The phyllosphere is densely colonised by microbial communities, despite sparse and heterogeneously distributed resources. The limitation of resources is expected to drive bacterial competition resulting in exclusion or coexistence based on fitness differences and resource overlap between individual colonisers. We studied the impact of resource competition by determining the effects of different bacterial colonisers on the growth of the model epiphyte Pantoea eucalypti 299R (Pe299R). Resource overlap was predicted based on genome-scale metabolic modelling. By combining results of metabolic modelling and pairwise competitions in the Arabidopsis thaliana phyllosphere and in vitro, we found that ten resources sufficed to explain fitness of Pe299R. An effect of both resource overlap and phylogenetic relationships was found on competition outcomes in vitro as well as in the phyllosphere. However, effects of resource competition were much weaker in the phyllosphere when compared to in vitro experiments. When investigating growth dynamics and reproductive success at the single-cell resolution, resource overlap and phylogenetic relationships are only weakly correlated with epiphytic Pe299R reproductive success, indicating that the leaf’s spatial heterogeneity mitigates resource competition. Although the correlation is weak, the presence of competitors led to the development of Pe299R subpopulations that experienced different life histories and cell divisions. In some in planta competitions, Pe299R benefitted from the presence of epiphytes despite high resource overlap to the competitor strain suggesting other factors having stronger effects than resource competition. This study provides fundamental insights into how bacterial communities are shaped in heterogeneous environments and a framework to predict competition outcomes.

Bacterial growth is classically assessed by measuring the increase in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions as it is not possible to measure the growth of individual strains within mixed cultures. Here we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures, but also in mixed bacterial cultures and determine the impact of competitors on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescent microplate readers that allow for high throughput and replication. Importance We expand on an important limitation of the concept of measuring bacterial growth which is classically limited to one strain at a time. By adopting this approach, it is possible to measure growth of several bacterial strains simultaneously in high temporal resolution and in a high throughput manner. This is important to investigate bacterial interactions such as competition and facilitation.

The phyllosphere is colonized by rich microbial communities, despite sparse and heterogeneously distributed resources. This resource limitation is expected to drive bacterial competition, resulting in either exclusion or coexistence based on the fitness differences and resource overlap between species. Here, we investigated the impact of competition in bacterial colonization and growth of the epiphyte Pantoea eucalypti 299R (Pe299R). To that end, pairwise competition experiments between Pe299R and diverse phyllosphere-colonizing bacteria were performed in vitro and in the Arabidopsis thaliana phyllosphere. Resource overlap was determined as the similarity in resource utilization in vitro. We found an effect of both resource overlap and phylogenetic relationships in the competition outcome between Pe299R and individual competitors in vitro. To account for bacterial individuality in the phyllosphere, we employed a single cell bioreporter to determine the number of divisions that individual cells from Pe299R populations underwent when challenged with individual bacterial competitors. We observed that at the single-cell level, resource utilization similarities and phylogenetic relationships were weakly correlated with Pe299R reproductive success. We observed contrasting results for two strains (Arthrobacter sp. Leaf145 and Sphingomonas melonis Fr1) that, despite sharing either a high or low resource overlap with Pe299R, both negatively affected the reproductive success of Pe299R. Interestingly, we also observed facilitative effects of Methylobacterium sp. Leaf85. This work furthers the understanding of bacterial assembly processes in heterogenous environments. Our high-resolution observations are important to build an ecological framework to predict competition outcomes in the phyllosphere and to design future bacterial biocontrol applications.