– Biomimetic surfaces help detecting micro-scale effects of topography on bacterial leaf colonization

This chapter was prepared as a manuscript for submission to The ISME Journal Esser D.S.¹, Meyer K.M.¹, Wiegand K.¹, Tecon R.²,

Gilmore S.F.³, Parikh A.N.³, Leveau J.H.J.²

1 Department of Ecosystem Modelling, University of Göttingen, Germany,

2 Department of Plant Pathology, University of California, Davis, CA, USA

3 Department of Chemical Engineering and Materials Science, University of California, Davis, CA, USA

Abstract

Microbial life is ruled by a wide variety of interactions that operate at different spatial and temporal scales. Intraspecific interactions co-occur with interspecific interactions with other microbes, with hosts, and with predators, as well as factors and processes of the non-living environment. Often the relative effect of a single process is difficult to isolate from others. For example, it is known that leaf-colonizing bacteria often aggregate in the grooves between epidermal cells of plant leaves but it is unclear if this phenomenon is the result of some process connected to leaf topography or if it is the result of a leaf biological process such as increased leaching of nutrients in the grooves. In our study, we analyzed the spatial distribution of two wide-spread leaf-colonizing bacteria on synthetic replicas of bean leaf surfaces. Comparison of our results to a similar study on real leaves suggests that the frequently reported aggregation of bacteria near grooves between epidermal cells is driven by physical processes connected to leaf topography rather than leaf biological processes. Our study highlights the importance of micro-topographic effects on bacterial surface colonization and illustrates the suitability of soft-lithographically manufactured micro-landscapes for studying these effects.

Introduction

The microbial colonization of surfaces has been intensely studied and has been regularly reviewed from different scientific perspectives such as the general process of biofilm formation (O’Toole, Kaplan and Kolter 2000; Danhorn and Fuqua 2007), particular aspects of adhesion (Katsikogianni and Missirlis 2004), the effect of physical stress on biofilm structure (Otto 2014), the description of chemical compounds and signaling molecules (Petrova and Sauer 2012), surface detection by microbes, changes in gene expression patterns, and cell morphology (Tuson and Weibel 2013; Wozniak and Parsek 2014) as well as the development of anti-fouling surfaces (Salta et al. 2010). Surface colonization involves complex interplays of the microbes with the surface (e.g. attachment, modification, detachment), with each other (e.g. competition, antibiosis, collaboration), and with the medium that interfaces the surface (e.g. air or water). Adding to this complexity is the fact that many surfaces feature spatial variability in the physical, chemical and biological conditions at scales that are relevant to microorganisms. One of the major challenges in microbial ecology is to understand this complex interplay as well as the individual and combined impact of these processes on surface colonization. One tried and proven approach to disentangle the complexity of surface colonization is experimental deconstruction, i.e. selectively removing one or more parts of the complexity in order to assess the impact of physical, chemical or biological condition. Examples are the use of single species experiments in order to exclude interspecies interactions or the use of artificial flat surfaces that eliminate potential effects of surface topography.

If, in contrast, such topographical effects, e.g. of biological surfaces, are of interest, these effects could be targeted by comparing the microbial colonization of the biological surface to the colonization patterns on a biomimetically patterned surface (BPS), i.e. a manufactured exact replica of the biological surface. For example, Zhang et al. (2014) and colleagues recently showed that soft-lithographic replicas of spinach leaves made from polydimethylsiloxane (PDMS) are well able to replicate spinach leaf surface structure.

They also found that wettability of the PDMS replicas was similar to real spinach leaves.

Finally, they pointed out that BPS made from agarose gel and supplemented by 'suitably controlled nutrient mixtures' allow studying the effect of surface topography on bacterial colonization (Zhang et al. 2014).

The spatial distribution of colonizers of the plant leaf surface, i.e. the colonization of the

phyllosphere (Last 1955), has long been studied (e.g. Leveau and Lindow 2001; Monier and Lindow 2004; Remus-Emsermann et al. 2012, 2014). A wide variety of environmental factors and processes was found to contribute to or to hamper the successful establishment of epiphytic microbes (Leben 1970; Roos and Hattingh 1983; Rodenacker et al. 2000; Fett and Cooke 2003; Monier and Lindow 2004; Hunter et al. 2010; Tecon and Leveau 2012;

Remus-Emsermann et al. 2014; Esser et al. 2015). Many environmental factors are difficult to measure at micrometer-scale resolution but for an increasing number of substances, e.g. carbon, water, or iron, such information became available in the form of bioreporters (Joyner and Lindow 2000; Leveau and Lindow 2001; Axtell and Beattie 2002;

Remus-Emsermann and Leveau 2009; Remus-Emsermann et al. 2012). They showed the heterogeneous distribution of these factors which affects local success of bacterial colonization in the phyllosphere. Additionally, interactions between the microbial individuals also contribute to the complexity of the observable colonization patterns (Yu et al. 2014). Finally, bacterial population density was found to be spatially correlated to the occurrence of certain leaf morphological structures such as veins, stomata, and trichomes (Marcell and Beattie 2002; Monier and Lindow 2004; Hunter et al. 2010; Esser et al.

2015). For example, glandular trichomes are known to excrete carbon-rich substances (Ascensão and Pais 1998) that can be utilized by bacteria and the crevices (grooves) between epidermal cells have frequently been reported to harbor large numbers of microbial colonizers. Especially for such plant-microbe interactions it is often unclear if they are based solely on some effect of the leaf surface topography or if also a biological effect is active. For example, some trichomes have the ability to better retain water than undifferentiated, smooth leaf areas (Brewer, Smith and Vogelmann 1991). Those areas that retain water the longest are likely to concentrate water-soluble nutrients during times of evaporation. This solely topographical (or physical) process could be amplified by the fact that improved water availability increases nutrient leakage from the leaf interior (Tukey 1970). We would consider such a trans-cuticular process as a leaf biological process, because it would not be present on an artificial biomimetic surface.

The manufacturing of artificial biomimetic leaf surfaces is a growing field of research in materials science. Here, scientists are often interested in producing self-cleaning surface coatings that mimic the superhydrophobic properties of leaves of different plant species such as Nelumbo nucifera or Triticum aestivum (Sun et al. 2005; Koch et al. 2007; Schulte et al. 2009). Microbial colonization of such artificial surfaces has also become a vivid field

of research. Active fields are the development of sterile surface coatings for medical tools (Ionescu et al. 2012; Cerqueira et al. 2013; Manabe, Nishizawa and Shiratori 2013; Cole et al. 2014; Depan and Misra 2014) or marine applications (Salta et al. 2013), or the study of bacterial communities in water treatment facilities (Liu et al. 2013; Al Ashhab, Herzberg and Gillor 2014). But only recently, microbial colonization of biomimetic leaf surfaces has been considered for the study of effects of plant leaf topography on bacterial growth and survival on leaves (Zhang et al. 2014; Doan and Leveau 2015).

Here, we present the first in-depth spatial analysis of bacterial colonization patterns on such artificial leaf surfaces. The aim of our study was to isolate the effect of topography on bacterial leaf colonization. Our main focus was on detecting differences in the spatial distribution of bacteria growing on bean leaves and on biomimetic bean leaf surfaces made from PDMS. Given the fact that such surfaces have similar physical properties as the biological surface (Zhang et al. 2014), we hypothesized that differences between experiments conducted on both surfaces are caused by processes that cannot be explained by surface microstructure alone. Conversely, microbial colonization patterns that do not differ between biological and biomimetic surfaces would suggest that microbial colonization is either surface-independent or at least independent from processes linked to the biology of the surface. We used spatial statistics for a detailed spatial description of bacterial colonization patterns on the artificial surfaces and compared the results to the results of an equivalent study on real bean leaves (Esser et al. 2015).