UC Merced

The aims of my dissertation research were to (1) refine culture techniques to maximize recovery of bacterial endophytes from conifer tissues and (2) identify these bacterial isolates through the sequencing of 16S rRNA and genome sequencing. Bacterial endophytes are found in many plant hosts and in every plant tissue studied thus far. Generally thought to be neutral or to have a beneficial impact on their hosts, these bacteria have been widely studied in agricultural plants. Our understanding of how bacterial endophytes associate with and impact plants in natural ecosystems lags behind and therefore studying endophytes from conifer hosts is an important approach. I improved current culture techniques to capture bacterial endophyte isolates from Pinus contorta, Pinus ponderosa and Pinus nigra bud/shoot tissue, and Pinus radiata needle and bud/shoot tissue. I identified the 16S rRNA sequences of these isolates through Sanger sequencing and picked four isolates for genome sequencing and further study. I found that liquid nitrogen tissue preparation (with or without cryopreservation) was the most effect tissue preparation method for maximum isolate recovery and that that Tris-buffered, R2A agar with multiple carbon sources was the most effective culture media for maximum isolate growth. Alphaproteobacteria dominated isolates from Pinus contorta. Actinobacteria dominated the isolates from Pinus nigra. Firmicutes dominated isolates from Pinus ponderosa. The isolates from Pinus radiata were dominated by Gammaproteobacteria, as was the culture-independent study completed to identify the community of bacterial endophytes present. Four isolates from Pinus radiata were chosen for genome sequencing with Illumina MiSeq. Genome BLAST searches against a database of NCBI completed bacterial genomes revealed that one isolate is likely an Erwinia spp. and that another isolate is likely a Sodalis spp. or represents a new genus in the family Enterobacteriaceae. A third isolate likely represents a new family in the order Rhizobiales, and the fourth isolate was identified to the family level as Enterobacteriales and is likely a new genus in the Enterobacteriaceae. Some known beneficial genes were identified however; more sequencing is needed to finalize identification of these isolates and to clarify their relationships with their host.