Plant microbial ecology and synthetic communities of duckweed-associated bacteria
Creators: name: Acosta, Kenneth
Publisher: name: No Publisher Supplied
Year: 2024

description: Bacteria are abundant plant colonists with the potential to promote plant growth. However, efforts to utilize these bacteria for plant growth promotion in agriculture have failed due to their displacement by resident soil and plant microbiota. Ultimately, harnessing the beneficial effects of plant-growth-promoting bacteria (PGPB) requires an understanding of community-level processes and underlying ecological interactions. To understand this community ecology, community profiling and synthetic community approaches are applied to terrestrial plant microbiomes. However, these approaches are complicated in terrestrial plants, which undergo sophisticated development and require a soil environment that is chemically and physically heterogeneous. Alternatively, duckweeds are a family of floating aquatic monocots with a reduced morphology, uniform clonal development, and a relatively homogenous freshwater environment. In this dissertation research, I hypothesized that synthetic communities of duckweed-associated bacteria (DAB) could elucidate mechanisms underlying community-level processes and functions present in the broader context of the plant microbiome. I addressed this by first asking whether bacterial communities associated with duckweed are similar to those found in terrestrial plants. Secondly, I investigated whether synthetic communities of DABs could elucidate molecular mechanisms underlying community assembly.In Chapter 2, I took a culture-independent approach and generated community profiles of DAB communities to determine their structure and whether there were structural similarities between bacterial communities of duckweed and terrestrial plants. By sampling wild DAB communities, I show that DAB communities are predominantly composed of particular families of Proteobacteria and Bacteroidetes and demonstrate a taxonomic overlap between DAB communities and terrestrial phyllosphere communities. By inoculating axenic duckweed with a wastewater microcosm, I demonstrate that duckweed influences the bacterial community in the surrounding environment, similar to the “rhizosphere effect” observed in terrestrial plants.In Chapter 3, I focused on the development of essential molecular techniques for culture-dependent investigations of DAB communities. Among the molecular tools developed was a versatile DNA fingerprinting assay. This assay serves multiple purposes, including the validation of the axenic status of duckweed, the detection of various bacterial genera associated with duckweed, and the assessment of bacterial contamination in duckweed samples. Additionally, I developed a computational pipeline designed for creating strain-specific PCR primers to detect specific bacteria within a community context and enable the quantification of their colonization levels. These molecular tools were validated by performing confocal microscopy on duckweed inoculated with PGPB known to display epiphytic and endophytic colonization patterns with terrestrial plants. The microscopy results not only corroborated the PCR findings but also illustrated colonization patterns of PGPB on duckweed that mirrored those observed on terrestrial plants.In Chapter 4, I established an initial culture collection of DABs and applied the insights gained in Chapter 1 regarding DAB community structure to construct a synthetic community (SynCom) of 30 members. I introduce an alternative approach using freezer stocks to inoculate complex synthetic communities and validate a 16S rDNA amplicon sequencing workflow to characterize SynCom colonization dynamics. I then leveraged genomic approaches available with SynComs to investigate competitive interactions observed in the DAB SynCom by integrating correlation networks, metabolic networks, and the strain-specific PCR approach introduced in Chapter 3. Together, these approaches pointed towards gluconolactone, a recently identified plant prebiotic, as a possible mediator of resource competition in DAB communities. In conclusion, my dissertation research uncovered numerous similarities in bacterial community structure and assembly between duckweed and terrestrial plants, supporting the proposition of utilizing duckweed to understand plant microbiome processes. Addressing the challenges associated with SynComs and expanding the DAB culture collection holds the potential to bridge more molecular features with community-level processes, thereby advancing our understanding of plant microbial ecology and its utilization to meet future agricultural needs.