The Isolation, Cuture And Molecular Identification Of Fraknkia And Non-Frankia Sctionayetec Forom Hipooha Rehanmde Root Nodules

Hippophae rhamnoides L. Elaeagnaceae is a pioneer plant in the northern semi-arid region of China. The root of Hippophae rhamnoides can fix nitrogen from atmosphere for plant growth in symbiosis with Frankia. This species has a wide adaptability and can easily grow in barren lands with harsh environments. Because of its ability in increasing water and soil conservation, adjusting the climate of micro-enviroment and effectively controlling water flow and soil erosion, Hippophae rhamnoides is considered as an excellent eco-econimic tree species. In addition, Hippophae rhamnoides fruits, leaves and seeds contain large amounts of nutrients and biological activity materials which have important significance for the human body.In order to exploit and develop efficiently symbiotic nitrogen fixation characteristics of Hippophae rhamnoides, a preliminary study was carried out on isolation and culture conditions and the phylogenetic position of Frankia in H. rhamnoides root, several main results were summarized as follows.(1) The phylogeny of endophytes from nodules of Hippophae rhamnoides. Nodules from different ecological environment and different varieties of Hippophae rhamnoides were collected. Using a direct PCR amplification protocol on nodules, 39 complete 16SrDNA sequences of endophytes were obtained, which inclueded 20 haplotypes, 19 complete 16SrDNA and 4 haplotypes were found in Hippophae rhamnoides nodules from Dengkou, whlie 20 complete 16SrDNA and 16 haplotypes were found from Sunwu. Endophytes from nodules of the two regions share no haplotype, suggesting zero gene flow between endophytes from the two different regions. Based on all 16SrDNA sequences, downloaded from NCBI and obtained in corrent study, Frankia strains from different regions and different host sources were subdivided into four major clades: Unisolated strains, Elaegnaceae infective strains, Alnus-Myrica-Casuarina infective strains,Atypical strains. All endophytes from Hippophae rhamnoides nodules were clusered into the Elaegnaceae infective group with a bootsrap support value 52%, all belonged to Frankia. (2) Isolation of endophytes from root nodules of Hippophae rhamnoides. With fresh young nodules, adopting nodule microtomy and homogenization, using BAP and QMOD medium, 18 pure cultures were obtained from surface sterilized root nodules of Hippophae rhamnoides, 14 strains were isolated by microtomy, while 4 isolates obtained by homogenization. It wsa found that, microtomy method and BAP medium showed more suitable for separation of isolates, liquid culturate can effectively separated stains, too,and strains colony appeared earlier than solid separation 7 to 10 days.(3) Morphology and molecular identification of pure culture strains from root nodules of Hippophae rhamnoides. Most isolates have developed aerial hyphae, under a microscope all strains were branched, but none had typical Frankia structures like diazovesicle or multiocular sporangia.Therefore, these stains were non-Frankia actinomycetes. According to morphological and culturing characteristics, such as development of substrate mycelium and aerial mycelium, degree of combination between mycelium and medium, velumor produced in liquid culture or not, hyphae septate or not and the differentiation of spores, the 18 isolates obtained from Hippophae rhamnoides nodules were assigned to Nocardia and Micromonospora genera. Phylogenetic tree based on all 16SrDNA gene sequences, downloaded from NCBI and obtained in corrent study , also divided these 18 isolates into two groups:Nocardia group and Micromonospora group, comfirmed the results based on morphology.