VALIDATING APPROACHES FOR STUDYING MICROBIAL DIVERSITY TO CHARACTERIZE COMMUNITIES FROM ROOTS OF Populus deltoides

Microbial (archaeal, bacterial, and fungal) communities associated with plant roots are central to its health, survival, and growth. However, a robust understanding of root microbiota and the factors that govern their community structure and dynamics have remained elusive, especially in mature perennial plants from natural settings. Although the advent of Next Generation Sequencing (NGS) technologies have changed the scale of microbial ecological studies by enabling exhaustive characterization of microbial communities, the accuracy of taxonomic and quantitative inferences are affected by multiple experimental and computational steps and lack of knowledge of the true ecological diversity. To test for inaccuracies and biases, I assembled diverse bacterial and archaeal ‘synthetic communities’ from genomic DNAs of sequenced organisms. I tested and compared different approaches that included metagenomic and small subunit rRNA (SSU rRNA) amplicon sequencing. The outcome was dependent on primer pairs, analysis parameters, and sequencing platforms. Nevertheless, new approaches in processing and classifying amplicons were able to recapitulate microbial diversity with high reproducibility within primer sets, even though all tested primers sets showed taxon-specific biases. Consequently, inferences from ‘synthetic communities’ study were implemented in experimental design and analysis of microbial communities from roots of naturally occurring mature riparian plants of Populus deltoides. Thaumarchaeota, Proteobacteria and Ascomycota dominated the overall archaeal, bacterial, and fungal communities respectively. Further, I investigated relationships of bacterial and fungal communities in rhizosphere and endosphere with soil and environmental properties, host genotype, season, and geographic setting. The variation of bacterial and fungal communities between each sampled roots were explained on the basis of seasonal, soil properties, and geographical settings (4% to 23%), however, most variations remain unexplained. I also tested if rhizosphere of P. deltoides and mature trees in general select for higher diversity of archaea than surrounding soil. I discovered a slightly higher diversity of archaea in the trees compared to corresponding bulk soil, but the results were not specific to P. deltoides. In summary, this dissertation validates current microbial diversity approaches, characterizes microbial communities of an important plant, and decipher drivers that are controlling root associated community structure.