Study On Green Fluorescent Protein Gene Marker And Infection Of Tea Tree Anthracnose Pathogen

Tea Camellia sinensis(L.)O.Ktze.,is an evergreen plant.Tea originated in China and spread to the whole world,becoming one of the world's favorite health drinks.Tea anthracnose is one of the leaf diseases on tea trees.Colletotrichum Corda is globally distributed phytopathogenic fungus with bristles on the conidia.When the fungus seriously occurs on the tea tree,it will lead to a decline in tea quality and quality,causing some economic damage,so anthrax occupies a more important position in plant pathogens.The mechanism of fungal invasion into the host is mainly divided into direct invasion,natural orifices(stomata,drainage holes,skin pores,stigmas,nectaries,etc.),wound invasion,and some pathogens first invade dead tissue and dying tissue,live,reproduce for a period of time and reinfect healthy leaves.The studies on GFP-labeled pathogens infecting tea leaves have rarely reported.In this paper,the main research results are as follows:1.The leaf diseases of anthracnose symptoms on tea trees were collected from a tea garden.After tissue separation,purification culture,the pathogenicity test of the isolated strain shows that it is pathogenic,the strain was identified as Colletotrichum gloeosporioides by morphological identification combined with a multi-gene phylogenetic tree(ITS?ACT?CAL?CHS-1?TUB2?GAPDH).GZCC20-0218 was conducted pathogenicity determination according to Koch's law,indicating that the Colletotrichum gloeosporioides was pathogenic.2.The strain of Colletotrichum gloeosporioides were tested for HygB resistance.The hygromycin concentration was 450?g/ml,which could be used as the initial screening concentration for GFP-labeled transformant strains.In this study,under the conditions of 0.2 M acetosyringone,the OD600 of the Agrobacterium liquid reached the range of 0.5 to 0.6,and the concentration of the conidia suspension was 10~6spores/ml,etc.The GFP genes in Agrobacterium tumefaciens were transformed into strain of Colletotrichum gloeosporioides with AS induced culture.The transformants were tested for fluorescence under a fluorescence microscope.The products obtained by PCR amplification of hygromycin-specific primers were subjected to electrophoretic detection.The fluorescent protein was successfully expressed in the test strain.After the transformants were continuously cultured for 6?7 generations,the GFP genetic stability of the transformants was determined,indicating to obtain GFP marker strains with a stable inherited character.3.The biological characteristics of the GFP-tagged strain and the wild-type test strain were compared with the morphological characteristics and the help of SAS analysis software,the P=0.05 level,indicating that there was no significant difference in the average growth rate.At the same time,GFP-labeled strains are still pathogenic.4.The live tea seedlings were infected by using a stable inherited GFP-tagged strain.The incidence rate reached to 100%after 7 days.Tissue section observation of the diseased leaves revealed that the conidia of Colletotrichum gloeosporioides could colonize the leaf stomata and tissues.