Phyllosphere

Cindy E Morris, Institut National de la Recherche Agronomique, Avignon, France

The phyllosphere is the microbial habitat extending from the surface of the cuticle covering the leaf epidermis to the outer edge of the boundary layer surrounding the leaf and inward into the leaf tissues.

Secondary article
Article Contents
. Introduction . Overview of the Phyllosphere Habitat . The Microbiology of the Phyllosphere . The Phyllosphere of Aquatic Plants

Introduction
The phyllosphere is the microenvironment extending from the leaf surface outward to the outer edge of the boundary layer surrounding the leaf and inward into the leaf tissues. A wide range of microorganisms survives and proliferates in the phyllosphere. Natural openings in the leaf tissue, such as stomata and hydathodes, allow ready passage of microorganisms between the external and internal domains of the phyllosphere. However, the environmental conditions associated with these domains and the strategies exploited by microorganisms in colonizing these domains are very dierent. This article will focus on the part of the phyllosphere external to the leaf surface; this will be the sense of the term phyllosphere used throughout this article unless indicated otherwise. The phyllosphere is characterized by a multitude of topographical features, sources of nutrients and water, and a range of microclimatic conditions that are often conducive to the survival and/or growth of diverse microorganisms. There are microenvironments analogous to the phyllosphere that involve other aerial organs of plants (stems, fruits, owers, etc). Knowledge of the properties of the phyllosphere per se is, in part, based on an understanding of the aerial phytosphere in general. The phyllosphere has been studied as a microbial ecosystem since the 1950s and has been the theme of a regular series of scientic meetings and books since the early 1970s. Hundreds of species of fungi and bacteria have been found on leaf surfaces as well as some yeasts and protozoa, and many have well-described roles in environmental processes or in the biology of the host plant. These include plant pathogens, animal pathogens and pathogens of insects; ice nucleation-active bacteria; microorganisms that produce active phytohormones, allergens, toxins or aromatic compounds; and antagonists of plant pathogens. There is evidence that phyllosphere microorganisms also participate in global cycling of carbon and nitrogen and are involved in fossilization processes of plant material. Hence, the phyllosphere harbours microorganisms having signicant roles in plant health and general plant biology, in the hygienic and market qualities of food products derived from plants, in global climatological processes and in recycling of elements. Furthermore, insects and microscopic animals such as nematodes that are plant pests also inhabit the phyllosphere.

Overview of the Phyllosphere Habitat

Plants inhabit 1.35 108 km2 of the 1.5 108 km2 of terrestrial surface of the Earth. The total leaf surface area of these plants has been estimated to be about 2 6 108 km2. At the scale of a 2-mm-long bacterial cell or a 10-mm-long fungal spore, the phyllosphere is a vast habitat for colonization. From the perspective of a microbial inhabitant, the phyllosphere is comparable to a forested island whose subsurface is replete with burrows and tunnels. To continue this analogy, at the oor of this microscopic forest is the cuticle. The topographical features of the leaf, such as leaf hairs and glandular trichomes, may be considered to be the forest itself, and the burrows and tunnels are represented by intercellular leaf spaces that communicate with the cuticular surface via stomata, wounds and other leaf openings. As in the case of a veritable forest, the microscopic forest of the phyllosphere has specic atmospheric conditions and the overall state of the environment is eected by herbivory and by agricultural practices and other human activities. However, unlike a veritable forest, the phyllosphere habitat undulates and turns in three dimensions. A schematic representation of the major components of the phyllosphere habitat is presented in Figure 1.

The cuticle
The cuticle serves as the principal surface for contact and attachment of microorganisms and insects to the external surface of leaves. It is composed of soluble and polymeric lipids. The hydrocarbons that constitute the wax component of the cuticle are generally C21 chains or greater and are saturated. This results in a substrate that is dicult for many microorganisms to absorb and metabolize. The primary function of the cuticle is to create a highly hydrophobic coat protecting plants from water loss. However, the cuticle has secondary functions that may inuence the capacity of microorganisms and insects to colonize leaf surfaces. The cuticle is involved in the reectance of incident radiation (visible, UV and infrared) by leaves. Thick cuticles promote greater reectance of infrared radiation and allow leaves to maintain a cooler temperature than leaves with thinner cuticles. The compo1

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Turbulent boundary layer

Microbial biofilm Germinating fungal spore Stomate Bacteria

Leaf hair

Cuticle Trichome

Bacteria Substomatal cavity

Figure 1 Schematic representation of the major components of the phyllosphere habitat depicted in cross-section. The boundary layer around the leaf may be several millimetres thick, leading to marked differences in environmental conditions between the phyllosphere and the ambient air. Epidermal cells are covered by a cuticle, which is the first point of contact between microorganisms and the leaf surface proper. The phyllosphere has topographical features such as leaf hairs, glandular trichomes and stomata (depicted here), as well as junctions of epidermal cells, grooves over leaf veins and hydathodes, some of which are favourable sites for microbial colonization. Stomata, hydathodes, wounds and other openings in the leaf allow passage of microorganisms between the inner and outer domains of the phyllosphere. The features depicted in this schema have been drawn to scale.

related to the phylogeny of the plant. Cuticles may be as thin as about 0.05 mm on the leaves of curled pondweed (Potamogeton crispus) or beet (Beta vulgaris), about 0.1 0.4 mm thick in the case of fava bean (Vicia faba), tobacco (Nicotiana tabacum) and apple (Malus pumila) leaves, 2 mm thick as on coee (Coea arabica) leaves, 5 mm thick on the leaves of black pine (Pinus nigra) and over 11 mm thick on leaves of scented hakea (Hakea suaveolens). But for any given species, cuticle thickness can be modied by exposure to light (increased exposure to visible light increases cuticle thickness) and by the ambient relative humidity and temperature. Cuticle thickness also changes with the age of the plant.

Radiation, temperature and the gaseous environment

On completely exposed abaxial (upper) leaf surfaces, the incident intensity of sunlight is that of the non-shaded surrounding environment. Hence, the phyllosphere is among the most light-intense of the terrestrial microbial habitats. Protection of the phyllosphere from direct sunlight occurs for adaxial leaf surfaces and for shaded leaves at lower points in the canopy or in understories. Furthermore, certain leaf features such a trichomes may oer shade for some microorganisms such as bacterial cells. Leaves are surrounded by a boundary layer, a zone within a few millimetres of each leaf in which the diusion resistance and heat transfer rates of the air are dierent from those of the ambient air. This leads to microclimatic conditions in the phyllosphere that are dierent from those of ambient conditions. The boundary layer of leaves may range from less than 1 mm to as much as 10 mm. Thick boundary layers are favoured by low wind speeds of ambient air, by large leaf width, by the presence of prominent leaf features (trichomes, ridges, etc.) that impede air ow, and when the dierences between ambient and leaf temperature are small. Wind speed at the leaf surface is considered to be zero except in the case of strong gusts. There may, nevertheless, be convective air movement near the leaf surface arising from temperature dierences at dierent sites on a leaf. Within the boundary layer, most gaseous exchange occurs via diusion, whereas outside of the boundary layer it can occur due to air movement. Relative humidity within the boundary layer is generally greater than that of ambient air. Much of the water vapour in the boundary layer originates from stomata as leaves transpire. Leaf volatile compounds may also diuse into the boundary layer, serving either as potential sources of nutrients or antimicrobial compounds. When air temperature is at or below about 308C, the temperature at the leaf surface may be as much as 578C greater than that of the air. Dierences in leaf and air temperature are greatest when wind speeds are low and

sition and structure of the cuticle can also inuence the wavelengths of visible light that are reected, which, in turn, can inuence the attractiveness of leaves to insects (in general, insects are attracted to leaves that appear the most green). Cuticles may also be involved in the germination of fungal spores by either triggering germination of certain fungi, or by absorbing self-inhibitors of germination. Cuticles are signicant barriers to the diusion of watersoluble compounds from the apoplast of the leaf out onto the external surface of the leaf. The most supercial layer of the cuticle, the epicuticular waxes, may be either crystalline or amorphous in structure, which inuences the wettability of the leaf surface. Furthermore, these waxes also provide a defence against insect herbivory as the attachment of insects to leaf surfaces is generally inhibited by a loose crystalline structure of the waxes. On a leaf, a cuticle is formed on all external surfaces of epidermal cells and on the epidermal surfaces lining the substomatal cavity. However, the cuticle in the substomatal cavity is much thinner than that on the epidermal cells on the outer surface of the leaf. The exposed surfaces of the mesophyll and palisade cells in the substomatal cavity are smooth and hydrophilic and not covered by a cuticle. The thickness of the cuticle varies across the leaf surface. In general, it is thickest on the outer radial walls of the epidermis, is thinnest or almost absent on glandular structures such as glandular trichomes, and it is absent on the cells of the hydathodes at the leaf margins. Cuticle thickness varies greatly among plant species, but is not
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incident solar radiation is high. Temperature can also vary by as much as about 48C across a single leaf, the leaf edges generally being cooler than the central part of the leaf. For ambient temperatures above about 308C, leaf temperature has been observed to be below that of the ambient air. It is believed that this is due to signicant changes in the internal resistance of the leaves to evaporation of water, allowing the leaf to cool itself, thereby avoiding lethal damage. The precise temperature at which plants make this transition depends on their adaptation to high temperatures. Fluttering of leaves is also a factor in regulation of the temperature in the phyllosphere. Fluttering poplar leaves were observed to be as much as 248C cooler than leaves that were prevented from uttering (Roden and Pearcy, 1993). Such leaf movement may lead to increased heat transfer and a decrease in the interception of light. Furthermore, uttering may increase the conductivity of the boundary layer to heat exchange.

Water, nutrients and the chemical environment

The quantity of free water on leaf surfaces is one of the most dynamic parameters of the physicalchemical environment of the phyllosphere. Condensation and impaction of water droplets on leaf surfaces alternate with evaporation and run-o of water in cycles that are generally diurnal. Furthermore, the ebb and ow of free water in the phyllosphere lead to variations in local pH, as substances such as ammonia, sulfur dioxide and calcium carbonate that may be present on leaf surfaces are dissolved, and also lead to variations in local concentrations of leaf surface nutrients. Free water on leaves comes from several sources: dew formation, precipitation (rain, mist or fog), irrigation and guttation. On average, a leaf can retain about 530 mg cm 2 2 of water on its surface, roughly equivalent to the amount of water falling in a rainfall of 12 mm. However, this water-holding capacity depends on the size of the water droplets forming or depositing on the leaf, the wettability of the leaf surface as well as the inclination and the movement of the leaf. Drops of mist or fog, for example, of about 10 mm in diameter are less likely to run o leaves than are larger drops of rain or irrigation water. The duration of free water on a leaf also depends on the wettability of the leaf, as this can inuence the rate of evaporation. On relatively wettable leaf surfaces, droplets will spread, thereby forming thin lms that evaporate rapidly. On highly hydrophobic surfaces, droplets will not spread and may even coalesce around trichomes. As for all living organisms, water and nutrients are essential for the growth and metabolic activity of microorganisms in the phyllosphere. It is well accepted that both of these resources are distributed heterogeneously in space

and time in any given phyllosphere. However, whereas the sources and quantities of free water on a leaf surface are relatively easy to describe, the sources and quantities of nutrients available for microorganisms in the phyllosphere are more dicult to dene. There are multiple possible origins of nutrients in the phyllosphere. These include diusion of volatile and solid compounds out of the leaf tissue, nutrients in guttation uids, compounds contained in trichomes, debris, pollen and honeydew, as well as dead microorganisms and insect larvae. Volatile compounds including alcohols, aldehydes, esters and monoterpenes can be emitted from leaves, usually via stomata. Conifers, for example, may emit up to 10 mg of terpenes per gram of leaf per minute. But little is known about how these volatiles aect microbial populations. It is likely that these compounds are antimicrobials as well as substrates for growth. Leaves also emit considerable amounts of methanol (Nemecek-Marshall et al., 1995). Methanol in the phyllosphere may be a ready source of carbon for the pink-pigmented facultative methylotrophic bacteria (mainly Methylobacterium and Mesophilicum spp.) that are ubiquitous in the phyllosphere (Hirano and Upper, 2000). Plant metabolites can leach out of leaf tissues across the cuticle. For intact cuticles, this can occur by simple diusion. Cuticular permeability for lipophilic molecules is relatively high, whereas it is relatively low (but not null) for inorganic ions and polar metabolites. In general, measures of cuticular permeability suggest that few sugars, amino acids and organic acids of plant origin can leach to the leaf surface. However, labelling of plant metabolites with radioisotopes has shown that sugars, amino acids and organic acids of plant origin do, in fact, leach to the leaf surface. On corn leaves, for example, glucose has been detected in leachates at concentrations of hundreds to thousands of nmol cm 2 2. It is likely that leaching of plant metabolites is enhanced by wounds in the cuticle. Leaching may also be enhanced by the application of pesticides to leaves. Pesticide formulations often contain surfactants to promote contact of the pesticide with the leaf surface. Although the pesticide itself may be deleterious to certain components of the microora and insect populations in the phyllosphere, the surfactant may solubilize lipophilic components of the cuticle, thereby increasing cuticular permeability and enhancing availability of nutrients. Physiological activities associated with hydathodes and trichomes may lead to the deposition of nutrients or other chemical substances on leaf surfaces. Plant uids can be forced out of leaves via hydathodes when there is signicant hydrostatic pressure in the xylem. This phenomenon, referred to as guttation, occurs when the transpiration of the plant is low, in particular at times of high relative humidity and low air movement. Guttation uids deposited on the leaf margin contain dissolved organic and inorganic substances. Up to tens of mg L 2 1 of sugars, for example, have been detected in these uids.
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Glandular trichomes contain a wide variety of substances including sugars, amino acids and organic acids as well as essential oils that can leak out of intact trichomes and that are liberated abundantly when trichomes are broken. Trichomes of halophilic plants such as Atriplex hamilus can also accumulate salt, which crystallizes on the leaf surface of this plant when the bladder cells of their saltaccumulating trichomes break. This leads to a diurnal cycle of salt concentration in the phyllosphere as dew forms and dries, with salt concentrations on leaves increasing up to 0.4 mol L 2 1. Bacteria are the principal epiphytic colonizers of the surfaces of these leaves, attaining population densities of about 104 cells cm 2 2. Some bacterial strains from these plants tolerate concentrations of sodium chloride up to 20% in laboratory media (Simon et al., 1994). In spite of the diversity of sources of nutrients on leaf surfaces and the presence of measurable quantities of nutrients, phyllosphere microbiologists are not in agreement concerning the presence of sucient quantities of nutrients to assure numerous cycles of multiplication of microorganisms in this habitat. From a theoretical perspective, it is likely that adequate nutrients are available to assure a considerable amount of multiplication of bacteria, for example. The dry weight of a bacterial cell is in the range of 0.1 to 1 pg. Hence, the doubling of a bacterial population of 106 cells cm 2 2 would require up to 1 mg of the molecules constituting the dry matter of these cells per cm2 of leaf surface. Such quantities are within the amounts observed in leachates from leaf surfaces. However, little is known about the spatial distribution of sources of nutrients relative to that of microorganisms in the phyllosphere and the coincidence of nutrients and microorganisms in time. Numerous chemicals deleterious to microorganisms may be present in the phyllosphere such as the volatiles and pesticides cited above. Major air pollutants such as sulfur dioxide, ozone, ammonia and oxides of nitrogen, originating from the combustion of fossil fuels and intensive agricultural practices, as well as heavy metals from smelting factories, have also been detected in the phyllosphere. These chemicals have been shown to have signicant eects on the composition of the microora in the phyllosphere, with yeasts in general being the most sensitive and lamentous fungi the most tolerant to pollutants.

Figure 2 Scanning electron micrograph of the abaxial surface of a leaf of cantaloupe (Cucumis melo). Leaf hairs, stomata and junctions between epidermal cells are evident. Bar, 100 mm.

(Cucumis melo) leaves. Leaves have tens to hundreds of stomata per mm2. Densities vary between adaxial and abaxial leaf surfaces and with plant ecotype. The pores of these stomata, when open, are 10100 mm2, large enough for the passage of bacteria cells and fungal hyphae. Stomatal openings may constitute up to 1% of the leaf surface. The density of trichomes is in the same order of magnitude as that of stomata. Furthermore, trichomes may be up to 100 mm long, providing colonization sites up into the boundary layer. Leaf topography at the nanometre scale has been evaluated with atomic force microscopy (Mechaber et al., 1996). Roughness of the leaf surface is highly variable across the leaf at this scale and changes with leaf age owing, in part, to senescence and weathering.

The Microbiology of the Phyllosphere

The diversity of microorganisms in the phyllosphere makes it impractical to list or even to summarize a list of the species known to be present in this habitat. Such a list is in continuous ux in response to new developments in taxonomy and to the honing of techniques allowing the identication of uncultured or unculturable microoganisms. Moreover, on a given leaf it would not be uncommon to nd a hundred or more species of bacteria, yeasts and fungi present simultaneously. Microbiologists have been particularly curious about the interaction that a given microbial species has with the plant and about the potential interactions among the microorganisms coinciding on the same leaf. This interest is driven by the importance of these organisms for the quality and productivity of cultivated plants and has led to the elucidation of numerous examples of how phyllosphere microorganisms aect plant biology in general. On the other hand, many of the microorganisms

Leaf topography
The topography of leaf surfaces is dened by relatively large features such as stomatal openings, trichomes, leaf veins and the junction between epidermal cells as well as by smaller features such as the crystalline structure of epicuticular waxes. Figure 2 is an example of the topographical features of the abaxial surface of cantaloupe
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present on leaves have no known interaction with the plant or no known role in the dynamics of phyllosphere microbial populations. Furthermore, microorganisms colonize discrete sites on leaf surfaces, leaving up to 98% of the surface uninhabited, and hence might not have the potential for antagonistic, synergistic or commensal interactions with many of the other co-inhabitants.

Significant effects of phyllosphere microorganisms on plant biology

Of primary importance to plant biology are the phytopathogenic microorganisms having an epiphytic phase as part of their life cycle and ultimately inducing disease symptoms on aerial plant parts. These include innumerable species of bacteria (species in the genera Clavibacter, Erwinia, Pseudomonas and Xanthomonas, for example), of lamentous fungi in the kingdoms Eumycota and Chromista causing rusts, mildews, sooty moulds and leaf spots, and of yeasts such as Albugo spp. The diseases caused by these organisms lead to losses in quality and quantity of plant products at harvest. Furthermore, plant pathogens as well as saprophytes are responsible for the degradation and changes in colour and odour of plant-derived food products after harvest. Some phyllosphere microorganisms can also enhance disease development induced by pathogens. For example, the production of biosurfactants by plant surface bacteria can lead to enhanced eciency of the pectolytic activity (breakdown of the intercellular cement) of soft-rot organisms by increasing surface wettability of plant tissues. Phyllosphere microorganisms can also have indirect, but important, eects on disease epidemiology. Epiphytic bacteria harbour plasmids containing the genetic determinants of resistance to heavy metals that can be transferred to bacteria of the same or dierent species, thereby contributing to the reduced eciency of pesticides containing these metals. Horizontal transfer of the genetic determinants of plant pathogenicity among microorganisms is also likely, but has not yet been clearly demonstrated in the phyllosphere. Another remarkable deleterious eect of microorganisms in the phyllosphere concerns their activity as catalysts for the freezing of supercooled water (Wolber, 1993). Strains of epiphytic bacteria in the genus Pseudomonas, as well as the other genera cited above, produce cellmembrane-bound proteins with a crystalline structure mimicking that of ice crystals. These ice nuclei catalyse the freezing of supercooled water at temperatures as warm as 2 28C, although most strains of ice-nucleation-active bacteria found in nature are active at temperatures colder than 2 48C. Epiphytic ice-nucleation-active bacteria are considered to be the most abundant and ubiquitous form of naturally occurring ice nuclei. These bacteria play a role in frost damage of leaves of herbaceous plants and of owers. When cultivated in the absence of ice-nucleation-

active bacteria (under experimental conditions in growth chambers, for example), frost-sensitive plants such as tomato, beans and cucumbers do not freeze when exposed to temperatures as low as 2 7 or 2 88C. However, when the leaves of these same plants are colonized by ice-nucleationactive bacteria active at 2 48C, these bacteria induce freezing of the water on the leaf surfaces and the plants subsequently manifest symptoms of frost damage at this temperature. Application of antibacterial compounds or non-ice-nucleation-active bacteria competitive with icenucleating bacteria to leaves and ower surfaces of eldgrown plants has led to reduced population sizes of epiphytic ice-nucleation-active bacteria on these plant parts and to subsequent reduction in frost damage, further illustrating the role that these bacteria play in frost damage to plants. Phyllosphere microorganisms also have benecial eects on plants. Free-living nitrogen xers such as Klebsiella spp. and Beijerinckia spp. reside in the phyllosphere. When inoculated onto plants in the eld as a form of fertilizers, strains of Beijerinckia spp. have induced yield increases in rice and jute similar to those obtained with conventional fertilizers. Epiphytic bacteria may also produce active phytohormones. Although certain of these may have negative eects on plant health, positive eects on growth and owering have been observed. Phyllosphere microorganisms are also very important as antagonists of other microbial plant pathogens or of insect pests. Bacteria, fungi and yeasts have been shown to limit the proliferation of plant pathogenic microorganisms via interaction with the pathogen itself (competition for space or nutrients, hyperparasitism, or antibiosis) or, in some cases, via induction of local or systemic plant defence mechanisms that, in turn, inhibit the pathogen. The phyllosphere also harbours entomopathogenic fungi such as Beauveria bassiana or the bacterium Bacillus thuringensis that produces spores toxic to lepidopterous insects. Microorganisms colonizing the phyllosphere of fallen leaves have important eects on the recycling of plant nutrients. In particular, fungi and bacteria on leaf detritus are responsible for the decay of these tissues. However, in some cases this decay process can be signicantly altered by other microorganisms. In particular, leaves falling into water may be colonized by dense biolm consortia that are eective mineralizers. It is likely that this process favours the fossilization of leaves that would otherwise decay.

Strategies for adaptation to the phyllosphere habitat

For successful survival in the phyllosphere, microorganisms must be able to adapt to periods of drought and intense light, to the ebb and ow of nutrient reserves, to uctuating pH, osmotic conditions and temperature, and to the presence of antimicrobial compounds. Spore
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formation by fungi is one of the principal means of resistance to the diverse stresses of aerial environments. Although some spore-forming bacteria inhabit the phyllosphere, the majority of prokaryotes in this habitat are Gram-negative nonsporulating bacteria. A wide spectrum of the adaptation strategies of these bacteria has been investigated (Beattie and Lindow, 1999). Production of exopolysaccharides by phyllosphere bacteria enhances resistance to water stress. Production of such polysaccharides might lead to the formation of biolms on leaf surfaces (Morris et al., 1997); these may in turn provide resistance to a wide range of stress and may also foster accumulation and recycling of nutrients. The presence of DNA repair systems and the abundant production of pigments by both fungi and bacteria is thought to be an adaptation to the high UV intensities characteristic of the phyllosphere. However, there is insucient evidence for a direct and major role of these pigments in the tness of microorganisms in the phyllosphere. Microbes on leaf surfaces produce a diverse array of compounds such as wetting agents, pectolytic enzymes, the plant growth regulator indolacetic acid and cuticle-degrading cutinases that may enhance cuticular permeability to nutrients and may facilitate leaf wetting, thereby fostering the metabolic activity and growth of these microorganisms. Although the phenotypes cited here may be found in microorganisms in a wide range of habitats, many of the microorganisms in these habitats are apparently incapable of surviving in the phyllosphere. Likewise, a large part of the bacteria and fungi adapted to the phyllosphere survive poorly in aquatic or soil ecosystems, for example. The key phenotypes essential for epiphytic tness have not been fully elucidated.

geneity. For bacteria, the most common sites on leaves for colonization are the junctions of epidermal cells, the grooves along leaf veins, the base of trichomes and around stomata. The number of microorganisms in the phyllosphere changes signicantly during diurnal periods as well as throughout the growing season of a crop. Over relatively long periods such as a growing season, total microbial densities on leaves tend to increase from leaf emergence until senescence. However, on a daily scale there is considerable positive and negative uctuation of population sizes, which suggests that these populations are resilient to climatic factors.

Leaves as islands for colonization

The processes governing the dynamics of microbial populations on leaves are considered to be analogous to those inuencing the colonization of islands as described in the theory of island biogeography. Hence, the number of individuals on leaves, as on islands, is the sum of the individuals added or removed from a leaf via immigration, emigration, birth (multiplication) and death. Direct measures of immigration rates of microorganisms to leaf surfaces have been accomplished by surface-disinfecting leaves of plants in the eld and then measuring subsequent increases in population sizes on these leaves. Such measures must be made in time periods shorter than the generation times of the microora common to these phyllospheres to eliminate the eect of multiplication on the observed increases in population size. Via such experiments, it was estimated that the population sizes of Aureobasidium pullulans and Cladosporium cladosporoides arriving on apple leaves via immigration within 12-h periods were 0.03% to over 100% of the population sizes of these microorganisms established on the surrounding leaves (Kinkel et al., 1989). For bacteria, about 103 bacteria-containing particles per day have been observed to arrive on leaves in bean elds during periods of dry deposition of aerosol particles (Lindemann and Upper, 1985). Microorganisms can also arrive on leaves via rainfall and subsequent splashing of raindrops or irrigation water. Wind and rain also contribute to the emigration of microorganisms from leaves. Wind movement across leaves contributes to the creation of dry aerosol particles that may contain microorganisms. The net upward ux of bacteria, for example, above plant canopies is usually much greater than that above fallow soil, suggesting that plants are the source of these aerosolized bacteria. Canopies harbouring bacterial densities of 106 bacteria cm 2 2 of leaf may contribute up to 106 aerosolized particles containing bacteria for each m2 of canopy (Lindemann et al., 1982; Lindemann and Upper, 1985). Rain water may also wash o up to 50% of the bacterial population on a leaf.

Variability of microbial populations in space and in time

One unifying feature of phyllosphere microbial populations is their variability at a wide range of spatial and temporal scales. This variability is driven in part by the eciency of the adaptation strategies described above, by the dierence in habitat quality among leaves and among dierent sites on a leaf, and by the impact of birth, death, immigration and emigration of microorganisms as described below. Little is known about the dynamics of the structure and composition of phyllosphere microbial communities. However, particular attention has been paid to changes in numbers of given species in the phyllosphere over time and to patterns of their localization on leaves. The number of bacteria, for example, on dierent leaves in a given canopy or among square-millimetre sites on a single leaf can vary by a factor of 10 to 1000 (Hirano et al., 1982; Kinkel et al., 1995). Aggregation of epiphytic microorganisms into biolm-like structures and the colonization of only specic sites on the leaf can enhance this hetero6

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The movement of phyllosphere microorganisms to and from leaves can lead to dissemination of those microorganisms over short distances or up to several hundreds of kilometres. Leaf-to-leaf contact leads to dissemination of phyllosphere microorganisms across several centimetres to metres; rain can move microorganisms across several kilometres, whereas the formation of aerosols can transport microorganisms into the stratosphere. Factors favouring airborne movement of phyllosphere microorganisms may dier markedly among dierent species. Wet conditions may be favourable for the spores of certain fungi to become airborne, while dry conditions favour others. Survival and spread of microorganisms in the air depend on microclimatic conditions (rain, patterns of air movement) and the capacity of microorganisms to resist periods of drying or absence of the host plant. Overall particle size is also an important factor. The theoretical drift of particles in a wind of about 2 m s 2 1 with no turbulence is almost 200 m for a particle 50 mm in diameter and 400 km for a particle 5 mm in diameter. Phyllosphere microorganisms can also be moved across a wide range of distances as contaminants on farm equipment, on animals, and on plants and plant products that are transported. All of these processes contribute to the immigration and emigration of phyllosphere microorganisms to and from leaves. Whereas there is relatively direct evidence for the eect of immigration and emigration on the population dynamics of phyllosphere microbial communities, it is more dicult to assess directly the importance of multiplication and death. Studies of immigration have given clues about the importance of cell death in the population dynamics of epiphytic microbes. In particular, the sum of the number of microorganisms immigrating to leaves over periods of days or weeks often greatly overestimates the size of epiphytic microbial populations on these leaves, suggesting that emigration and cell death signicantly limit population sizes. Multiplication of microorganisms on leaf surfaces has been discerned on leaves protected from the eects of immigration and emigration, generally under experimental conditions in greenhouses or growth chambers. Furthermore, establishment of large populations of clonal lines of microorganisms on leaf surfaces also suggests that these organisms multiplied on leaves, but multiplication per se is dicult to demonstrate in situ on leaves in open environments.

thickness of this boundary layer depends on water ow, ambient temperature and leaf morphology, as for the boundary layer of aerial phyllospheres. This boundary layer has a marked impact on oxygen and carbon dioxide concentrations and on pH gradients in the phyllosphere. However, unlike the boundary layer of aerial phyllospheres, the boundary layer of aquatic phyllospheres promotes diusion of water-soluble solid compounds and inhibits the diusion of gases. The incident radiation and temperature in the phyllosphere of aquatic plants are inuenced by the position of the leaves in the plant canopy and the density of the canopy as for aerial phyllospheres, although the solar radiation to which submerged leaves are exposed is attenuated by water. Surfaces of submerged leaves are colonized by an abundant microora that is composed primarily of heterotrophic bacteria and autotrophic algae. Total bacterial population densities, for example, on these leaves are within the range of about 104 107 bacteria cm 2 2. Epiphytic bacteria and algae on aquatic plants are metabolically very active. The heterotrophic activity of the epiphytic bacteria on plants in densely vegetated streams has been observed to surpass that of the bacterioplankton in the same streams. As for aerial leaves, the spatial distribution of epiphytic microorganisms on submerged leaves is not uniform, and the population densities change with leaf age and numerous environmental factors. The main processes governing population dynamics on leaves of aerial plants immigration, emigration, multiplication and death can also be described for aquatic plants. In particular, microorganisms are scrubbed from leaf surfaces by water currents, dispersed into the bulk water and then disseminated to other leaves. Some of the microorganisms in the phyllosphere of aquatic plants are deleterious to these plants and are considered as fouling agents of aquatic systems. The regulation of microbial populations on aquatic plants by the plant itself has been demonstrated for the red alga Delisea pulchra. This plant produces halogenated furanones that are analogues of acylated homoserine lactones, the chemical signal important for triggering attachment, biolm formation and other densitydependent phenotypes of bacteria. These analogues interfere with normal cell-to-cell signalling in bacteria, thus inhibiting bacterial attachment and colonization on this plant.

The Phyllosphere of Aquatic Plants

This article has focused on the phyllosphere of leaves surrounded by air. However, submerged leaves of aquatic plants are associated with a phyllosphere that has properties similar to that of aerial leaves. A boundary layer forms at the solidliquid interface of submerged leaves. The

References
Beattie GA and Lindow SE (1999) Bacterial colonization of leaves: a spectrum of strategies. Phytopathology 89: 353359. Hirano SS and Upper CD (2000) Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae a pathogen, ice nucleus, and epiphyte. Microbiology and Molecular Biology Reviews 64: 624653.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net

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Hirano SS, Nordheim EV, Arny DC and Upper CD (1982) Lognormal distribution of epiphytic bacterial populations on leaf surfaces. Applied and Environmental Microbiology 44: 695700. Kinkel LL, Andrews JH and Nordheim EV (1989) Fungal immigration dynamics and community development on apple leaves. Microbial Ecology 18: 4558. Kinkel LL, Wilson M and Lindow SE (1995) Eects of scale on estimates of epiphytic bacterial populations. Microbial Ecology 29: 283297. Lindemann J and Upper CD (1985) Aerial dispersal of epiphytic bacteria over bean plants. Applied and Environmental Microbiology 50: 12291232. Lindemann J, Constantinidou HA, Barchett WR and Upper CD (1982) Plants as sources of airborne bacteria, including ice-nucleation-active bacteria. Applied and Environmental Microbiology 44: 10591063. Mechaber WL, Marshall DB, Mechaber RA, Jobe RT and Chew FS (1996) Mapping leaf surface landscapes. Proceedings of the National Academy of Sciences of the USA 93: 46004603. Morris CE, Monier JM and Jacques MA (1997) Methods for observing microbial biolms directly on leaf surfaces and recovering them for isolation of culturable microorganism. Applied and Environmental Microbiology 63: 15701576. Nemecek-Marshall M, MacDonald RC, Franzen JJ, Wojciechowski CL and Fall R (1995) Methanol emission from leaves. Plant Physiology 108: 13591368. Roden JS and Pearcy RW (1993) The eect of utter on the temperature of poplar leaves and its implications for carbon gain. Plant, Cell and Environment 16: 571577.

Simon RD, Abeliovich A and Belkin S (1994) A novel terrestrial halophilic environment: the phylloplane of Atriplex halimus, a saltexcreting plant. FEMS Microbiology Ecology 14: 99110. Wolber PK (1993) Bacterial ice nucleation. Advances in Microbial Physiology 34: 203237.

Further Reading
Andrews JH and Hirano SS (eds) (1991) Microbial Ecology of Leaves. New York: Springer-Verlag. Blakeman JP (ed.) (1981) Microbial Ecology of the Phylloplane. New York: Academic Press. Dickinson CH and Preece TF (eds) (1976) Microbiology of Aerial Plant Surfaces. New York: Academic Press. Fokkema NJ and van den Heuval JE (eds) (1986) Microbiology of the Phylloplane. Cambridge: Cambridge University Press. Kerstiens G (ed.) (1996) Plant Cuticles. Oxford: Bios Scientic Publishers. Lindow SE, Poinar E and Elliot V (eds) (2002) Phyllosphere Microbiology. St Paul, MN: American Phytopathological Press. Morris CE, Nicot PC and Nguyen-the CE (eds) (1996) Aerial Plant Surface Microbiology. New York: Plenum Press. Preece TF and Dickinson CH (eds) (1971) Ecology of Leaf Surface Micro-organisms. New York: Academic Press.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net