to understand the defensemechanisms of host and pathogen from different angles.The main results were as following:1. The callus was inducted with mature jinmi pear. Through the subculture, the callusthat was yellow, uniform, half density, vigorous growth and increasing at the moderate speedwere selected as the materials for the study. Compared with the infection situation betweenpear callus and fruit after them been inoculated by Botryosphaeria berengriana f.sp. piricola(BBP) and Monilinia fructigena Honcy (MFH), founding that the callus is same with the fruiton susceptible and resistant to the two kinds of pathogens, namely callus is susceptible to thebrown rot disease and resistant to the Pear ring rot disease.2. MDA content, relative electrical conductivity and cells protective enzymes were determined in callus infected by BBP and MFH. The MDA content and cell membranepermeability were determined to describe the difference of callus infected by BBP and MFH.The results showed that there was similar trend of increasing relative conductivity rate andMDA content of the callus infected by two kinds of fungi, but the change was smaller inBBP-infected callus than that in MFH-infected callus. It showed bigger range of the changeson MFH-infected callus than on BBP-infected callus. In order to understand the role of cellsprotective enzymes, we analyzed the activity of peroxidase (POD), superoxide dismutase(SOD) and catalase (CAT). The SOD、POD and CAT enzyme activity were higher inBBP-infected callus than in MFH-infected callus, and lasted for a long time in BBP-infectedcallus. THE more new isozymes were induced in BBP-infected callus. These results indicatethat there was a close relation between changes of enzyme activity and isozyme and callusresistance, it plays an important roal in defense mechanisms.3. In order to understand the adaptability of pear callus to fungal pathogens, we analyzedthe activity of several defense enzymes, including polyphenol oxidase (PPO), phenylalanineammonia-lyase (PAL), chitinase and β-1,3-glucan enzymes in callus inoculated by both BBPand MFH. This study mainly focuses on analyzing the relationship between the defenseenzyme activity and their roles in disease resistance at the cell level. Within120h afterinoculation, the activities of PPO, PAL, chitinase and β-1,3-glucan enzymes showed similartrend, increasing at first and followed by a decreasing. The peak value appeared in differenttime for each kind of enzyme, but the enzyme activity peaks were always higher inBBP-infected callus than that in MFH-infected callus. So it showed that defensive enzymesshould play an important role in the physiological defense mechanism to the fungi infectionand there is a positive correlation between the changes of the defensive enzymes and theresistance.4. The content of IAA, GA3, ZR and ABA changed differently after pear callus beeninoculated by both BBP and MFH. The tendency of change was the content of IAA and CTKincreased, but the content of GA and ABA declined. The IAA content increased in the BBPearly infection, especially in36h after inoculation, it reached a peak together with the GAcontent. The ZR content is also higher than control at the same time but the ABA contentdeclined. Compared with the control, the content of IAA and ZR increased in the early time ofMFH infection. The content of ZR and ABA changed significantly between two pathogens,but the content of IAA and GA3did not changed. The ZR content was significantly higherthan control but ABA content was lower than control in BBP-infected callus, and this wascontrasted with MFH-infected callus. It was suggested that the changes of plant hormonecontent were related to plant resistance, and there was a significant correlations and coordination between these hormones in plant defense response.5. There was a similar trend on change of soluble protein content after infections of twofungi, and appeared a rising peak in the early infection and the content reduced in the latetime. The soluble protein content was always higher in BBP-infected callus than inMFH-infected callus. SDS-PAGE protein electrophoresis results show that, a new protein wasinduced by BBP infection, and the expression of it increased with infection time gradually. Itinfers that the specific protein may be a PR and it plays an important role in the defensemechanisms.6. In different time after pear callus was inoculated by both BBP and MFH, nucleus weredyed by DAPI and observed under fluorescence microscopy. The results showed that somenucleus of BBP-infected callus was uneven and shrunken in chromatin24hours afterinoculation， which was stick or half-moon in shape， whereas most nucleus wereapproximately round. Most nucleuses of callus cells become abnormal48h after inoculation.In the cells of MFH-infected callus48h after inoculation，intercellular hyphae was found andmore branched at72h with vacuolar neighbor cells without evident chromatin condensation.DNA extracted from bark tissue by1.6%agarose-gel electrophoresis indicated there wereevident DNA ladder at24h after inoculation by BBP，but no the same phenomenece inMFH-infected callus and no systematic HR in it. There was tailed DNA in both callus inInfection later，which was thought as a necrosis by cytology results.7. In order to clone defence-related genes during infection process, in vitro systems basedon pear callus infected by M. fructicola were constructed and gene expression levels werecharacterized by cDNA-SRAP (cDNA sequence-related amplified polymorphism) at differenttime point (control,12,24,36,48and60h). We observed that457fragments were amplifiedfrom30SRAP primer groups, out of which16fragments (3.5%of the total) weredifferentially expressed in polyacrylamide gel electrophoresis (PAGE) detection. Those16fragments were re-amplified for strip recovery by agarose gel electrophoresis. Only five weresuccessfully recovered and subsequently sequenced. Sequence homology analysis of thosefragments by Blastn showed that: one had no homology to any known sequences in the NCBIdata base; two of them are of identical sequence; the three different sequences were of highsimilarity to cinnamyl alcohol dehydrogenase (CAD), DNA binding-protein (DBP) andoligopeptide transporter protein (OPT), with the identity being about96%,85%and78%,respectively. They were hence named as PbDBP, PbOPT and PbCAT. Differential expressionpatterns of PbDBP, PbOPT and PbCAT were observed at different time points (0-60h) usingqRT-PCR. Calli were co-cultured with M. fructicola for12–24h, PbCAD expression levelwas2.94–2.66-fold higher than the control. The higher expression levels of PbOPT gene was observed after co-cultured with M. fructicola for12–36h, they were2.46-,2.17-, and2.34-fold higher than the control, respectively. However, there were no significant higherexpression levels detected for PbDBP at all infected period. These results suggested thatPbCAD and PbOPT could be defence-related genes during the process of brown rot infectionin pear fruit callus.