Preliminary Study On The Isolation Of Ericoid Mycorrhizal Fungi And Dark Septate Endophytes And Their Interaction

Ericoid mycorrhizal fungi(EMF)and dark septate endophyte(DSE)can co-colonize in the hairy roots of Rhododendron plants and promote nutrient uptake,but the interaction between them is unclear.Exploring the interaction between the two fungi is of great significance for the growth and development of Rhododendron plants.In this study,Vaccinium uliginosum and Vaccinium vitis-idaea were used as experimental materials.Ericoid mycorrhizal fungi and dark septate endophytic fungi were isolated from the hairy roots of Vaccinium uliginosum and Vaccinium vitis-idaea.The in vitro culture interaction ability of the two fungi was analyzed.The effects of single inoculation and mixed inoculation of the two fungi in soil matrix medium on plants and the dynamic changes of the two fungi in plant roots were further explored.The main results were as follows:(1)A total of 68 strains of fungi were isolated from the hairy roots of Vaccinium uliginosum.After morphological and molecular biological identification,23 species of fungi were obtained,including one species of ericoid mycorrhizal fungi,Cryptosporiopsis ericae,and two species of dark septate endophytic fungi,Phialocephala fortinii and Cadophora sp.,respectively.A total of 74 strains of fungi were isolated from the hairy roots of Vaccinium vitisidaea,and 9 species of fungi were obtained by morphological and molecular biological identification.Among them,one species of ericoid mycorrhizal fungi was Oidiodendron maius,and two species of dark septate endophyte fungi were Phialocephala fortinii and Leptodontidium sp.(2)The isolated ericoid mycorrhizal fungi Oidiodendron maius strain H14 and dark septate endophyte Phialocephala fortinii strain H5 were used for enzyme production in shake flasks.The pectinase activities of the two fungi were high,and the polygalacturonase activity of strain H5 reached 170 U/m L.The activities of polymethylgalacturonase produced by the two fungi were similar,up to 180 U/m L.The laccase,polyphenol oxidase and lignin peroxidase activities of strain H14 were higher than those of strain H5.Two strains can also produce chitinase and neutral protease,and the enzyme activity is similar.The inactivated mycelia of Oidiodendron maius strain H14 could promote the production of laccase,polyphenol oxidase,chitinase and lignin peroxidase from Phialocephala fortinii strain H5.The inactivated mycelium of strain H5 improved the laccase production ability of strain H14,and the laccase activity increased by 3 times compared with the control group.Inactivated mycelia of strain H5 also promoted the production of polyphenol oxidase by strain H14.In addition,the presence of inactivated mycelia inhibited the activity of fungal neutral protease.The soluble metabolites of Oidiodendron maius strain H14 significantly inhibited the growth of Phialocephala fortinii strain H5,while the soluble metabolites of Phialocephala fortinii strain H5 promoted the growth of Oidiodendron maius strain H14.Under co-culture conditions,Oidiodendron maius strain H14 was the dominant strain.(3)The Oidiodendron maius strain H14 and the dark septate endophyte Phialocephala fortinii strain H5 were inoculated in the sterile tissue culture seedlings of blueberry,respectively,to analyze and compare the colonization of fungi and their effects on physiological and biochemical indexes of plants.The result showed that single inoculation and double inoculation of fungi could increase the activities of catalase and superoxide dismutase in blueberry plants,and increase the contents of soluble sugar,chlorophyll and free proline in leaves of plants.There was no significant difference in the effect between single inoculation and double inoculation on plants.Primers were designed according to the IGS specific fragment of the two fungi,and the fluorescence quantitative PCR detection method was constructed.The standard curve was established to quantitatively detect the fungi in different treatment plants,and the fungal DNA copy number was calculated.By calculating the sum of relative DNA copies,the results showed that double inoculations increased the colonization of fungi,indicating that the two fungi had synergistic effect in plants.According to the calculation of competitive intensity index,Phialocephala fortinii strain H5 was the dominant strain under co-colonization conditions.The results showed that Oidiodendron maius strain H14 and Phialocephala fortinii strain H5 were competitive under in vitro co-culture conditions,and Oidiodendron maius strain H14 was the dominant strain,which had a certain inhibitory effect on the growth of Phialocephala fortinii strain H5.Under the condition of co-colonization in plants,Oidiodendron maius strain H14 and Phialocephala fortinii strain H5 showed a synergistic effect.The colonization of strain H5 was significantly increased compared with that of single inoculation,while the variation of DNA copy number of strain H14 was small,and the competition intensity was lower than that of strain H5.