The Research On Cytology Of The Infection Of Oil Tea By Colletotrichum Gloeosporioedes And Physiological Response

Camellia oleifera is a particular woody edible oil tree in China,and it has high economic value.In recent years,Camellia oleifera anthracnose has occurred widespread in major Chinese producing areas such as Hunan,Jiangxi and Guangxi.It seriously damaged the quality and total yield of Camellia oleifera.So far,the pathogenic mechanism of Colletotrichum gloeosporioedes and Camellia oleifera's resistance mechanism were not very clear.The first task of controlling Camellia oleifera anthracnose was cognizing pathogenesis of disease,which including pathogenicity of pathogenic fungi to plant and resistance of plant to pathogenic fungi.What counts was blocking the invasion of pathogens.Therefore,understanding the cellular and biochemical basic biological processes of interaction between oil tree and Colletotrichum gloeosporioedes were of significance to effectively prevent Camellia oleifera anthracnose.In this study,we studied the interaction between plant and pathogen from the aspects of cytology,enzyme and biochemistry.We observed the the way and process of infecting host by Colletotrichum gloeosporioedes,detected the kinds and activity of extracellular enzymes produced by pathogens and explored physiological and biochemical changes of host resist to pathogens.The main results were as followed:(1)Study on cytology of inf-ecting host by Colletotrichum gloeosporioedes.We studied the process of infecting host by Colletotrichum gloeosporioedes using microscopic inspection,whole-stain clearing,and frozen tissue section.According to the morphological characteristic of Colletotrichum gloeo.sporioedes,its infection process could be divided into 3 main stages,namely,preinfection period,infection period and post invasive extension.The early stage of infection is the germination of spore on the surface of leaves.2 hours after inoculation,the spore began to germinate,coming into one or two thin and short germ tubes.Between four hours and six hours after inoculation,large of spores germinated and its germ tubes's length were different.At ninth hour,the germination rate reached as high as 90%.The spore germinated and formed germ tubes and the tips of germ tubes gradually began to form appressorium at 9th hour after inoculation.At 12th hour,appressorium reached maturity and was dark brown with two oil globules in the middle.During the invasion stage,appressorium attached to the host epidermis,seeking the appropriate site of invasion.Most appressoria invaded into host cell directly by producing penetration,but few appressoria invaded into host cell through stoma.In the extension period,pathogens entered into epidermal cell after puncturing cuticula,and gradually spread to palisade tissue.Then,when hypha entered into spongy tissue cell,they would gradually kill surrounding cells.At last,leaves showed symptom.(2)Study on the role of key enzymes in the pathogenic processes of Camellia oleifera anthracnose caused by Colletotrichum gloeosporioides.By culturing in vitro,different carbon source(including sucrose,pectin,bran,CMCNa,Camellia oleifera leaves)all could induce Colletotrichum gloeosporioides to produce cellulase,pectinase and laccase.in vitro with carboxymethyl cellulose sodium(CMCNa)for induction of the substrate,the activities of carboxymethyl cellulase and laccase were the highest,were respectively 73.20 U/mg and 4.50 U/mg.Whereas citrus pectin was the best for pectinase.Colletotrichum gloeosporioides could produce high activity of cellulase,pectinasen and laccase induced by Camellia oleifera leaves.The effects of 5 kinds of enzyme degrading on leaves were obvious different.The enzyme produced by CMCNa had a high degree of degradation on leaves,leding whole leaf to black.The enzyme produced by bran came next.The enzymes produced by sucrose,pectin,Camellia oleifera leaves had a light degree of degradation on leaves,and the local of leaf browning with water stain symptom.Under the treatment by spore suspension,the activity of the CWDEs around the boundary between diseased and healthy areas was the highest.The activities of the CWDEs rapidly increased 4 days after inoculation when the leaves emerged symptom.Filter paper enzyme(FPA),?-glucosidase and laccase showed maximum activities in the sixth day after inoculation,were respectively 4.53 U/mg,7.44 U/mg,1.21 U/mg.But carboxymethyl cellulase(Cx)and pectinase reached at the peak in the eighth day after inoculation.The activities of the CWDEs were very stably from 10th and16th day after inoculation.(3)Early defense,physiological and biochemical responses of Camellia oleifera to Colletotrichum gloeosporioides.By inoculating spores,We detected the burst of reactive oxygen species and determined the PAL,POD,PPO activities and the content of phenol and total soluble sugar at different times.The results of DAB color indicated that the cell around pathogens invading sites produced large reactive oxygen species,collecting brown precipitate.Besides,the longer the infection time was,the less the brown precipitate was on the surface of cell.After inoculated by Colletotrichum gloeosporioides,the activities of PAL,POD and PPO increased and the content of phenol and total soluble sugar raised.In the early inoculation,activity of PAL and POD increased slowly.Afterwards theyincreased quickly and reached their peak at 5th day.Subsequently,their activities decreased.Until 9th day,the enzymes activity were still higher than control group.While PPO activity was greatest at 3th day,then gradually decreased.The content of phenol in early inoculation was higher than the later stage,but the content of total soluble sugar had been rising.