Biology of the Plant Cuticle Edited by Markus Riederer, Caroline Müller Copyright © 2006 by Blackwell Publishing Ltd Biology of the Plant Cuticle Edited by Markus Riederer, Caroline Müller Copyright © 2006 by Blackwell Publishing Ltd 11 Microbial communities in the phyllosphere Johan H.J. Leveau 11.1 Introduction The term phyllosphere was coined by Last (1955) and Ruinen (1956) to describe the plant leaf surface as an environment that is physically, chemically and biologically distinct from the plant leaf itself or the air surrounding it. The term phylloplane has been used also, either instead of or in addition to the term phyllosphere. Its two- dimensional connotation, however, does not do justice to the three dimensions that characterise the phyllosphere from the perspective of many of its microscopic inhab- itants. On a global scale, the phyllosphere is arguably one of the largest biological surfaces colonised by microorganisms. Satellite images have allowed a conservative approximation of 4 × 108 km2 for the earth’s terrestrial surface area covered with foliage (Morris and Kinkel, 2002). It has been estimated that this leaf surface area is home to an astonishing 1026 bacteria (Morris and Kinkel, 2002), which are the most abundant colonisers of cuticular surfaces. Thus, the phyllosphere represents a significant refuge and resource of microorganisms on this planet. Often-used terms in phyllosphere microbiology are epiphyte and epiphytic (Leben, 1965; Hirano and Upper, 1983): here, microbial epiphytes or epiphytic microorganisms, which include bacteria, fungi and yeasts, are defined as being cap- able of surviving and thriving on plant leaf and fruit surfaces. Several excellent reviews on phyllosphere microbiology have appeared so far (Beattie and Lindow, 1995, 1999; Andrews and Harris, 2000; Hirano and Upper, 2000; Lindow and Leveau, 2002; Lindow and Brandl, 2003). This chapter aims to provide a brief but broad and updated synthesis of research activities and findings related to phyllo- sphere microbiology. As the next chapter in this volume (Chapter 12) will focus in greater detail on phyllosphere fungi, the present chapter is biased, in some sections at least, towards bacterial colonisers of the leaf surface. 11.2 Methodologies in phyllosphere microbiology 11.2.1 Sampling techniques Sampling the phyllosphere is at the basis of many observations or experiments in leaf surface microbiology, and thus deserves its own and in-detail section Publication 3593 NIOO-KNAW Netherlands Institute of Ecology. MICROBIAL COMMUNITIES IN THE PHYLLOSPHERE 335 at the beginning of this chapter. Several reviews are available that describe the advantages and disadvantages of different methods for sampling and quantification of microorganisms from the phyllosphere (Donegan et al., 1991; Jacques and Morris, 1995; Dandurand and Knudsen, 1996). One of the simplest techniques is leaf printing (Corpe, 1985), by which a plant leaf is pressed onto the surface of an agar plate for a defined period of time and carefully removed again. The agar plate is then incubated to allow growth of the microorganisms that transferred from the leaf surface. Depending on the medium and the abundance and composition of the microflora, the result is a collection of bacterial and fungal growth foci (‘colonies’) that often follow the contours of the leaf. Obvious disadvantages of the method are its low resolution of observation and its limited ability to provide a quantitative appreciation for the phyllosphere community composition. However, leaf printing can provide a first indication that the distribu- tion of microorganisms on leaf surfaces is not uniform (Leben, 1998). For example, more growth often occurs where the veins of the leaf touch the agar, suggesting that these structures are more densely populated areas on the leaf (Manceau and Kasempour, 2002). Furthermore, leaf printing has been an extremely useful and popular laboratory experiment for teaching purposes (Holland et al., 2000). The outcome of a leaf printing experiment will depend in large part on the medium that is used to prepare the agar plate. Medium composition can be varied and exploited to look only at a subset of the microbial community on the leaf. By incorporation of compounds with antifungal (e.g. cycloheximide) or antibacterial (e.g. penicillin) activity, fungal or bacterial colonisers, respectively, can be excluded from the analysis. As micro- organisms differ in their nutritional capabilities, it is also possible to include specific nutrient sources in the medium to select for a nutritionally defined subpopulation of the phyllosphere. For example, agar plates that contain methanol as the sole source of carbon have been used to demonstrate the ubiquity of methylotrophic bacteria on plant leaf surfaces (Corpe, 1985). More quantitative than leaf printing is the method of leaf washing. One or more leaves are placed in a tube or flask containing a specified volume of wash solution (e.g. saline solution or phosphate buffer), and microbes are removed from the cuticular surface by vortexing and/or sonication. The number of bacteria in the wash solution is then determined by the most probable number (MPN) tech- nique (Oblinger and Koburger, 1975) or by plating, directly or after dilution of the wash solution, onto nonselective or selective solid medium to determine the num- ber of colony-forming units (CFUs). The plating can be done using, for example, a Drigalski spatula, glass beads or a spiral plater (Gilchrist et al., 1973). The latter has the advantage that quantitation of CFUs is possible without prior dilution of the leaf wash sample. As with the leaf printing technique, medium composition of the agar plate determines what subpopulation of the phyllosphere will be analysed. This analysis should also take into consideration the fact that different organisms from the leaf surface grow at different rates, so that those bacteria or fungi that grow fast and appear on agar plates first will be more likely to be included in the analysis 336 BIOLOGY OF THE PLANT CUTICLE than those that grow slowly and appear late or not at all due to overgrowth by fast appearing strains. From the number of CFUs per plate the number of bacteria that were present on the leaf can be estimated quantitatively by taking into account the volume of the leaf washing solution, the dilution factor and the volume of the aliquot that was spread onto the agar plate or used to inoculate the medium in the MPN method. CFU numbers are usually expressed as the number of bacteria per leaf, per gram of leaf weight or per square centimetre of leaf surface. Often the number is expressed as 10log(CFU), as this allows comparison of population counts from different leaves, which are often distributed not normally but lognormally (Hirano et al., 1982; Kinkel et al., 1995; Woody et al., 2003). The leaf washing method can be modified to obtain answers to different ques- tions. For example, by not vortexing or sonicating the leaf in a wash solution, bacteria that occupy the leaf surface as aggregates can be removed as intact aggregates and be separated by filtration from those bacteria that live solitary. In this way, Morris et al. (1998) were able to estimate the fraction of bacteria that live in bacterial aggregates on the leaf surface. Incidentally, this aggregated lifestyle of bacteria may be a reason for underestimation of total bacterial populations on leaf surfaces: an aggregate of two or more bacterial cells will produce a single CFU on plates, which stresses the need for breaking up of these aggregates as much as possible (Miller et al., 2000). Another reason for underestimating microbial populations from leaf washing data is the resistance of some microorganisms to be readily removed from the leaf surface by leaf washing (Romantschuk, 1992). A good example is the group of pink-pigmented facultatively methylotrophic bacteria (Holland, 1997). This is probably one of the reasons why these particular bacteria are often missed in phyl- losphere composition studies (Holland and Polacco, 1994). Also, plant-pathogenic fungi, such as downy and powdery mildews, produce structures that anchor the fungal hyphae into the plant leaf making them hard to remove (Section 11.3.2, and Chapter 12). To check the efficiency of any leaf washing method, one can use microscopy (Section 11.2.3) to validate the degree of removal from the leaf surface, for example, by staining with the fluorescent DNA stain 4,6-diamidino-2- phenylindole (DAPI). In one instance this was done to check the removal efficiency of a less common sampling method which involves placing a leaf on sterile water, rapidly freezing the water, then carefully removing the leaf and collecting the ice which contains the transferred leaf microflora (Heuser and Zimmer, 2002). Whereas washing by vortexing and sonication usually leaves the cuticular surface covered by residual microorganisms (Jacques and Morris, 1995; Heuser and Zimmer, 2002), the freezing method almost completely removes microorganisms from the leaf (Heuser and Zimmer, 2002). A method with similar removal efficiency is leaf maceration, by which a single leaf or a collection of leaves is macerated, for example, with a mortar and pestle, in a defined volume of buffer, which is then analysed by plate counting. However, since leaf maceration liberates in addition to those microorganisms that live on MICROBIAL COMMUNITIES IN THE PHYLLOSPHERE 337 the leaf surface also those that grow inside the plant tissue, phyllosphere population sizes may be overestimated by this method. One final point of consideration when using methods such as MPN or plate counts is that they provide estimates only for the culturable subset of the micro- bial population. Microorganisms that resist growing under laboratory conditions are not included in these analyses (also see Section 11.4.1). Furthermore, viable- but-not-culturable-microbial cells (VBNC) that experience prolonged periods of starvation may enter a physiological state which renders them not-culturable, that is, they cannot form a colony on an agar plate, but they are still viable (Bogosian and Bourneuf, 2001).