Susan S. Hirano

Bacteria in the Leaf Ecosystem with Emphasis on Pseudomonas syringae—a Pathogen, Ice Nucleus, and Epiphyte

SUMMARY The extremely large number of leaves produced by terrestrial and aquatic plants provide habitats for colonization by a diversity of microorganisms. This review focuses on the bacterial component of leaf microbial communities, with emphasis on Pseudomonas syringae—a species that participates in leaf ecosystems as a pathogen, ice nucleus, and epiphyte. Among the diversity of bacteria that colonize leaves, none has received wider attention than P. syringae, as it gained notoriety for being the first recombinant organism (Ice− P. syringae) to be deliberately introduced into the environment. We focus on P. syringae to illustrate the attractiveness and somewhat unique opportunities provided by leaf ecosystems for addressing fundamental questions of microbial population dynamics and mechanisms of plant-bacterium interactions. Leaf ecosystems are dynamic and ephemeral. The physical environment surrounding phyllosphere microbes changes continuously with daily cycles in temperature, radiation, relative humidity, wind velocity, and leaf wetness. Slightly longer-term changes occur as weather systems pass. Seasonal climatic changes impose still a longer cycle. The physical and physiological characteristics of leaves change as they expand, mature, and senesce and as host phenology changes. Many of these factors influence the development of populations of P. syringae upon populations of leaves. P. syringae was first studied for its ability to cause disease on plants. However, disease causation is but one aspect of its life strategy. The bacterium can be found in association with healthy leaves, growing and surviving for many generations on the surfaces of leaves as an epiphyte. A number of genes and traits have been identified that contribute to the fitness of P. syringae in the phyllosphere. While still in their infancy, such research efforts demonstrate that the P. syringae-leaf ecosystem is a particularly attractive system with which to bridge the gap between what is known about the molecular biology of genes linked to pathogenicity and the ecology and epidemiology of associated diseases as they occur in natural settings, the field.

Bacterial Community Dynamics

The term community has been used in a number of different ways by ecologists (cf. Krebs, 1985; Diamond and Case, 1986). Indeed, Roughgarden and Diamond (1986) state that “... a natural unambiguous definition of communities does not exist.” They further state that the most inclusive definition of a community is “all the organisms in a prescribed area,” a definition that is usually restricted by the criteria used to describe the organisms and the boundaries delimiting the area (Roughgarden and Diamond, 1986). If we follow this definition, the set of organisms that we address is restricted to bacteria; the area to that of a leaf or leaflet. Because a leaf is discrete in space, the boundary of the community is explicitly delineated. Unlike many terrestrial habitats in which the “edge” of the area of interest may be more arbitrarily defined, the leaf margin clearly describes the edge of the phyllosphere habitat. However, each of the many plants in a canopy produces many leaves; each is, in turn, separated in space and inhabited by a distinct assemblage of bacteria (i.e., a community). In a canopy, there exists not a single habitat, but rather populations of habitats. Thus, bacterial communities in the phyllosphere are best described in terms of populations of communities on populations of leaves.

Factors that Affect Spread of Pseudomonas syringae in the Phyllosphere

ABSTRACT Successful spread of an organism to a new habitat requires both immigration to and growth on that habitat. Field experiments were conducted to determine the relative roles of dispersal (i.e., immigration) and bacterial multiplication in spread of Pseudomonas syringae pv. syringae in the phyllosphere. To study spread, individual plots consisted of three nested concentric squares with the inner 6 m(2) planted to snap beans serving as the sink. Each sink, in turn, was surrounded by a barrier zone, usually 6 m wide, which was surrounded by a 6-m-wide source area. The source areas were planted with snap bean seeds inoculated with doubly marked strains derived from wild-type P. syringae pv. syringae B728a. The treatments were designed to test the effects of the nature and width of the barrier zone and suitability of the habitat in the sinks on spread of P. syringae pv. syringae. The marked strains introduced into the source areas at the time of planting were consistently detected in sink areas within a day or two after emergence of bean seedlings in the sources as assessed by leaf imprinting and dilution plating. The amounts of spread (population sizes of the marked strain in sinks) across barrier zones planted to snap bean (a suitable habitat for growth of P. syringae pv. syringae), soybean (not a favorable habitat for P. syringae pv. syringae), and bare ground were not significantly different. Thus, the nature of the barrier had no measurable effect on spread. Similarly, spread across bare-ground barriers 20 m wide was not significantly different from that across barriers 6 m wide, indicating that distance on this scale was not a major factor in determining the amount of spread. The suitability of the sink for colonization by P. syringae pv. syringae had a measurable effect on spread. Spread to sinks planted to clean seed was greater than that to sinks planted with bean seeds inoculated with a slurry of pulverized brown spot diseased bean leaves, sinks planted 3 weeks before sources, and sinks planted to a snap bean cultivar that does not support large numbers of P. syringae pv. syringae. Based of these results, we conclude that the small amount of dispersal that occurred on the scale studied was sufficient to support extensive spread, and suitability of the habitat for multiplication of P. syringae pv. syringae strongly influenced the amount of spread.

[54] Bacterial nucleation of ice in plant leaves

Publisher Summary This chapter discusses the procedures to detect the most efficient bacterial ice nucleus associated with a given plant part and procedures to quantitate bacterial ice nuclei associated with individual leaves or other individual plant parts. The most efficient heterogeneous ice nuclei known are certain bacterial cells. Only four bacterial species include strains that are ice nucleation active: (1) Pseudomonas syringae Van Hall, (2) P. fluorescence biotype G Migula, (3) P. viridiflava , and (4) Erwinia herbicola . These four bacterial species have in common the ability to colonize leaves and other plant parts. Not all strains within an ice nucleation-active (INA) bacterial species are active. The relative ice-nucleating activity of a bacterial suspension is expressed in terms of nucleation frequency (NF)—the ratio of the number of ice nuclei per unit volume to the number of viable bacterial cells per unit volume. The drop-freezing technique is modified in various ways to quantitate ice nuclei associated with plant material. Tube nucleation test is developed for detecting and in some cases quantitating bacterial ice nuclei associated with individual leaves, leaflets, or other individual plant parts. The ways in which the tube nucleation test is prepared and used is described in the chapter.

Use of an Intergenic Region in Pseudomonas syringae pv. Syringae B728a for Site-Directed Genomic Marking of Bacterial Strains for Field Experiments

ABSTRACT To construct differentially-marked derivatives of our model wild-type strain, Pseudomonas syringae pv. syringae B728a (a causal agent of bacterial brown spot disease in snap bean plants), for field experiments, we selected a site in the gacS-cysMintergenic region for site-directed insertion of antibiotic resistance marker cassettes. In each of three field experiments, population sizes of the site-directed chromosomally marked B728a derivatives in association with snap bean plants were not significantly different from that of the wild-type strain. Inserts of up to 7 kb of DNA in the intergenic region did not measurably affect fitness of B728a in the field. The site is useful for site-directed genomic insertions of single copies of genes of interest.

Predicting Behavior of Phyllosphere Bacteria in the Growth Chamber from Field Studies

Greenhouse and growth chamber have served the plant sciences well. Use of a controlled environment facilitates experimental manipulation, diminishes environmental variability and provides the means to separate variables and test hypotheses. Such fundamental studies as those that led to the discovery and/or elucidation of mechanisms of phenomena such as photoperiodism, phototropism, photosynthesis, phytohormones and many others were greatly facilitated by use of controlled environmental facilities. The eminent success of such an approach for many aspects of plant-microbe interactions is well documented. For example, screening for resistance to a number of diseases has been successfully conducted in controlled environments for decades.

Transposon Insertion in the ftsK Gene Impairs In Planta Growth and Lesion-Forming Abilities in Pseudomonas syringae pv. syringae B728a

A Tn5 insertion in the ftsK gene of Pseudomonas syringae pv. syringae B728a impaired brown spot lesion formation on Phaseolus vulgaris, the ability to grow within bean leaves, and swarming ability on semisolid agar. Plasmids containing the ftsK gene were sufficient to complement the original Tn5 mutant for lesion formation and swarming and partially restored in planta growth.