Isolation And Analysis Of Colonization Of Mycorrzozal Fungi Of Wild Blueberries From Greater Khingan Mountains

Blueberry is mutualistic with mycorrhizal fungi, and this kind of mycorrhizal belongs to Ericoid mycorrhizal (ERM). The ERM can obviously improve nutrient absorption, disease resistance, and adversity of resistance of blueberry. ERM has a significant influence on the growth of blueberries. In this study, we isolated and identified of mycorrhizal fungi from wild blueberries in Greater Khingan Mountains. The colonization of mycorrhizal fungi in blueberries root is also analyzed by in situ PCR. Understanding the population diversity and the characteristic of colonization for mycorrhizal fungi, it is a foundation for the development of fungal fertilizer and the cultivation for wild blueberries in Greater Khingan Mountains, and it also has an important significance to the application of mycorrhizal technology in agricultural production. The results are showed as follows:(1) One hundred and thirty-nine strains of endophytic fungi from wild blueberries roots in Greater Khingan Mountains are isolated using the MEA and MMN mediums. By the morphology and molecular biology identification the fungi are classified as follows: Oidiodendron, Crypyosporiopsis, Phialocephale, Lachnum, Yarrowia lipolytica, Chaetomium globosum, Sordariomycetes sp., Mucoromycotina sp..(2) Using pCAMBIA1300 as backbone, the enhanced GFP expression cassette from the plasmid pCT74 was cloned into the pCAMBIA1300 multiple cloning site (MCS). The vector pXBTCEH-GFP is built after the hygromycin resistance gene (HYG) expression cassette from the plasmid pCT74 was cloned into the pCAMBIA1300 to replace invalid HYG expression cassette.(3) GFP gene is expressed in Crypyosporiopsis ericae and Sordariomycetes sp. by agrobacterium tumefaciens-mediated transformation. The hyphae displaying fluorescence observed by fluorescent microscope indicates that this hyphae remained stable after five times subculture and the GFP fragments was acquired from the genome of transformant by PCR.(4) Using tissue culturing seedlings of blueberries with mycorrhizal fungi as experimental material, transverse and longitudinal frozen sections were prepared. The DNA probe of mycorrhizal fungi 18S rDNA was prepared by digoxin kit with with ITS1/ITS4 primers, the fungi colonization in the blueberries root was analysis by in situ PCR. The results showed that five types of mycorrhizal fungi are effective to colonization in root and most of mycorrhizal fungi distributed in epidermis and the cell gap of cortex parenchyma near the epidermis, and it had many invade sites in the epidermis, the number of mycorrhizal fungi in epidermis was more than one in cortex.