Individual-based microbial ecology of the phyllosphere

Individual-based microbial ecology is a new and exciting field of study (Kreft2013) that aims to record and quantify the individual experience of microorganisms in their natural habitats. Data collection at this microscopic scale leads to a better understanding of how environmental heterogeneity along micrometer distances acts on individual microbes, how it impacts the ability of microorganisms to survive, reproduce, co-exist and interact, and how individual micrometer experiences translate into outcomes and observations at the macroscopic level. In Lab Leveau, the model system for this research is the plant leaf surface, or phyllosphere (Meyer2012), which is home to a diverse and abundant community of microorganisms (Lindow2002, Leveau2009, Rastogi2013). At micrometer dimensions, it represents a heterogeneous environment in terms of physical, chemical, and biological conditions. The leaf surface offers great conceptual and experimental amenability for exploring the question how heterogeneity determines the fate of individual leaf colonists. Operating at the interface of microbial ecology and plant pathology, our research has societal relevance by contributing to our basic understanding of microbes associated with plant foliage, which include plant pathogens of commercial crops and enteropathogenic contaminants on leafy greens.



GFP bacteriaA key component of this project is the development of bioreporters (Leveau2002) -more specifically bioreporters based on the green fluorescent protein, or GFP (Leveau2001)- that allow us to interrogate individual bacteria for their experience of the plant leaf surface. We have developed several types of bioreporters, for example one that responds to the availability of fructose and sucrose, two of the main sugars found on leaf surfaces that sustain bacterial populations (Leveau2001). This bioreporter was used to estimate the rate (vanderWal2011) and spatial resolution (RemusEmsermann2011) at which photosynthates such as fructose, diffuse through the plant leaf cuticle to the plant leaf surface. Another bioreporter is based on the predictable dilution of GFP from dividing bacterial cells. We refer to it as CUSPER (RemusEmsermann2010), as it measures REProductive SUCcess (REPSUC) based on the reverse interpretation of GFP cell content to estimate past doublings (REPSUC in reverse reads CUSPER). Foliar application of CUSPER showed that the plant leaf surface offers microbial immigrants a wide range of fates and abilities to produce offspring. CUSPER also led us to redefine the bacterial carrying capacity of plant leaves to be understood as the sum of many ‘local carrying capacities’ in order to better explain and predict the course and outcome of bacterial leaf colonization (RemusEmsermann2012). Our results were consistent with a leaf environment that is characterized by a few sites where individual bacterial immigrants can produce high numbers of offspring, while most other parts of the leaf represent sites with only low or medium chance of reproductive success. We also used CUSPER to demonstrate that during the first hours after immigration to a leaf surface, individual bacteria may divide but their offspring may not all stay together to form clusters, which suggests that bacterial relocation is an important process very early on in the process of leaf colonization (Tecon2012). In another study, we used CUSPER to demonstrate that while the establishment of secondary immigrants on a leaf surface was negatively correlated with the level of pre-population by primary colonizers, even under conditions of heavy pre-population a small fraction of secondary immigrants still produced offspring (RemusEmsermann2013). This observation has direct relevance for biocontrol strategies that are based on preemptive exclusion of foliar bacterial pathogens: even at seemingly saturating levels of primary inoculum, few secondary colonizers may still reproduce enough to trigger behaviors that enhance survival or virulence. As a research tool, CUSPER has broad potential for the field of individual-based microbial ecology, allowing us to quantify single-cell competitive and facilitative interactions, to assess the role of chance events in individual survivorship, and to reveal causes that underlie individual-based environmental heterogeneity.


Simulated and artificial leaf surfaces

We have also been developing a spatially-explicit simulation model (vanderWal2013) of how microorganisms colonize the phyllosphere (PHYLLOSIM), which we fed with experimentally derived estimates for processes that occur in the phyllosphere, such as the leaching of photosynthetic sugars from the inside of the leaf as a function of free water on the leaf surface (vanderWal2011). The model revealed, and we confirmed experimentally, that detachment of single bacterial cells from clusters and re-attachment elsewhere along the surface is an important mechanism underlying bacterial exploration of the phyllosphere. A more recent direction of the project involves the development of artificial leaf surfaces. For this, we are collaborating with Prof. Atul Parikh at UC Davis to construct defined landscapes that allow us to tease out, among other things, the effect of leaf topography on water retention and bacterial survival. This subproject takes a landscape approach to individual-based microbial ecology and explores the mechanisms and consequences of connectivity in the leaf landscape from a microbe’s perspective, with a focus on bacteria representing the smallest organismal scale and the greatest challenge in terms of trying to link micro-scale activities to macro-scale effects.