Isolation Of Endophytic Mycorrhizal Fungi From Blueberry And Its Promoting Effects On Growth

Blueberry(Vaccinium spp.),as a perennial shrub or small shrub species,belongs to Ericaceae family,which has attracted much attention because of its rich anthocyanin and other functional ingredients.the surface of blueberry roots is not covered by root hairs,and its nutrient absorption mainly depends on the symbiotic mycorrhizal fungi.However,the mechanism involved in blueberry root interacting with the symbiotic fungi and applications of symbiotic fungi in field practise still lack systematic research.In this study,we isolated and purified the endomycorrhizal fungi from blueberry,then,we screened and identified the endomycorrhizal fungi which can promote the growth of blueberry.Meanwhile,the mechanism involved in endophytic mycorrhizal fungi promoting the growth and development of blueberries were also analyzed.Furthermore,after re-inoculated with endophytic mycorrhizal fungi,the full c DNA length of PHT1 were isolated and their expression pattern were assayed through RTPCR.Finally,their biofuctions were testified by transgenic Arabidopsis thaliana.We aimed to provide theoretical basis and new ideas for the modern production and cultivation of blueberries.The main results were as following:Five-year-old seedling roots of ’Sharp Blue’,’O’Neill’ and ’Jietu’ Blueberry were used as plant materials.The symbiotic fungi were isolated and purified from root segments and further identified through ITS sequence amplification.Then,the isolated symbiotic strains were re-inoculated to ‘star’ blueberry.A total of 15 strains were isolated and identified from the roots of ’O’Neill’,’Sharp Blue’ and ’Jie Rabbit’.Except for strains of X5 and X10 with unknown classification status,4 strains belonged to Fusarium,6 strains belonged to Penicillium,1 strain belonged to Aspergillus,Talaromyces and Gibberella,respectively.Ninety-three pencentage of the isolated fungi strains were ascomycetes,suggesting that ascomycetes are the dominant resource of mycorrhizal fungi resources for blueberry,which is consistent with the characteristics of rhododendron-like mycorrhizal fungi with ascomycetes as dominant fungi.The reinoculation of strain X2,X6,X11,X24,A2 and A4 fungi significantly promoted the biomass growth of blueberry seedlings.After inoculation with symbiotic fungi,blueberry was subjected to fungal infection observation,measurement of rhizosphere p H and root viabilityt,nutrient content determination,and analysis on expression pattern of PHT1 gene family.The results showed that all six fungi could form endophytic mycorrhizal structures after inoculation to the blueberry root system,and improved the root vitality of blueberries to different degrees.Among them,X6,X11,and X24 had the most significant promoting effects,respectively with 94.52%,127.61% and 125.87% increments.Inoculated with mycorrhizal fungi also increased the nitrogen and phosphorus content in blueberry seedlings by11-49%,indicating that nitrogen and phosphorus absorption efficiency having been improved through enlargement of root absorption area.The RTPCR results showed that the transcripts of PHT1-1～4 were up-regulated or downregulated in blueberry leaves and roots,indicating that the expression patterns of these genes are tissue-specific.The expression of PHT1-1 in the blueberry root group was up-regulated,and the P content in the blueberry root was increased,This indicated that PHT1-1 may be expressed in the root in large quantities to promote the absorption of phosphorus by the root.Similar result was obstained for PHT1-2,with increased expression level in leaves and higher P content simultaneously,indicating that the large expression of PHT1-2 gene in leaves promoted the upward transport of P into the leaves.The full-length of c DNA sequences of PHT1-1,PHT1-2,PHT1-3 and PHT1-4were successfully cloned from blueberry.The sequences were 1611,1611,1617,and1005 bp in length,encoding 537,537,The amino acids of 539 and 335 have isoelectric points of 8.87,8.87,6.57,and 8.35,respectively.After constructing and analysing the evolutionary tree and comparing the amino acid sequences,it was found that PHT1-1and PHT1-3 are closely related to the Rhododendron sylvestris,and PHT1-2 and PHT1-4 are closely related to the species such as Chinese kiwi and tea tree.The results of TMHMM analysis showed that there may be 12,12,12,and 6 transmembrane domains respectively in PHT1-1,PHT1-2,PHT1-3,and PHT1-4,which suggested that all of them are membrane proteins.Through genetic transformation experiments of Arabidopsis thaliana,it was further verified that PHT1-1 and PHT1-3 have the function of absorbing and transporting phosphorus elements.Under low-phosphorus conditions,wild-type Arabidopsis plants have short plant development,small leaf discs,small leaf numbers,and low vitality.Overexpression of PHT1-1 and PHT1-3 greatly alleviated this phenomenon,indicating that these two genes may have functions of P absorption and transport to alleviate the symptoms induced by P deficiency in plants.However,the relative mechanisms still need further researches.