Interactions between microbes is a substantial field of research spanning from experimental evolution studies (e.g. Barrick & Lenski, 2013; Elena & Lenski, 1997; Griffin et al., 2004; Rainey & Rainey, 2003; Rainey & Travisano, 1998) to field experiments (e.g. Hansen et al., 2007; Huang
et al., 2011; Pastar et al., 2013; Tong et al., 2007). Although interactions between microbes, particularly those leading to the maintenance of diversity in itself, is a comprehensive field (Fierer & Lennon, 2011; Hibbing
et al., 2010; Kassen & Rainey, 2004; Rainey et al., 2000), bacterial interactions discussed below are limited to those relevant to the phyllosphere, to align with the aim of this thesis.
The phyllosphere is an environment where multipartite interactions occur: interactions between microorganisms and the plant host, among pathogenic bacteria, between pathogenic and commensal microbes and between other microorganisms and bacteria (Vorholt, 2012). The interactions between plant and bacteria are manifold and have been described in section 1.1.2. Microbial interactions can have wide-reaching consequences, from affecting the local tissue to the entire host (Stubbendieck et al., 2016) and influencing the community structure. Interplay among bacteria occurs at the level of individuals, populations and communities and represents an essential driving force to evolution and function. Understanding how, where and between which microorganisms these interactions take place and how environmental factors shape
interactions will support our understanding of what drives the divergence, emergence or extinction of certain bacterial lineages (Hibbing et al., 2010).
A bacterial ‘community’ constitutes different populations of a number of species, with bacterial ‘populations’ defined as a subset of bacteria of the same species. The community structure is highly dynamic and evolves over time, and is influenced by the number and types of interactions amongst its members and the influence of abiotic and biotic factors. Given that a suitable microbial habitat is typically densely populated (e.g. 106 - 107 per cm2 leaf
surface, Hirano & Upper, 2000), the level of interactions must be quite high and associated with a high degree of variability.
Such interactions range from cooperative to antagonistic, whereas neutral interactions are not to the benefit or detriment of either party. Cooperative interactions occur when at least one gains a profit, whereas the other is not affected (commensalism), or both benefit from the interaction (mutualism). Amensalism, on the other hand, involves one member being negatively affected from the interaction, which is a form of competition. Competition among bacteria, as in every living being, is probably the most common type of interaction. It is broadly distinguished between exploitative and interfering competition (Birch, 1957), with nutritional resources typically being a major limitation in a natural environment. Exploitation refers to competition for resources by depleting the environment for e.g. nutrients, water, light, or simply two-dimensional in space. Interfering competition involves the secretion of toxins, enzymes or antibiotics that kill or inhibit growth of the competitor. The ecological
similarity (e.g. identical metabolic needs) of competing species can increase the level of the competition. The leaf surface is home to a comprehensive and diverse number of bacteria occupying a small space. As previously mentioned, the structure and composition of this bacterial community is shaped by a variety of environmental factors like nutrient and water availability, UV radiation, or pollution (Hirano & Upper, 2000; Lindow & Brandl, 2003; Vorholt, 2012). Given the physical and physiological surface properties of the leaf, only restricted areas are available for colonization, hence pathogenic and non-pathogenic microbes must be in close contact with each other. The spatial proximity facilitates HGT events, which is a major influence on genotypic variation (described in section 1.3). In terms of infectious diseases, synergistic interactions have been shown, where a pathogen does not necessarily act independently, but virulence was affected by interaction with co-inhabiting strains (Singer, 2010). For example, this has been demonstrated for olive knot disease, where P. savastanoi pv.
savastanoi is the causative agent, but Erwinia sp. and Pantoea sp. were discovered as endophytes co-colonizing the cankers and impacting the severity of disease (Marchi et al., 2006; Moretti et al., 2011). Related pathogen species can co-exist on an individual plant host (Fittet al., 2006). Another interesting example of synergistic interactions displays when bacterial strains lacking virulence factors benefit from the coexisting pathogenic isolate and reap their benefits, e.g. population sizes of hrp
mutants were smaller when inoculated alone, as compared to co-inoculation with the wildtype P. syringae pv. phaseolicola B728a strain (Hirano et al., 1999).
Clearly co-residing strains are involved in complex interactions with plant pathogens, but the outcome of these interactions remains to be elucidated. In fact, in diseased hosts the diversity of non-pathogenic isolates has been neglected in the past. There is a need for studies in which a properly designed sample scheme allows discovery of the diversity of the natural populations as a prelude to the investigation of potential interactions. But first, it is necessary to define the level at which interactions are studied. It is important to ask, what constitutes a bacterial population? Do we consider only members of the same species that engage with each other? Within that population, what roles do commensals play, and what is the outcome of competition with the pathogenic strain? What is the evolutionary potential of these commensal bacteria and what ecological factors drive interactions between pathogenic and non-pathogenic isolates?