Dynamic Changes Of Reactive Oxygen Species And Differential Protein Expression In The Spores Of Botrytis Cinerea Respond To Oligochitosan Treatments

Fruits decomposing caused by invasion of Botrytis cinerea is one of the mostimportant reasons for postharvest losses. At present, chemical fungicides are usedmost controlling plant diseases. In recent years, the abuse of chemical pesticidesleading to pesticide residue and environment pollution have extensively drawn thepublic attention. Therefore, research and development of alternative biopesticides isbecoming a research focus after the agricultural production process. Chitosan is aalkaline polysaccharide containing reactive amino-groups and hydroxyl-groups,which has many biological functions. Many researches have found that chitosan andits degradation products oligochitosan can effectively inhibit the occurrence anddevelopment of gray mould. It has many great advantages such as ecological friend,easily degradation and biocompatibility, which is superior than those traditionalpesticides. So it has broad application prospects. So far, there are many thoroughlyresearches on the inhibiting laws of chitosan and oligochitosan, but few on theantibacterial mechanism limiting its popularization and application in agriculture. Thepresent paper utilizing the fluorescent marking and proteomics technology carries outresearch about the antifungal mechanism of oligochitosan from both cytology andbiochemistry perspectives. The results are as follows.Using fluorescent marking technology, we separately observed ROS signalsinside the spores of gray mould inducing by oligochitosan on0,15,30,45,60,90,120,150,180minute reaction time point, which included different concentrations ofoligochitosan, external source of hydrogen peroxide inducing and applying reducingagent catalase. In the different concentrations experiment, we chose0.5%and0.05%as high and low concentrations, finding that there were both weak ROS signals in allof the groups. But the differences were that ROS signals were lost after15minutes in the control group, while the signals were quickly decrease and then gradually increasewith time in the experiment group. The intension of signal was better in lowconcentration than the high. After120minutes, both signals were nearly same. In thehydrogen peroxide experiment, we first disposed gray mould spores with hydrogenperoxide in50mmol/L for one hour. The result was that ROS signals in theexperiment group were obviously lower than the control, but the gap reduced withtime. After120minutes, both signals were nearly same. In the catalase experiment,the chosen concentration of catalase was3mg/ml. We found that ROS signals in theexperiment group were obviously lower than the control, the most remarkable gapappeared between60to90minutes, then the gap became narrowing until180minuteswhen both having the same ROS signals. According to previous result, we used laserscanning confocal microscope to observe the distribution of ROS inside spores afterinducing by oligochitosan and found that ROS signals was weak and uniform in thecontrol group, but the signals mainly distributed around the spores in the experimentgroup. The above results demonstrate oligochitosan remarkably inducing ROS signalsin gray mould spores, and the level of ROS having positive correlation with reactiontime. The effect of oligochitosan in low concentration is better than the highconcentration. For certain time ranges, induction of hydrogen peroxide in lowconcentration can increase the endurance capacity of gray mould spores to ROS.Catalase can apparently decrease ROS level in spores. After experiment witholigochitosan, there are not only more ROS produce, but also uneven ditricutionhappens. The experimental spores have different inner structure.In the proteomics experiment, we induced spores using oligochitosan in0.05%for8hours. Proteins in the spores were collected using the tris-phenol extraction way.After two-dimensional electrophoresis and gel comparison, we found61differentexpression protein spots. After identifying28spots among them utilizing massspectrum, analyzing and searching data base, we successfully identified23differentexpression protein spots, which belong to18proteins and four spots expressed higherand14lower after inducing. Their functions were separately about materialmetabolism, protein synthesis, protein folding, proteolysis and signal transduction besides those unclear functions. After inducing with oligochitosan, there were moredown-regulation proteins than up-regulation ones. The above results demonstrate thatoligochitosan can limit the production of protein in the spores, further disturb thefolding of them, which leads to the accumulation of misfolding proteins.Consequently, oligochitosan disorders the normal metabolism of spores, moreoverinducing the occurrence of apoptosis.