| Endophytes are microorganisms living inside of a plant without causing pathogenesis or inducing symbiotic structures. The focus of most endophyte research has been on crops, with an emphasis on diazotrophic endophytes. Populus is an economically-important, early-successional genus of trees. A better understanding of the endophytes of Populus will help increase our knowledge of the roles of the microbial community in tree plantations and in their native environment. Research in the Doty lab of the University of Washington has led to the discovery of dozens of endophytic microorganisms isolated from both wild and hybrid Populus species. Many of these isolates seem to be capable of fixing nitrogen. Three of the isolates are endophytic yeasts.; A WPB strain isolated from wild cottonwood (Populus trichocarpa ) was characterized. Sequences of the recA gene revealed that WPB was most closely related to species Burkholderia vietnamiensis. Phylogenetic analysis of the nifHDK cluster shows that WPB shares high sequence similarity with a Burkholderia vietnamiensis G4 strain. Nitrogen fixation activity of WPB was further characterized using 15N2 incorporation and acetylene reduction assays. The WPB strain can also produce indole-3-acetic acid (IAA), a phytohormone, when incubated with L-tryptophan. A grass growth promotion assay showed that inoculation of WPB can increase Kentucky bluegrass biomass by 42% over WPB-uninoculated controls in nitrogen free medium in 50 days.; Via examining three genes, the 18S rDNA, internal transcribed spacer region, and 26S rDNAD1/D2 region, the two yeast isolates from hybrid poplar, PTD2 and PTD3, were identified to be species Rhodotorula mucilaginosa . Phenotypic characteristics of the yeast isolates were also obtained using a yeast identification kit and used for assisting species identification. WP1 was identified to be most closely related to species Rhodotorula graminis by analyzing phylogenetic and phenotypic characteristics. Additionally, the three yeast isolates can produce IAA when incubated with L-tryptophan.; Two yeast isolates, WP1 and PTD2, were observed to be able to grow in nitrogen free medium. In a series of experiments, including the total nitrogen mass balance assay, 15N2 incorporation assay, nifH gene isolation and acetylene reduction assay, important evidence indicated that the yeast strains may be diazotrophic yeast. |
