Isolation,Identification And Diversity Analysis Of Root Symbiotic Fungi Of Different Cultivars Of Blueberry

Blueberry belongs to the family of Ericaceae,genus of Vaccinium.It is the small berry tree with high economic value and broad development prospects.We used the roots of lowbush blueberry ‘Blomidon',half-highbush blueberry ‘Northland' and northern highbush blueberry ‘Sweetheart' collected in the berry orchard of Jilin Agricultural University.The study observed the mycorrhizas types of three different blueberry roots,analyzed the diversity of symbiotic fungi isolated from blueberries,and established the symbiosis system between Vaccinium and fungi.The result lays the foundation for further study on the symbiotic interaction mechanism between Vaccinium and mycorrhizal fungi and provides scientific basis for the research and industrial development of microbial inoculum.The main results are as follows:(1)The roots of three different cultivars were taken as samples and observed by acid fuchsin staining method.It was found that ericoid mycorrhiza(ERM)was the main type of mycorrhizas in ‘Blomidon' roots,and ectotrophic mycorrhiza existed in a few root samples.We observed ericoid mycorrhiza and dark septate endophytes in ‘Northland' roots at the same time and only detected ericoid mycorrhiza in the roots of ‘Sweetheart'.(2)The study applied root to culture fungi,and utilized the eukaryotic universal primer pair ITS1/ITS4 to amplify rDNA ITS sequences of fungus strains,then compared with sequences in GenBank through a BLAST research after sequencing.A total of 49,126,37 colonies were isolated from the roots of ‘Blomidon',‘Northland' and ‘Sweetheart',respectively.We classified the colonies into 40 types according to the morphological characteristics.The BLAST analyses showed that 31 symbiotic fungi were identified at genus level,including 15 genera,4 symbiotic fungi were identified at order level,including 2 orders and 5 uncultured fungi.Therefore,the symbiotic fungi isolated from blueberry roots have rich species,including advantage or typical ericoid mycorrhizal fungi Oidiodendron.(3)We analyzed the ITS sequence on the basis of sequence in GenBank,then used unweighted group average method(UPGMA)to construct phylogenetic tree.The result showed that ITS sizes of 40 symbiotic fungi ranged from 414~538 bp with GC contents of 45.42%~59.14%;ITS1 were 103~246 bp with GC content of 39.83%~64.63%;5.8S were 158~161 bp with GC content of 44.72%~48.13%;and ITS2 were 121~192 bp with GC content of 46.45%~74.42%.The phylogenetic tree showed that 40 symbiotic fungi were divided into four groups.Symbiotic fungi isolated from ‘Blomidon',‘Northland' and ‘Sweetheart' distributed in three groups,which belong to Leotiomycetes,Hypocreales and Pleosporales.Symbiotic fungi NO08 isolated from ‘Northland' clustered into Clade ? alone.(4)Re-inoculation experiments were conducted with advantage or typical fungi BL01,N022 and SW17.The consequence indicated that BL01 and SW17 could form hyphal coils and NO22 could form dark septate hyphal and microsclerotia.Three symbiotic fungi could infect the root of blueberries,and improve the growth of roots.