Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae-a pathogen, ice nucleus, and epiphyte

Abstract

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.

FIG. 1

(A) Three-dimensional schematic of a leaf. Phyllosphere bacteria colonize the surface of the epidermis and the intercellular spaces (apoplast). Reprinted from reference with permission. (B) Low-magnification electron micrograph of the adaxial surface of a pea (Pisum sativum) leaf. Reprinted from reference with permission. (C) Electron micrograph of a carbon replica of the adaxial surface of a corn (Zea mays) leaf. Note the waxy protuberances. Reprinted from reference with permission.

FIG. 2

(A) Scanning electron micrograph of bacteria on the surface of a corn (Z. mays) leaf. Photograph taken by J. Lindemann and M. Garment; reprinted from reference with permission. (B) Imprint of a corn leaf. Segments of a field-grown corn leaf were gently pressed onto the surface of King's medium B (146), a nonselective medium.

FIG. 3

Qualitative variability in bacterial populations on individual leaflets of field-grown snap bean (Phaseolus vulgaris) plants. Leaflets, sampled on the same day, were gently pressed onto King's medium B (146).

FIG. 4

P. syringae—pathogen, ice nucleus, and epiphyte. (A) Foliar and (B) pod symptoms of bacterial brown spot disease of snap bean (P. vulgaris) caused by P. syringae pv. syringae. (C) An ice nucleation event occurred in the test tube on the right due to the large numbers of ice nucleation-active P. syringae present on the leaf. (D) Symptoms of frost injury to snap bean plants in the field. (E) Asymptomatic snap bean leaves—habitats for P. syringae. Figures B, C, and E are reprinted from reference with the permission of the publisher.

FIG. 5

Ice nucleation spectrum of a strain of P. syringae. Cells of P. syringae were suspended in phosphate buffer (0.1 M, pH 7.0) to ca. 10⁸ CFU/ml. Tenfold serial dilutions were prepared from this suspension. Droplets from the original and each diluted suspension were placed on an aluminum block. The block was cooled, and the temperatures at which droplets froze were recorded. Each symbol represents determinations from one of the five suspensions. The concentration of ice nuclei N at temperature T [N(T)] in the suspension was calculated by the method of Vali (283). Nucleation frequency is the ratio of the number of ice nuclei to bacterial cell density.

FIG. 6

Quantitative variability in population sizes of P. syringae on individual leaves. (A) Each plate represents an equivalent dilution from washings of different individual rye leaves. (B) Lognormal distribution of population sizes of P. syringae on two sets of individual bean leaflets. The mean population size for both sets of leaflets is approximately 5.0 log CFU/leaflet. The population variances are 2.5 for set A (●) and 0.23 for set B (○). Reprinted from reference with the permission of the publisher.

FIG. 7

Immigration and emigration of bacteria to snap bean canopies. (A) Emigration rates are based on measurements of concentrations of air-borne bacteria at canopy height and 1.5 m above the canopy using six-stage Andersen viable samplers (171). (B) Immigration was measured by the deposition of air-borne bacteria onto petri dishes filled to the rim with King's medium B (146). Measurements of immigration and emigration were made over canopies of snap beans during three growing seasons. Data from J. Lindemann; reprinted from reference with the permission of the publisher.

FIG. 8

Insects as dispersal agents of P. syringae in the field. (A) Nonrandom distribution of P. syringae colonies on a semiselective medium (208) for P. syringae. The petri dishes, deployed at canopy height in a bean field, were exposed from 0600 to 0900 h, when leaves were wet with dew. (B and C) An insect (Glischrochilus quadrisignatus) was trapped in a sterile empty petri dish exposed in a bean canopy in the early morning. The trapped insect was transferred to a petri dish containing King's medium B (146) and allowed to walk over the surface of the medium. The white colonies in panel B are P. syringae. (C) The colonies fluoresced under UV light. Panel B is reprinted from reference with the permission of the publisher.

FIG. 9

Population dynamics of P. syringae and total culturable bacteria in association with snap bean leaves. (A and B) Plants at about 20 and 60 days after planting, respectively. (C and D) Population sizes of P. syringae and total CFU on bean cultivars that differ in susceptibility to brown spot disease. Cultivar ‘Eagle’ is more susceptible than cv. ‘Cascade’. Reprinted from reference with the permission of the publisher. (E) Bacterial population dynamics on cv. ‘Cascade’ during a growing season with little rain and high temperatures. (C, D, and E) At each sampling time, 30 individual leaflets were collected from the top of the canopy at 0800 h. Each sample was processed individually by dilution plating of leaf homogenates. The data are the means and standard error (SE) for each set of 30 leaflets. Variability in population sizes among the individual leaflets within a set is exemplified by the data shown in Fig. 6B.

FIG. 10

Effect of raindrop momentum on population sizes of P. syringae. (A) Modification of the microclimate with polyethylene shelters and inert fiberglass screens. The shelters were used to shield bean plants from rain when rain was imminent; the screens were used to decrease the momentum of raindrops as they passed through the screens and dripped onto the plants. (B) Phyllosphere population sizes of P. syringae on bean plants exposed to natural rains (control), plants shielded from rains (shelter), and plants under screens. Each datum point represents the mean log CFU per sample and SE based on three replicate plots per treatment with 10 individual samples per plot. Reprinted from reference with the permission of the publisher.

FIG. 11

Population dynamics of PPFMs, P. syringae, and total culturable bacteria in association with leaves of field-grown snap bean plants (cultivar Cascade). Under the relatively hot and dry weather conditions that prevailed during the experiment, the PPFMs flourished while P. syringae did not. Each datum point represents the mean and SE of bacterial population sizes based on 30 samples, collected at 0800 h each morning, and processed individually by dilution plating of leaf homogenates.

FIG. 12

(A) Population dynamics of P. syringae pv. syringae B728a (○) and Tn5 mutant derivatives 14 (▿) and 22 (▵), methionine auxotroph MX7 (□), and tryptophan auxotroph 94 (◊) on bean plants under field conditions in June. Each point represents the mean and SE of 12 leaf samples. Periods of daylight are indicated by dashed lines. Because means could not be estimated for data sets with 10 or more leaves harboring undetectable populations, means for these data sets (∗) were calculated using log (CFU per gram [fresh weight]) values of 0 for leaves with no detectable population. Reprinted from reference with permission. (B) Population dynamics of P. syringae pv. syringae B728a (○) and Tn5 mutant derivatives 14 (▿) and 22 (▵), methionine auxotroph MX7 (□), and tryptophan auxotroph 94 (◊) after vacuum infiltration into the intercellular spaces of leaves. Each point represents the mean and SE of eight samples, each composed of two 6.5-mm leaf disks. The values indicating the number of lesions induced by each strain are the mean and SE of seven plants, with the lesion number per plant represented by the mean number of lesions enumerated in three randomly chosen 1.2-cm² leaf regions. Reprinted from reference with permission.

FIG. 13

Organization of the hrp gene cluster in P. syringae pv. syringae strain 61. The gene designation employs the unified nomenclature for widely conserved hrp genes (hrc) (27). Arrowheads indicate the direction of transcription for each operon. Genes encoding proteins predicted to be associated with the inner or outer membrane of the type III secretion system are stippled and hatched, respectively, but HrcJ may be associated with both membranes. The cluster of hrp genes from hrpK to hrpR is similar in strains 61 (weakly virulent) and B728a (highly virulent). Flanking regions are not as highly conserved in the two strains. Reprinted from reference with permission.

FIG. 14

Population dynamics of P. syringae pv. syringae B728a hrp mutants (ΔhrpZ::nptII, hrpJ::ΩSpc, and ΔhrcC::nptII) in association with field-grown snap bean plants. The bacterial strains were inoculated onto seeds immediately before planting. Samples were seeds or germinating seedlings collected on or before days 7 (A) and 9 (B) after planting; primary leaves were collected between 9 and 14 (A) and 12 and 20 (B) days after planting; and single leaflets from trifoliolate leaves were collected at all other times. Each datum point represents the mean log CFU per sample and SE based on three (A) or four (B) replicate plots with six or eight individual samples per plot. Inverted solid triangles indicate rain events with sustained rates of >1 mm min⁻¹. Reprinted from reference with the permission of the publisher.

FIG. 15

gacS/gacA regulon in P. syringae pv. syringae B728a and phenotypes affected.

FIG. 16

Population dynamics of a gacS mutant of P. syringae pv. syringae B728a. (A) Bacterial strains (10⁶ CFU/ml) were infiltrated into primary leaves of growth chamber-grown bean plants. At each sampling time, leaf disks (6-mm diameter) were removed with a cork borer from each of five leaves. Each datum point represents the mean log CFU per sample and SE (n = 5). Unlike the hrpJ mutant, growth of the gacS mutant was indistinguishable from that of the wild type. (B) Population dynamics of a gacS mutant under field conditions. The bacterial strains were inoculated onto the foliage of 25-day-old snap bean plants. Population sizes were estimated by dilution plating of leaf homogenates. Each datum point represents the mean log CFU per sample and SE based on three replicate plots with eight individual leaf samples per plot. Reprinted from reference with the permission of the publisher.

FIG. 17

Interactions of P. syringae and leaf habitats—putting the puzzle together. An immigrant arrives on a leaf, either from growth on a germinating seed (bottom) or through the air. Conducive conditions such as a susceptible plant, intense rain, and early vegetative stage of plant development favor growth and establishment of large population sizes of P. syringae. Dry, hot conditions or an unfavorable plant will diminish growth and drive bacteria toward survival rather than growth. Some bacterial genes in the gac and hrp regulons and several housekeeping genes are necessary for sufficient growth to achieve large bacterial population sizes. Genes necessary to produce type IV pili may also enhance bacterial growth and survival. The larger the population size, the more bacteria are available to emigrate, either on insects when leaves are wet or as aerosols in dry sunny weather, or to be washed off by rain. As population sizes approach 10⁶ cells per leaflet, disease becomes likely. Genes in the gac regulon (gacS/gacA and salA) are required for lesion formation. Lesions may provide a site for enhanced survival of P. syringae during unfavorable weather. These processes are repeated throughout the growing season on each of the 10⁶ or more leaf habitats per hectare, with bacterial generation times on the order of 2 to 5 h. If the plants are allowed to mature, bacteria present on pods may move to seeds, where the bacteria can survive until the seeds are planted to begin the next plant generation. ↑ and ↓, positive and negative effects, respectively.