Multiple forces shape the phyllosphere microbiome: The importance of vertical transmission, environmental selection, and bacteriophages

Author(s): Morella, Norma Marie | Advisor(s): Koskella, Britt; Lindow, Steven | Abstract: As our understanding of host-associated microbial communities (microbiomes) deepens, there is a simultaneous revelation of key gaps in our understanding of these systems. Among these is knowledge of the forces underlying the assembly of, selection within, and dynamics among microbiota. These questions relate to broad principles that are shared across host species, and synthesis across these systems will identify conserved principles in the larger field of microbiome research. The work presented here seeks to identify and explore the relative importance of multiple forces simultaneously shaping the microbiome, specifically, that of the phyllosphere (above-ground surfaces of plants). This research begins by investigating the importance of vertically transmitted (parent to offspring) microbes in seedling health. Currently, there is little understanding of the ecological importance of commensal or mutualistic bacteria that are transmitted on or within the seeds. Using a tomato study system and combination of classic microbiological techniques and next generation sequencing, we found that the vertically transmitted seed microbiome is capable of protecting seedlings against a common plant pathogen. This work provides evidence that the seed microbiome plays an important ecological role in early seedling life, and very likely shapes the development of the microbiome both directly through priority effects and indirectly through interactions with the plant host. This work then explores the importance of host genotype and environmental selection in shaping the phyllosphere microbiome in tomato plants. A successive passaging experiment was used to address this question by selecting upon the phyllosphere microbiome. Beginning with a diverse microbial community generated from field-grown tomato plants, replicate plants across five plant genotypes were inoculated for four eight-week long passages, and the microbial community was sequenced at each passage. We observed consistent shifts in both the bacterial (16S amplicon sequencing) and fungal (ITS amplicon sequencing) communities across replicate lines over time, as well as a general loss of diversity over the course of the experiment suggesting that much of the naturally observed microbial community in the phyllosphere in an open environment in outdoor setting is likely transient or poorly adapted. We found that both host genotype and environment shape microbial composition, but the relative importance of genotype declines through time. Furthermore, using a community coalescence experiment, we found that the bacterial community from the end of the experiment was robust to invasion by the starting community. These results highlight that selecting for a stable microbiome that is well adapted to a particular host environment is indeed possible, emphasizing the great potential of this approach in agriculture and other systems. In the final chapters of this work, the importance of bacteriophages (viruses that infect bacteria) in the phyllosphere is examined. This question was addressed by transferring microbial communities from field-grown tomato plants to juvenile plants grown under mostly sterile conditions in either the presence or absence of their associated bacteriophage (phage) community. In three separate experiments, we found that the presence of phages affects overall bacterial abundance during colonization of new host plants. Furthermore, bacterial community analysis (16S amplicon sequencing) shows that phages significantly alter the relative abundance of dominant community members and can influence both within- and among-host diversity. This is then extended through the measuring of the impact of phages on bacterial communities weeks after initial inoculation. This work describes the impact of disrupting bacteria-phage temporal dynamics on bacterial communities. Together, these results underscore the importance of both lytic and lysogenic phages in host-associated microbiomes but how they can have fluctuating impacts over relatively short timescales. Together, these results contribute to fundamental knowledge gaps by demonstrating the ecological importance of vertically transmitted microbes, determining that microbiomes can be adapted to their host and environment, and uncovering the temporal variability of key driving forces underlying microbiome structure.